WO2000075184A9 - Modulation of protein levels using the scf complex - Google Patents

Abstract

This invention encompasses various methods of modulating protein levels using the SKP1, CDC53/Cullin, F-box(SCF) protein complex. More specifically, the present invention provides various methods of target protein degradation using targeted ubiquitination techniques. The present invention also provides various compositions and assays associated with the disclosed modulation of protein levels using the SCF complex as well as various methods of detecting, monitoring and treating cancerous cells.

Description

MODULATION OF PROTEIN LEVELS USING THE SCF COMPLEX

INVENTORS

Hui Zhang, Lyuben M. Tsvetkov and Takeshi Kondo

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application No. 60/137,494 filed June 4, 1999 which is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention pertains, in general, to the field of protein knockout technology. In particular, the present invention pertains to protein knockout technology using targeted ubiquitination techniques.

BACKGROUND OF THE INVENTION

All publications, patents and patent applications discussed herein are incorporated by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference. The paper by Tsvetkov et al, (1999) Current Biology 9, 661-664 including S1-S2 is fully and completely herein incorporated by reference.

Cyclin-Dependent Kinases (CDKs)

Entry into each phase of the eukaryotic cell division cycle is regulated by proteins known as cyclin-dependent kinases (CDKs). In mammalian cells, seven different CDK protein subtypes have been described, each of which has been associated with particular phases of the cell cycle (Matsushime et al, (1992) Cell 71, 323-334; Xiong et al, (1992)

Cell 71, 505-514; Meyerson et al, (1992) EMBO J. 11, 2909-2917; Fisher & Morgan,

(1994) Cell 78, 713-724; Meyerson & Harlow, (1994) Mol. Cell. Biol. 14, 2077-2086).

Activation of each CDK in the cell cycle is regulated by its association with an equally diverse family of regulatory subunits known as cyclins. Multiple cyclin-CDK associations have been implicated in cell cycle control during cell proliferation in mammals. For example, cyclin D-CDK4 is associated with cell cycle progression through Gi phase while both cyclin E-CDK2 and cyclin A-CDK2 facilitate Gi to S phase transition (Nasmyth & Hunt, (1993) Nature 366, 634-635).

CDKs are regulated at several different levels including phosphorylation and interaction with other proteins. Activation of CDKs is initially dependent on complex formation with their cognate cyclin subunits and is regulated at this stage by fluctuations in the levels of these subunits (Sherr, (1993) Cell 73, 1059-1065). Phosphorylation of a conserved threonine residue in CDK is essential for activation following cyclin-CDK complex formation (Solomon et al, (1993) EMBO J. 12, 3133-3142; Makela et al, (1994) Nature 371, 254-257). Studies have also focused on the role of CDK inhibitor proteins such as pi 6, p21 or p27, which act as another level of cell cycle regulation by preventing unscheduled entry into another phase of the cell cycle (Hunter & Pines, (1994) Cell 79, 573-583; Sherr, (1996) Science 274, 1672-1677). These proteins interact with specific domains surrounding the phosphorylated threonine residue on the CDK. p27 for example, inhibits cyclin E-CDK2 and has been characterized in detail (Polyak et al, (1994) Cell 78 59-36; U.S. Patent No. 5,688,665).

Transformed cells differ from normal cells in their ability to proliferate indicating that alterations in pathways which control cell cycle progression accompany cellular transformation. Alterations in the regulatory events underlying cellular proliferation pathways can translate into changes in the cyclin-CDK pathways controlling cell cycle progression and has long been implicated in cellular transformation. In normal cells each cyclin-CDK complex exists in a quaternary complex that also contains proliferating cell nuclear antigen (PCNA) and a CDK inhibitor protein. These quaternary complexes are absent in transformed cells because the CDK inhibitory protein is not expressed (Zhang et al, (1993) Mol. Biol. Cell 4, 897-906).

For example, studies in normal human fibroblasts demonstrated that cyclin A-CDK2 was associated with p21 and PCNA in a quaternary complex. p21 and PCNA were absent in other transformed cells or established tumor cell lines, and cyclin A-CDK2 was bound to three novel proteins to form a protein complex (Zhang et al, (1993) Mol. Biol. Cell 4, 897-906). The first two proteins in this complex were S-phase kinase associated proteins designated SKP1 and SKP2. The third protein, designated CUL1, is a member of the cullin/CDC53 family of proteins. The cyclin A-CDK2/SKP1/SKP2/CUL1 complex functions as a conserved ubiquitin E3 enzyme that regulates mammalian Gi to S phase transition by specifically targeting mammalian Gi regulators, such as p21 for ubiquitin-dependent degradation (Yu et al, (1998) Proc. Natl. Acad. Sci. USA 95, 11324-1 1329). Decreased levels of p21 in tumor cells confirm that p21 is being targeted for ubiquitin-dependent degradation in transformed cells (Xiong et al, (1993) Genes Dev. 7, 1572-1583; Yu et al, (1998) Proc. Natl Acad. Sci. USA 95, 11324-1 1329).

Ubiquitin-Dependent Protein Degradation Ubiquitin-dependent protein degradation functions to regulate protein turnover in a cell by closely regulating the degradation of specific proteins. Once a protein is tagged with ubiquitin it is degraded in an ATP-dependent reaction by the 26S proteosome. Ubiquitin is a small protein composed of seventy-six amino acids that serves only as a tag to mark proteins for degradation. Three distinct enzymes are required for protein ubiquitination (King et al, (1996) Science 274, 1652-1659). First, ubiquitin is activated in an ATP dependent reaction by forming a thioester bond with the ubiquitin activation enzyme designated El . The activated ubiquitin is then transferred from El to the ubiquitin conjugating enzyme designated E2. This enzyme mediates the transfer of ubiquitin to protein substrates in conjunction with a ligase enzyme designated E3. The ubiquitinated protein substrates are then degraded by the 26S proteosome.

S-Phase Kinase Associated Proteins

In many DNA viral oncoprotein transformed or other established tumor cells that are deficient in p53 expression, p21 and proliferating cell nuclear antigen (PCNA) disappeared and cyclin A/CDK2 was prominently complexed with two novel proteins, S- phase kinase associated proteins 1 and 2 (SKP1 and SKP2, also known as pi 9 and p45, respectively) (Yu et al, (1998) Proc. Natl. Acad. Sci. USA 95, 11324-11329; Zhang et al, (1997) WO9711176). SKP1 and SKP2 have been isolated and the genes encoding these proteins have been sequenced (Zhang et al, (1997) WO9711176; which is herein incorporated by reference in its entirety). SKP2 expression has been shown to be highly induced in many transformed cells (Zhang et al, (1995) Cell 82, 915-925, which is herein incorporated by reference in its entirety).

The SKP1/SKP2/CUL1 E3 ligase complex has been implicated in the ubiquitin-dependent degradation of p21 during cell cycle progression. Furthermore, p27 has also been shown to be a target of ubiquitin-dependent degradation in a CDC34- dependent proteolytic process. CDC34 serves as a ubiqutin E2 conjugating enzyme for SCF (SKP1, CDC53/Cullin, F-box protein) complexes (Yu et al., (1998) Proc. Natl. Acad. Sci. USA 95, 11324-11329; Pagano et al, (1995) Science 269, 682-685; King et al, (1996) Science 274, 1652-1659). The ubiquitin-dependent p27 degradation occurs during the transition from GI to S phase as indicated by the increase in the level of SKP2 in late GI which corresponds with a decrease in p27 levels. p27 ubiquitin-dependent degradation is also dependent on cyclin E/CDK2 activity (Brandeis & Hunt, (1996) EMBO J. 15, 5280-5289; Sheaff et al, (1997) Genes Dev. 11, 1464-1478).

SUMMARY OF THE INVENTION

The present invention encompasses a method of altering the level of polypeptide in a cell comprising altering the amount of one or more of the proteins selected from the group consisting of SKP1 , SKP2, SKP2-like protein and CUL-1. In a preferred embodiment, the polypeptide is phosphorylated and the SKP2-like protein is selected from the group consisting of ZF1 (SEQ ID NO: 27), ZF3 (SEQ ID NO: 29), ZF4 (SEQ ID NO: 31), ZF5 (SEQ ID NO: 33), ZF6 (SEQ ID NO: 35), ZF7 (SEQ ID NO: 37), ZF8 (SEQ ID NO: 39), ZF9 (SEQ ID NO: 41), ZF11 (SEQ ID NO: 43), ZF13 (SEQ ID NO: 45), ZF16 (SEQ ID NO: 47), ZF18 (SEQ ID NO: 49), ZF19 (SEQ ID NO: 51), ZF20 (SEQ ID NO: 53), ZF23 (SEQ ID NO: 55), ZF24 (SEQ ID NO: 57), ZF25 (SEQ ID NO: 59) and ZF26 (SEQ ID NO: 61).

In yet another preferred embodiment the polypeptide in the method of the invention is selected from the group consisting of p27 (SEQ ID NO: 65), cyclin E (SEQ ID NO: 63), Max (SEQ ID NO: 9), Mad (SEQ ID NO: 11), c-Myc (SEQ ID NO: 13), MDM2 (SEQ ID NO: 15), p53 (SEQ ID NO: 17), Bax (SEQ ID NO: 19), Bad (SEQ ID NO: 21) and Bcl-2 (SEQ ID NO: 23). The method of invention may be used to increase the level of polypeptide by decreasing the amount of SKP2 or in the alternative the level of polypeptide is reduced by increasing the amount of SKP2.

In a yet another embodiment, the invention includes a method of altering the level of SKP2 comprising altering the amount of p27 polypeptide which is available for binding with SKP2. In a further embodiment, the invention includes a method of modulating the activity of SKP2 comprising contacting SKP2 with a peptide comprising a SKP2 interaction domain which is available for binding with SKP2. In a preferred embodiment, the peptide is phosphorylated and the SKP2 interaction domain is derived from p27 or cyclin E. In a preferred embodiment, the peptide comprises any one of the amino acid sequences of SEQ ID NO: 1, 2, 3, 4, 5 or 6.

The invention also includes a method of treating a tumor in a mammal comprising altering the level of SKP protein in the cells of said tumor. In a preferred embodiment the SKP protein is SKP2 or allelic variants thereof. In a related embodiment the invention includes a method of detecting a tumor in a mammal wherein the level of SKP2 is used as a diagnostic and prognostic indicator to determine the progression of said tumor. In a preferred embodiment, the invention encompasses a method of monitoring the treatment of a tumor in a mammal wherein the level of SKP2 is used as a diagnostic and prognostic indicator.

The invention also includes methods of testing an agent for the ability to modulate an interaction between SKP2 and a target protein wherein the method comprises (a) fusing SKP2 with a target protein interaction domain to produce a SKP2 fusion protein; (b) contacting the agent, the SKP2 fusion protein and the target protein; and (c) determining whether the interaction of the SKP2 fusion protein with the target protein has been modulated by the agent. The invention further encompasses a method of altering the level of a target protein in a cell comprising inserting a heterologous target protein interaction domain with SKP2 or a SKP2-like protein to produce a fusion protein, and contacting fusion protein with the target protein. In a preferred embodiment, the SKP-2 like protein is selected from the group consisting of ZF1 (SEQ ID NO: 27), ZF3 (SEQ ID NO: 29), ZF4 (SEQ ID NO: 31), ZF5 (SEQ ID NO: 33), ZF6 (SEQ ID NO: 35), ZF7 (SEQ ID NO: 37), ZF8 (SEQ ID NO: 39), ZF9 (SEQ ID NO: 41), ZF11 (SEQ ID NO: 43), ZF13 (SEQ ID NO: 45), ZF16 (SEQ ID NO: 47), ZF18 (SEQ ID NO: 49), ZF19 (SEQ ID NO: 51), ZF20 (SEQ ID NO: 53), ZF23 (SEQ ID NO: 55), ZF24 (SEQ ID NO: 57), ZF25 (SEQ ID NO: 59) and ZF26 (SEQ ID NO: 61).

In yet another embodiment, the invention includes a method of altering the level of a target protein in a cell comprising expressing a cDNA coding for a SKP2 fusion protein comprising a SKP2 protein fused with a target protein interaction domain which is specific for the target protein. In a related embodiment, the invention includes a method of ubiquitinating a target protein comprising fusing a target protein interaction domain with SKP2, and contacting the SKP2 fusion protein with the target protein. In preferred embodiments, the target protein is selected from the group consisting of p27 (SEQ ID NO: 65), cyclin E (SEQ ID NO: 63), Max (SEQ ID NO: 9), Mad (SEQ ID NO: 1 1), c-Myc (SEQ ID NO: 13), MDM2 (SEQ ID NO: 15), p53 (SEQ ID NO: 17), Bax (SEQ ID NO: 19), Bad (SEQ ID NO: 21) and Bcl-2 (SEQ ID NO: 23).

The invention also includes a method of modulating protein ubiquitination comprising altering the amount of SKP2 which is available to facilitate protein ubiquitination.

Finally, the invention encompasses a fusion protein comprising a first protein comprising at least one SKP2 C-terminal motif (SCM) capable of interacting with SKP1 and forming a complex with CUL-1 and a second protein which is capable of interacting with a heterologous target protein. In a preferred embodiment, the fusion protein contains only one SCM capable of interacting with SKP1. In another preferred embodiment, the SCM is selected from any one of the following proteins selected from the group consisting of SKP2 (SEQ ID NO: 67), ZF1 (SEQ ID NO: 27), ZF3 (SEQ ID NO: 29), ZF4 (SEQ ID NO: 31), ZF5 (SEQ ID NO: 33), ZF6 (SEQ ID NO: 35), ZF7 (SEQ ID NO: 37), ZF8 (SEQ ID NO: 39), ZF9 (SEQ ID NO: 41), ZF11 (SEQ ID NO: 43), ZF13 (SEQ ID NO: 45),

ZF16 (SEQ ID NO: 47), ZF18 (SEQ ID NO: 49), ZF19 (SEQ ID NO: 51), ZF20 (SEQ ID NO: 53), ZF23 (SEQ ID NO: 55), ZF24 (SEQ ID NO: 57), ZF25 (SEQ ID NO: 59) and ZF26 (SEQ ID NO: 61). BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 - Phosphorylation-dependent p27 degradation in HeLa extracts (A) In vitro translated, ³⁵S-labeled p27 or p27 TI 87G mutant was incubated with HeLa extracts for 3 hours at 30 °C. The addition of cyclin E/CDK2 and the 26S proteosome inhibitor, MG132 (20 μl) is indicated. The p27 reaction products were isolated by immunoprecipitation and visualized by autoradiography. (B) Time course of p27 degradation using baculovirus produced and ³⁵S-labeled p27 (0.5 μg). (C) Baculovirus-expressed, ³⁵S-labeled p27 was incubated with HeLa extracts in the absence or presence of cyclin E/CDK2 and MG132. The reaction products were treated with lambda phosphatase (PPTase). The phosphorylated and high molecular weight p27 species (in brackets) are indicated.

Figure 2 - Ubiquitination of p27 in the HeLa cytosolic extracts

Accumulation of ubiquitinated p27 in the presence of modified ubiquitins. p27 was incubated with HeLa extracts, cyclin E/CDK2, and methyl ubiquitin (UbM, 0.5 mg/ml) and ubiquitin aldehyde (UbA, 1 μM) as indicated. The ubiquitinated p27 ladders accumulated because methylated ubiquitin shortens the polyubiquitinated chain and thus slows down the rate of degradation while ubiquitin aldehyde inhibits de-ubiquitination of ubiquitinated proteins by isopeptidases.

Figure 3 - Inhibition of p27 degradation by depletion of the SCF^{S1 P2} complex

(A) Depletion of CUL-1 abolishes p27 degradation. HeLa extracts were passed through the affinity purified CUL-1 antibody or a control IgG column. The control and CUL-1 depleted extracts were assayed for p27 degradation activity at various times at 30 °C in the presence of cyclin E/CDK2. The left three lanes are p27 input and regular HeLa extracts and incubated for three hours. Reaction products were treated with lambda phosphatase.

(B) SKPl depleted extracts. The extracts were control depleted using IgG or depleted with an SK l antibody column and then incubated with p27 as described in A. The reaction products were not treated with the phosphatase so the phosphorylated p27 are shown. Ext: regular HeLa extracts. (C) SKP2 depleted extracts. SKP2 was immuno-depleted as described in A and B. The control and SKP2 depleted extracts were incubated for three hours at 30 °C and the reaction products were analyzed after phosphatase treatment. (D) Specific removal of SKP2, SKPl, and CUL-1 by the immuno-depletion processes. The regular HeLa (Ext), control depleted, and SKP2, SKPl or CUL-1 depleted extracts were Western-blotted by SKP2 (top), SKPl (middle), and CUL-1 (bottom) antibodies, respectively.

Figure 4 - SCF^SKP2 complex specific interactions with thr! 87 phosphorylated p27 peptide (A) Sequences of p27 carboxy-terminal peptides (amino acids 175-198) with or without threonine 187 phosphorylation. (B) SKP2 specifically binds to the threonine 187 phosphopeptide of p27. F-box proteins, SKP2, β-TrCP, and MD6 were in vitro translated as ³⁵S-labeled proteins. The proteins (10 μl each) were incubated for one hour with the p27 peptide or threonine 187 phosphopeptide beads. The proteins associated with the beads were purified and analyzed. (C) Selective binding of SKP2, SKPl and CUL-1 in the HeLa extracts to the p27 threonine 187 phosphopeptide. HeLa extracts (400 μg) were incubated with the p27 phosphopeptide or peptide beads for one hour. The proteins associated with the beads were analyzed by Western-blot analysis with either SKP2, SKPl, CUL-1 or β-TrCP antibodies, as indicated. The immunoprecipitated SKP2, SKPl and CUL-1 are included as a control as indicated. For lane four, β-TrCP, HeLa extracts (25 μg) were directly loaded without immunoprecipitation. HeLa extracts for SKP2 immunoprecipitation were 25 μg while for SKPl and CUL-1 were 100 μg. (D)

Association between SKPl, SKP2 and CUL-1 in the HeLa extracts. The SKPl , SKP2 and CUL-1 were immunoprecipitated by specific antibodies, respectively, as indicated. The immunoprecipitated proteins were examined for the presence of SKP2 by Western-blotting with SKP2 antibodies. (E) Association between SKPl and p27 phosphopeptide depends on the presence of SKP2. HeLa (Ext), Mock or SKP2 depleted extracts (100 μg each) were incubated either with p27 peptide (pept) or phosphopeptide (phophopept) beads. The proteins associated with the peptide beads were examined for the presence of SKP2 or SKPl by Western blot. SKP2 and SKPl in the mock and SKP2 depleted extracts (25 μg each) were also examined by direct Western blotting of the extracts. Depl: depletion of extracts by pre-immune IgG (Mock) or SKP2 antibodies. Figure 5 - SCF^SKP2 complex contains a p27 ubiquitination E3 activity (A) Restoration of p27 degradation activity in SKP2 depleted extracts by recombinant SCF^{SI P2}. Insect SF9 cells were co-infected with baculoviruses encoding GST-SKP1 and CUL-1, either in the presence (SCF^SKP2) or in the absence of SKP2 (SC) baculoviruses. The SCF^SKP2 and SC complexes were isolated by glutathione Sepharose. The recombinant SCF^SKP2, SC (one μg each in two μl), or the buffer (two μl) was added into the SKP2 depleted extracts (200 μg), as indicated, and p27 degradation was assayed in the presence of cyclin E/CDK2. HeLa extract (ext) was included as the control. (B) p27 ubiquitination using recombinant proteins. p27 was incubated with the recombinant SCF^SKP2 complex, in the presence of purified cyclin E and cyclin A/CDK2 kinases, human El ubiquitin activating enzyme, ATP, ubiquitin, and recombinant CDC34, an E2 ubiquitin conjugation enzyme (lane 2). The reactions in lanes 3 and 4 were conducted in the absence of either ubiquitin or CDC34, respectively.

Figure 6 - SKP2 specifically binds to the phosphorylated Thr380 in cyclin E

(A) Sequences of the cyclin E carboxy-terminal peptides (residues 371-394) with (TP-CP) or without Thr380 (TP-C) phosphorylation. (B) SKP2 is specific for cyclin E phosphopeptide binding. In v/tro-translated and ⁵S-labeled F-box proteins SKP2, FBL2, 5, 6, 7, and 8 (10 μl each) were incubated with the TP-C or TP-CP beads. Their associations with cyclin E peptide beads were analyzed. Lysates: translated lysate control. (C) Specific interaction between SKP2 and the Thr380-phosphorylated cyclin E peptide in HeLa cell extracts. 400 μg of HeLa cytosolic extracts were incubated with either TP-C or TP-CP beads (25 μl) for one hour. The proteins associated with the beads were Western- blotted with antibodies against SKP2, SKPl, or CUL-1. Left lane, HeLa lysate control. (D) Upper panel - The phosphorylated TP motif is required for specific SKP2 interaction. Mutant derivatives of cyclin E peptides were synthesized in which either Thr380 is converted into serine or phosphoserine (SP-C or SP-CP) or Pro381 is converted into alanine in TP-CP (TA-CP). The beads bearing the wild-type and the mutant cyclin E peptides were assayed for SKP2 binding as described in (C). Lower panel - competition of the interaction between SKP2 and cyclin E Thr380 peptide beads (TP-CP) with wild-type or mutant cyclin E phosphopeptides. 25 μl TP-CP beads were incubated with HeLa extracts (400 μg) in the absence or in the presence of either TP-CP (10, 50 or 250 μg/ml) or equal amounts of TA-CP or SP-CP. The association between SKP2 and the TP-CP beads was analyzed by Western-blot.

Figure 7 - SKP2 promotes cyclin E ubiquitination and degradation

(A) Dependency of SKP2-mediated cyclin E degradation on Thr380 in cyclin E. T7- cyclin E or cyclin E T380G mutant constructs were transfected into HeLa cells in the presence or absence of SKP2 expression vector. Cell lysates were prepared in an SDS- containing buffer and 40 μg of each lysate were loaded directly onto a protein gel. The proteins were detected by Western-blotting with anti-T7 (top), SKP2 (middle), and CDK2 (lower) antibodies. (B) SKP2-induced formation of high molecular-weight species of cyclin E is sensitive to the Thr380 mutation in cyclin E. One microgram of T7-cyclin E or cyclin E T380G mutant expression constructs was transfected into 293 cells in the absence or presence of increasing amounts of the SKP2 construct (0, 0.25, 0.5, 1 and 2.5 μg, respectively). The proteins were detected by anti-T7 (top) or SKP2 (lower) antibodies. (C) SKP2 and ubiquitin both induce high-molecular-weight species of cyclin E. Expression vectors encoding SKP2 (5 μg), T7-tagged cyclin E (1 μg), or HA-tagged ubiquitin (HAUb, 1 μg) were transfected into 293 cells as indicated. Twenty-four hours post-transfection, cells were treated with LLNL for six hours. The proteins were detected by either anti-T7 monoclonal (top and middle panels) or anti-SKP2 (lower panel) antibodies. The middle panel is a lighter exposure of the top panel. (D) SKP2 promotes polyubiquitination of cyclin E. Expression vectors encoding SKP2 (5 μg), T7-tagged cyclin E (1 μg), or HA-tagged ubiquitin (HAUb, 0.1 μg) or a combination of them were transfected into 293 cells as indicated. The proteins were immunoprecipitated with the anti-HA antibody (12CA5) for ubiquitinated proteins followed by Western-blotting with anti-T7 antibody for cyclin E. (E) SKP2-mediated cyclin E ubiquitination is p27- independent but requires Thr380. p27-/- mouse embryonic fibroblasts were transfected with T7-cyclin E, SKP2 expression constructs, or both as described in B. The proteins were detected by anti-T7 (top) or SKP2 (lower) antibodies.

Figure 8 - SKP2 affects cyclin E stability by directly binding to cyclin E (A) SKP2 shortens the half-life of the cyclin E protein. Tagged-cyclin E expression construct was transfected into HeLa cells in the absence or in the presence of SKP2. Twenty-four hours after transfection, the cells were pulse-labeled with ³⁵S-methionine for thirty minutes The labeling medium was removed and the cells were chased in fresh medium containing 1 mM unlabeled methionine. The cells were harvested at various points (0, 1, 2, 3 and 4 hours) in the chasing medium and the labeled cyclin E protein was immunoprecipitated and examined. (B) Association of cyclin E with SKP2 in vivo. p27-/- mouse embryonic fibroblasts were transfected with DNA expression constructs encoding LacZ (β-Gal), T7-cyclin E, or the T380G cyclin E mutant. The lysates were prepared and immunoprecipitated with anti-cyclin E (left) or anti-SKP2 antibodies. The presence of cyclin E in the immunoprecipitates was examined with the anti-T7 antibody by Western- blotting. (C) Cyclin E degradation is inhibited by p27. SKP2 (5 μg), T7-tagged cyclin E (1 μg), LacZ (β-Gal), or p27 TI 87G mutant (1 μg) expression constructs were transfected into HeLa cells as indicated. The levels of cyclin E and p27 T187G mutant were detected with T7 and p27 antibodies.

Figure 9 - Effects of SKP2 on endogenous cyclin E

(A) SKP2 decreases the levels of endogenous cyclin E. U87EcoR cells were infected with recombinant retroviruses encoding either LacZ (β-gal) or SKP2. Thirty-six or sixty hours after infection, cell lysates were prepared and 40 μg of lysates were used for examination of the levels of endogenous cyclin E, CDK2, p27 and the expression of SKP2 by Western- blotting using their specific antibodies. (B) SKP2 induces cyclin E down-regulation in S- phase cells. Thirty-six hours post-retrovirus-infection, cells were treated with 5 mM HU for twenty-four more hours to synchronize cells in S phase. The levels of endogenous cyclin E and CDK2 as well as the expression of SKP2 were examined. (C) Expression of a dominant negative SKP2 mutant causes the accumulation of endogenous cyclin E. Glioblastoma U87EcoR cells were infected with recombinant retroviruses containing either an empty vector or a SKP2 dominant negative mutant (SKP2DN). The levels of either endogenous cyclin E, p27, SKP2 as well as the exogenous SKP2DN were examined forty-eight hours after infection. (D) The dominant negative effect of the SKP2 mutant on cyclin E accumulation is p27-independent. The experiment was performed in essentially the same way as in C, except that p27-/- mouse embryonic fibroblasts were used.

Figure 10 - Alteration of the substrate-specificity of F-box proteins The β-TRCP and SKP2 hybrid protein was generated to alter the substrate-specificity of β- TRCP to that of SKP2. The cDNA encoding the amino-terminus domain of β-TRCP (residues 1-204, including the F-box) was amplified with PCR and cloned into Bluescript at Xhol site. The cDNA containing the carboxy-terminus region of SKP2 without the F- box but retaining the LRR region (residues 169-435, without the F-box) was similarly amplified and fused with the amino-terminal region of β-TRCP. The resulting cDNA encoding the TRCP.N/SKP2.C hybrid protein is cloned into pcDNA3 under CMV promoter control. The corresponding truncated SKP2 carboxy-terminal region (SKP2.C) or the amino-terminal region of β-TRCP (β-TRCP .N) was also cloned into pcDNA3.

Figure 11 - TRCP.N/SKP2.C hybrid induces formation of polvubiquitinated cyclin E One microgram of T7-tagged cyclin E were transfected into 293 human embryonic kidney cells in the presence of either the control empty vector, SKP2, SKP2 amino-terminal region (SKP2.N, residues 1-168), β-TRCP amino-terminal region (TRCP.N), TRCP.N/SKP2.C hybrid, or SKP2 carboxy-terminal region (5 μg each) by the calcium phosphate method. Both SKP2.N and TRCP.N contain the F-box. Cell lysates were prepared twenty-four hours post-transfection in an SDS-containing buffer and 40 μg of each lysate were loaded directly onto a protein SDS-PAGE gel. The proteins were detected by Western-blotting with anti-T7 antibodies for the transfected cyclin E.

Figure 12 - F-box amino acid sequence alignment

Homologies within the F-box region between various F-box containing proteins.

Figure 13. - SCM domain amino acid sequence alignment Homology between the α domain of the von Hippel-Linda protein (NHL) (SEQ ID NO: 73) and the SCM domain of SKP2. Figure 14 - Dependency of SKP2-mediated cyclin E degradation on Thr380 in cyclin E T7-cyclin E or cyclin E T380G mutant constructs were transfected into HeLa cells in the presence or absence of SKP2 expression vector. Cell lysates were prepared and 40 μg of each lysate was loaded directly onto a protein SDS PAGE gel. The proteins were detected by Western-blotting with anti-T7 (upper) and SKP2 (lower) antibodies.

Figure 15 - Isolated SCF^SKP2 complex contains ubiquitination activity

The immunoprecipitated complex was incubated for 1 hour at 30°C with 6 μM ubiquitin, 2 mM ATP, 50 mM creatine phosphate, 20 μg/ml creatine kinase, 1 μg purified ubiquitin activating enzyme El, 1 μg purified E2 conjugating enzyme CDC34 in a buffer containing 20 mM Hepes, pH 7.2, 10 mM MgCh, 1 mM DTT. The ubiquitin reaction was terminated by addition of 0.5% SDS and loaded directly in an SDS-PAGE protein gel. The ubiquitinated proteins were detected by Western-blotting with the anti-ubiquitin antibody (Chemcon International) .

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.

A. Definitions As used herein, the term "agent" means any molecule that is randomly selected or rationally designed. As used herein, an agent is said to be randomly selected when the agent is chosen randomly without considering the specific sequences involved in the association of the proteins under study or the known functions of the proteins under study. An example of randomly selected agents is the use a chemical library, a peptide combinatorial library, or a growth broth of an organism. As used herein, an agent is said to be "rationally designed" when the agent is chosen on a non-random basis which takes into account the sequence of the proteins under study and/or their conformation in connection with the agent's action. Agents can be rationally selected or rationally designed by utilizing the amino acid sequences that make up potential contact sites between the proteins. For example, a rationally selected peptide agent can be a peptide whose amino acid sequence is identical to an identified contact site on one of the proteins under study. Such an agent will reduce or block the association of the protein with its binding partner by binding to the contact site on the first protein.

The agents of the present invention can be, as examples, peptides, small molecules, nucleic acids, vitamin derivatives, as well as carbohydrates. A skilled artisan can readily recognize that there is no limit as to the structural nature of the agents of the present invention.

The peptide agents of the invention can be prepared using standard solid phase (or solution phase) peptide synthesis methods, as is known in the art. In addition, the DNA encoding these peptides may be synthesized using commercially available oligonucleotide synthesis instrumentation and produced recombinantly using standard recombinant production systems. The production using solid phase peptide synthesis is necessitated if non-gene-encoded amino acids are to be included.

Another class of agents are antibodies immunoreactive with one of the proteins under study. Particularly useful are antibodies immunoreactive with the extracellular domain of membrane proteins under study. As described above, antibodies are obtained by immunization of suitable mammalian subjects with peptides, containing as antigenic regions, those portions of the protein intended to be targeted by the antibodies. Critical regions include the contact sites between the two proteins as well as extracellular regions of membrane proteins .

As used herein, the term "agonist" includes those agents, compounds, compositions, etc. which when administered can up-regulate (increase, promote or otherwise elevate the level of) a particular protein.

As used herein, an "allelic variant" refers to a proteins having different amino acid sequences than those sequences listed herein or incorporated by reference. For example, the allelic variants of p27, the target protein interaction domain of p27, SKPl, SKP2 or SKP2-like proteins, or CUL-1, though possessing a slightly different amino acid sequence, such as a conservative amino acid substitution, than those disclosed herein or incorporated by reference, will still have the requisite biological activity of the native protein. As used herein, a "conservative amino acid substitution" refers to alterations in the amino acid sequence of a protein which do not adversely effect their native abilities. Allelic variants, conservative substitution variants and related proteins and protein fragments utilized herein preferably will have an amino acid sequence having at least about 75% amino acid sequence identity with the published sequences, more preferably at least about 80%, even more preferably at least about 90%, and most preferably at least about 95%. Thus, the peptides, variants and related molecules that are the subject of or utilized in this invention include molecules having the sequences disclosed; fragments thereof having a consecutive sequence of at least about 3, 5, 10, 15, 20, 25, 30, 50 or more amino acid residues from the corresponding native proteins and amino acid sequence variants of such proteins, or their fragments as defined above, that have been conservatively substituted by another residues.

As used herein, the term "altering the level" of a particular protein means either increasing or decreasing the amount of that protein. For example, "altering the level of SKP2" means either increasing or decreasing the amount of SKP2.

As used herein, the term "antagonist" includes those agents, compounds, compositions, etc. which when administered cause the down regulation (inhibition, prevention, reduction, etc.) of a particular protein.

As used herein, the term "fusion protein" means a hybrid protein including a synthetic or heterologous amino acid sequence. A fusion protein can be produced, for example, from a hybrid gene containing operatively linking heterologous gene sequences. As used herein, the terms "isolated DNA, RNA, peptides, polypeptides, or proteins" means DNA, RNA, peptides, polypeptides or proteins that are isolated or purified relative to other DNA, RNA, peptides, polypeptides or proteins in the source material. For example, "isolated DNA" that encodes SKP2 (which would include cDNA) refers to DNA purified relative to DNA which encodes polypeptides other than SKP2. As used herein, the term "modulating the activity" of a particular protein means affecting the covalent or noncovalent binding of that protein with another protein. For example, when referring to "modulating the activity" of SKP2 this means affecting the binding of SKP2 with another protein, such as p27 or a peptide which includes the SKP2 interaction domain of p27.

As used herein, the term "pharmaceutically acceptable" refers to molecular entities and compositions such as fillers and excipients that are physiologically tolerated and do not typically produce an allergic or toxic reaction, such as gastric upset, dizziness and the like when administered to a subject or a patient; the preferred subjects of the invention are vertebrates, mammals and humans.

As used herein, the term "polypeptide" refers to a peptide which on hydrolysis yields more than two amino acids, called tripeptides, tetrapeptides, etc. according to the number of amino acids contained in the polypeptide. The term "polypeptide" is used synonomously with the term "protein" and "peptide"throughout the specification.

As used herein, "SCF" refers to a triple protein ligase consisting of SKP, Cullin and F-Box. As used herein, "SCM" refers to a SKP2 C-terminal motif.

As used herein, "SKP" refers to a S-phase kinase associated protein. Specific examples of SKP proteins include, but are not limited to, SKPl, SKP2 and SKP2-like proteins.

As used herein, "SKP2-like protein" refers to a protein which can replace SKP2 to form a complex with SKPl and CUL-1 or their yeast homologs. SKP2-like proteins are proteins that contain a SKPl interacting domain that is homologous to the SKPl interacting domain of the SKP2 sequence. Specific examples of SKP2-like proteins include, but are not limited to, ZFl (SEQ ID NO: 27), ZF3 (SEQ ID NO: 29), ZF4 (SEQ ID NO: 31), ZF5 (SEQ ID NO: 33), ZF6 (SEQ ID NO: 35), ZF7 (SEQ ID NO: 37), ZF8 (SEQ ID NO: 39), ZF9 (SEQ ID NO: 41), ZFl 1 (β-TRCP) (SEQ ID NO: 43), ZF13 (SEQ ID NO: 45), ZF16 (SEQ ID NO: 47), ZF18 (SEQ ID NO: 49), ZF19 (SEQ ID NO: 51), ZF20 (SEQ ID NO: 53), ZF23 (SEQ ID NO: 55), ZF24 (SEQ ID NO: 57), ZF25 (SEQ ID NO: 59) and ZF26 (SEQ ID NO: 61).

As used herein the term "SKPl interacting domain" refers to the region on the SKP2 protein that interacts with the SKPl protein. This region is also called the F-box for SKPl binding. As used herein the term "SKP2 interacting domain" refers to the region on a protein other than SKP2 that interacts with the SKP2 protein.

As used herein, the term "target protein" refers to an autologous or heterologous protein other than SKP2 which is targeted for interacting with a SKP2 or a SKP2-like protein.

As used herein, the term "target protein interaction domain" refers to a sequence which when fused to SKP2 or a SKP2-like protein interacts with a target protein.

As used herein, the term "ubiquitin" refers to a polypeptide found in all eukaryotic cells that participates in a variety of cellular functions including protein degradation. As used herein, the terms "ubiquitinating" and "ubiquitination" refer to processes whereby ubiquitin is attached to a protein.

B. SKP2-mediated Degradation of Target Proteins

Applicants have identified SKP2 as an F-box protein that mediates ubiquitin- dependent degradation of p27 (SEQ ID NO: 65) and cyclin E (SEQ ID NO: 63). SKP2 (SEQ ID NO: 67) is an F-box protein that is expressed in late GI, S, and G2 phases, playing a role in S phase of the cell cycle (Zhang et al, (1995) Cell 82, 915-925). SCF^{S1 P2} binds and targets the CDK inhibitor p27 for ubiquitin-dependent degradation. In addition, SKP2 also interacts with cyclin E and plays a role in the ubiquitin-dependent degradation of cyclin E. The present invention therefore includes methods for SKP2-mediated degradation of autologous and heterologous proteins. This SKP2-mediated cyclin E ubiquitination and degradation is mostly dependent on the presence of Thr380 in cyclin E, although weak cyclin E ubiquitination in the absence of Thr380 was also promoted by SKP2 in vivo. Although cyclin E ubiquitination is independent of p27, in the presence of co- expressed CDK inhibitor p27, cyclin E degradation was inhibited even in the presence of SKP2 (Figure 8C). This observation indicates that p27 might inhibit cyclin E autophosphorylation on Thr380, leading to resistance to SKP2-mediated ubiquitin- dependent degradation of cyclin E. The effect of p27 is not to be due to a competition between p27 and cyclin E for SKP2 binding, since a non-phosphorylated mutant form of p27 in which the critical Thrl87 was converted into glycine (T187G) cannot bind to SKP2. This data is consistent with the previous report that p27 inhibits the Thr380- dependent cyclin E degradation (Clurman et al, (1996) Genes Devel. 10, 1979-1990) and indicates that SKP2-mediated cyclin E ubiquitination is p27-independent.

Applicants have also identified that SKP2 performs a dual function during the Gl/S transition. It is required for the ubiquitin-dependent degradation of p27 in late GI . The degradation of p27 by SCF^SKP2 activates cyclin E/CDK2 and promotes entry into the S-phase (Sutterluty et al, (1999) Nat. Cell. Biol. 1, 207-14; Coats et al, (1996) Science 272, 877-880). Once cells are in the S phase, cyclin E is degraded which may be required for terminating the S-phase initiation events, allowing the cells to progress from the S phase into the G2 phase (Clurman et al, (1996) Genes Dev. 10, 1979-1990; Won et al,

(1996) EMBO J. 15, 4182-4193). Applicants have identified that SKP2 is also involved in the ubiquitin-dependent degradation of cyclin E and therefore the invention encompasses modulation of SKP2 activity and expression as a means of regulating cell cycle progression. Applicants have determined that a number of phosphorylation dependent and ubiquitin-dependent degradation events occur during the Gl/S transition, which are temporally regulated. The expression of SKP2 in the late GI and S phases leads to assembly of the SCF^SKP2 complex. Previous reports suggest that the phosphorylation status of p27 and cyclin E could be temporally separated. p27 phosphorylation on the critical Thrl 87 has been shown to occur in the late GI phase and p27 ubiquitination has been reported to require its binding to the cyclin E/CDK2 complex (Montagnoli et al, (1999) Genes Dev. 13, 1181-1189). The phosphorylation of Thrl87 in p27 triggers the binding of SKP2, leading to the subsequent ubiquitin-dependent degradation of p27.

It has been shown that binding of p27 to cyclin E/CDK complexes inhibits the activity of cyclin E/CDK2 and cyclin E degradation (Clurman et al, (1996) Genes Dev. 10, 1979-1990). The binding of p27 therefore prevents phosphorylation on Thr380 in cyclin E or there is a competition between p27 and cyclin E for the binding of SKP2. Applicants have also demonstrated that p27 binding can also cause a conformational change in cyclin E so that Thr380 in cyclin E is not exposed for phosphorylation or SKP2 binding. Applicants have determined that SKP2 binds to the p27 phosphopeptide with higher affinity than that of cyclin E peptide. Thus the affinities between SKP2 and p27 or cyclin E may also affect the ubiquitination rate of p27 and cyclin E by SKP2. Once p27 is degraded, the cyclin E/CDK2 kinase activity is activated, leading to the S-phase entry. Activation of cyclin E also leads to its autophosphorylation in Thr380 (Clurman et al, (1996) Genes Dev. 10, 1979-1990; Won & Reed, (1996) EMBO J. 15, 4182-4193). The phosphorylation of Thr380 promotes the SKP2 binding which in turn results in the ubiquitin-dependent degradation of cyclin E. The invention therefore encompasses peptides capable of blocking the interaction of SKP2 and SKP2-like proteins with autologous and heterologous target proteins.

C. Alteration of Substrate-Specificity of Various F-box Proteins

F-box proteins are the substrate-targeting component of the SCF complex (SKPl, CUL-1, F-box proteins) (Zhang et al, (1995) Cell 82, 915-925; Bai et al., (1996) Cell 86, 263-274; Feldman et al, (1997) Cell 91, 221-230; Skowyra et al., (1997) Cell 91, 209- 219). The F-box is a 40-50 amino-acid motif that is commonly present in the otherwise diverse proteins (Zhang et al., (1995) Cell 82, 915-925; Bai et al, (1996) Cell 86, 263- 274). This motif mediates the interaction between an F-box protein and SKPl (SEQ ID NO: 69). Applicants have identified about 30 F-box proteins which share no apparent homology except in the F-box motif (Figure 12). In mammals, two F-box proteins, SKP2 and β-TRCP, have been well characterized. Applicants have also determined that SKP2 binds to p27, a CDK inhibitor, through the phosphorylated threoninel 87 and this interaction targets p27 for ubiquitin-dependent degradation. Applicants further determined that SKP2 interacts with and ubiquitinates cyclin E when the threonine380 of cyclin E is phosphorylated. Likewise it has been shown that β-TRCP (ZFl 1) binds to two critical serine residues in both β-catenin (serines 33 and 37) and IκB-α (serines 32 and 36) when they are phosphorylated (Maniatis, (1999) Genes Dev. 13, 505-510; Winston et al, (1999) Genes Dev 13, 270-283; Spencer et al, (1999) Genes Dev. 13, 284-94; Yaron et al, (1998) Nature 396, 590-594). This interaction leads to the ubiquitination and degradation of β-catenin or IKB-C.. The difference in the substrate binding and thus the substrate specificity by these two F-box proteins is that SKP2 contains a substrate interaction domain of leucine-rich repeats (LRR) at its carboxy-terminal region (residues 220-400) (Zhang et al, (1995) Cell 82, 915-25) while β-TRCP (ZF11) instead has a completely different substrate-interaction domain consisting of WD40 repeats (WD) in the similar position (residues 212-569) (Winston et al, (1999) Genes Dev 13, 270-283).

The substrate-specificity of these protein-protein interaction domains has been established through the analysis of yeast F-box proteins such as CDC4, a WD-repeat containing F-box protein, and GRR1, an F-box protein that has LRR at its carboxy terminus (Skowyra et al, (1997) Cell 91, 209-219). Although the F-box proteins containing the LRR and WD repeats preferentially bind to substrates only when the substrates are phosphorylated, the existence of many F-box proteins that contain diverse protein-protein interaction domains indicates that many interact with target proteins directly without phosphorylation of the targets Winston et al, (1999) Curr. Biol. 9, 1 180- 1182; Cenciarelli et al, (1999) Curr. Biol. 9, 1177-1179).

The fact that various F-box proteins contain completely different substrate- interaction domains indicates that these domains are specifically used to contact substrates. Once the substrates are in association with the F-box proteins, the presence of the F-box region in the F-box proteins promotes the binding of SKPl and CUL-1 (SEQ ID NO: 71), as well as additional SCF components such as the recently identified Rbxl/Rocl&2 (Ohta et al, (1999) Mol. Cell. 3, 535-541 ; Skowyra et al, (1999) Science 284, 662-665; Kamura et al, (1999) Science 284, 657-661), to form the SCF complexes. The assembly of the complete SCF ubiquitin E3 ligase complexes promotes the ubiquitin-transfer reaction to the SCF-interacting substrates by the ubiquitin conjugating E2 enzyme, CDC34, and the ubiquitin activating enzyme El (Koepp et al, (1999) Cell 97, 431-434). The polyubiquitinated substrate proteins are subsequently degraded by the 26S proteasome. In the case of SKP2 and β-TRCP, the effect of mutation in the F-box region has been examined. Expression of mutant forms of SKP2 or β-TRCP that contain a deletion in the F-box but retain the complete substrate-interaction domain of LRR or WD repeats causes the protection of their respective substrates, p27, cyclin E or β-catenin and IκB (Carrano et al, (1999) Nat. Cell. Biol. 1, 193-199; Winston et al, (1999) Genes Dev. 13, 270-283; Spencer et al, (1999) Genes Dev. 13, 284-294) (Figure 9). This is because these SKP2 or β-TRCP mutants are fully capable of binding to the substrates while defective in recruiting the SKPl/CUL-1 into the complex, producing a dominant negative effect for the stability of the target proteins in vivo.

The concept of altering the substrate specificity of the various F-box proteins can thus be extended to fuse a protein interaction domain or a ligand binding site, in the form of either a protein, a peptide, or a chemical, with the F-box motif of either SKP2, β-TRCP (ZFl 1) or other F-box proteins (ZF series). In this design, this hybrid protein or molecule can be used to bind its normal protein partner and targets the protein partner for ubiquitin- dependent degradation. For example, if the F-box protein is fused with Max or Mad, proteins that bind to Myc oncoprotein (Blackwood & Eisenman, (1991) Science 251, 1211-1217; Blackwood et al, (1991) Cold Spring Harb. Symp. Quant. Biol. 56, 109- 117), the F-box/Max or Mad fusion protein will bind to and target Myc for ubiquitination and degradation.

Thus in one aspect of the invention, the protein levels of Myc in a cell can be modulated by such an F-box/Max or Mad hybrid construct. Another example is fusion of the amino-terminus of MDM2 (residues 1-158), a region that is known to bind the tumor suppressor protein p53 (Chen et al, (1993) Mol. Cell. Biol. 13, 4107-4114), with the F- box region derived from SKP2, β-TRCP and other F-box proteins. A hybrid F- box/MDM2 protein could be generated that would target p53 for ubiquitination. Such a pairwise selection can be extended to the cyclin-CDK (Hunter & Pines, (1994) Cell 79, 573-582), Bcl-2-Bax/Bad (Yang et al, (1995) Cell 80, 285-291 ; Chao & Kors eyer,

(1998) Annu. Rev. Immunol. 16, 395-419), and many others for the selective degradation of the desired targets.

The concept of modulating protein levels by the alteration of SCF substrate- targeting specificity can be further extended to include fusing the protein-interaction domains with a peptide or a chemical that interact with SKPl or CUL1 or the SCF complex. A fusion protein is an expression product resulting from the fusion of two genes. Such a protein may be produced, e.g., in recombinant DNA expression studies or, naturally, in certain viral oncogenes in which the oncogene is fused to gag.

The production of a fusion protein sometimes results from the need to place a cloned eukaryotic gene under the control of a bacterial promoter for expression in a bacterial system. Sequences of the bacterial system are then frequently expressed linked to the eukaryotic protein. Fusion proteins are used for the analysis of structure, purification, function, and expression of heterologous gene products.

A fused protein is a hybrid protein molecule which can be produced when a nucleic acid of interest is inserted by recombinant DNA techniques into a recipient plasmid and displaces the stop codon for a plasmid gene. The fused protein begins at the amino end with a portion of the plasmid protein sequence and ends with the protein of interest.

The production of fusion proteins is well known to one skilled in the art (see U.S. Patent Numbers 5,908,756; 5,907,085; 5,906,819; 5,905,146; 5,895,813; 5,891,643; 5,891,628; 5,891,432; 5,889,169; 5,889,150; 5,888,981; 5,888,773; 5,886,150; 5,886,149; 5,885,833; 5,885,803; 5,885,779; 5,885,580; 5,883,124; 5,882,941 ; 5,882,894; 5,882,864; 5,879,917; 5,879,893; 5,876,972; 5,874,304; and 5,874,290). For a general review of the construction, properties, applications and problems associated with specific types of fusion molecules used in clinical and research medicine, see Chamow et al, (1999) Antibody Fusion Proteins, John Wiley.

D. Modulation of SKP2 Expression and Activity

The identification of SKP2 and SKP2-like proteins has led to the discovery of compounds that are capable of down-regulating expression of these proteins. Molecules that down-regulate SKP2 and SKP2-like proteins are therefore part of the invention. Down-regulation is defined here as a decrease in activation, function or synthesis of SKP2 and SKP2-like proteins, its ligands or activators. It is further defined to include an increase in the degradation of the SKP2 gene, its protein product, ligands or activators. Down-regulation is therefore achieved in a number of ways. For example, administration of molecules that can destabilize the binding of SKP2 and SKP2-like proteins with its ligands. Such molecules encompass polypeptide products, including those encoded by the DNA sequences of the SKP2 gene or DNA sequences containing various mutations. These mutations may be point mutations, insertions, deletions or spliced variants of the SKP2 gene. This invention also includes truncated polypeptides encoded by the DNA molecules described above. These polypeptides being capable of interfering with interaction of SKP2 and SKP2-like proteins with other proteins. A further embodiment of this invention includes the down-regulation of SKP2 function by altering expression of the SKP2 gene, the use of antisense gene therapy being an example. Down-regulation of SKP2 or SKP2-like protein expression is accomplished by administering an effective amount of antisense oligonucleotides. These antisense molecules can be fashioned from the DNA sequence of the SKP2 gene or sequences containing various mutations, deletions, insertions or spliced variants. Another embodiment of this invention relates to the use of isolated RNA or DNA sequences derived from the SKP2 gene. These sequences containing various mutations such as point mutations, insertions, deletions or spliced variant mutations of SKP2 gene and can be useful in gene therapy.

Molecules that increase the degradation of the SKP2 or SKP2-like proteins may also be used to down-regulate its functions and are within the scope of the invention. Phosphorylation of SKP2 or SKP2-like proteins may alter protein stability, therefore kinase inhibitors may be used to down-regulate its function. Down-regulation of SKP2 or SKP2-like proteins may also be accomplished by the use of polyclonal or monoclonal antibodies or fragments thereof directed against the SKP2 or SKP2-like proteins. Such molecules are within the claimed invention. This invention further includes small molecules with the three-dimensional structure necessary to bind with sufficient affinity to block SKP2 or SKP2-like protein interactions with p27 or cyclin E. SKP2 or SKP2-like protein blockade resulting in decreased degradation of p27 or cyclin E and other processes of transformed cells where it is expressed make these small molecules useful as therapeutic agents in treating tumors.

The agents discussed above represent various effective therapeutic compounds in treating tumors. Applicants have thus provided antagonists and methods of identifying antagonists that are capable of down-regulating SKP2 or SKP2-like proteins.

A further embodiment of the invention relates to antisense or gene therapy. It is now known in the art that altered DNA molecules can be tailored to provide a specific selected effect, when provided as antisense or gene therapy. The native DNA segment coding for SKP2 has, as do all other mammalian DNA strands, two strands; a sense strand and an antisense strand held together by hydrogen bonds. The mRNA coding for SKP2 has a nucleotide sequence identical to the sense strand, with the expected substitution of thymidine by uridine. Thus, based upon the knowledge of the SKP2 sequence, synthetic oligonucleotides can be synthesized. These oligonucleotides can bind to the DNA and RNA coding for SKP2. The active fragments of the invention, which are complementary to mRNA and the coding strand of DNA, are usually at least about 15 nucleotides, more usually at least 20 nucleotides, preferably 30 nucleotides and more preferably may be 50 nucleotides or more. There is no upper limit, other than a practical limit, on the maximal size of such a nucleic acid molecule in that the nucleic acid molecule can include a portion of a gene, an entire gene, or multiple genes, or portions thereof. The binding strength between the sense and antisense strands is dependent upon the total hydrogen bonds. Therefore, based upon the total number of bases in the mRNA, the optimal length of the oligonucleotide sequence may be easily calculated by the skilled artisan. The sequence may be complementary to any portion of the sequence of the mRNA. For example, it may be proximal to the 5 '-terminus or capping site or downstream from the capping site, between the capping site and the initiation codon and may cover all or only a portion of the non-coding region or the coding region. The particular site(s) to which the antisense sequence binds will vary depending upon the degree of inhibition desired, the uniqueness of the sequence, the stability of the antisense sequence, etc.

In the practice of the invention, expression of SKP2 or SKP2-like proteins are down-regulated by administering an effective amount of synthetic antisense oligonucleotide sequences described above. The oligonucleotide compounds of the invention bind to the mRNA coding for human SKP2 thereby inhibiting expression (translation) of these proteins. The isolated DNA sequences containing various mutations such as point mutations, insertions, deletions or spliced mutations of SKP2 are useful in gene therapy as well. Antisense oligonucleotides can also be used as tools in vitro to determine the biological function of genes and proteins. Oligonucleotide phosphorothioates (PS-oligos) have also shown great therapeutic potential as antisense-mediated inhibitors of gene expression. Various methods have been developed for the synthesis of antisense oligonucleotides. See Agrawal et al, (1993) Methods of Molecular Biology: Protocols for Oligonucleotides and Analogs, Humana Press; Eckstein et al, (1991) Oligonucleotides and Analogues: A Practical Approach, Oxford University Press). E. Diagnostic Assays

In another diagnostic embodiment, susceptibility to certain tumors associated with elevated levels of SKP2 or SKP2-like proteins in a human subject can be measured by the steps of: (a) measuring the level of SKP2 or SKP2-like proteins in a biological sample from said human subject; and (b) comparing the level of SKP2 or SKP2-like proteins present in normal subjects, wherein an increase in the level of SKP2 or SKP2-like proteins as compared to normal levels indicates a predisposition to certain tumors.

In another diagnostic embodiment, a therapeutic treatment of certain tumors associated with elevated levels of SKP2 or SKP2-like proteins in a human subject may be monitored by measuring the levels of SKP2 or SKP2-like proteins in a series of biologic samples obtained at different time points from said subject undergoing therapeutic treatment wherein a significant decrease in said levels of SKP2 or SKP2-like proteins indicates a successful therapeutic treatment. Diagnostic probes useful in such assays of the invention include antibodies to

SKP2 or SKP2-like proteins. The antibodies to SKP2 or SKP2-like proteins may be either monoclonal or polyclonal, produced using standard techniques well known in the art (See Harlow & Lane, (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press). They can be used to detect SKP2 or SKP2-like proteins by binding to the protein and subsequent detection of the antibody-protein complex by ELISA, Western blot or the like. The SKP2 or SKP2-like proteins used to elicit these antibodies can be any of the SKP2 or SKP2-like proteins variants discussed above. Antibodies are also produced from peptide sequences of SKP2 or SKP2-like proteins using standard techniques in the art (See Protocols in Immunology, John Wiley & Sons, 1994). Fragments of the monoclonals or the polyclonal antisera which contain the immunologically significant portion can also be prepared. Use of immunologically reactive fragments, such as the Fab, Fab', of F(ab')₂ fragments is often preferable, especially in a therapeutic context, as these fragments are generally less immunogenic than the whole immunoglobulin.

Assays to detect or measure SKP2 or SKP2-like proteins polypeptide in a biological sample with an antibody probe may be based on any available format. For instance, in immunoassays where SKP2 or SKP2-like proteins are the analyte, the test sample, typically a biological sample, is incubated with anti-SKP2 antibodies under conditions that allow the formation of antigen-antibody complexes. Various formats can be employed, such as "sandwich" assay where antibody bound to a solid support is incubated with the test sample; washed, incubated with a second, labeled antibody to the analyte; and the support is washed again. Analyte is detected by determining if the second antibody is bound to the support. In a competitive format, which can be either heterogeneous or homogeneous, a test sample is usually incubated with an antibody and a labeled competing antigen, either sequentially or simultaneously. These and other formats are well known in the art.

F. Methods to Identify Binding Partners

Another embodiment of the present invention provides methods for use in isolating and identifying binding partners of proteins of the invention. In detail, a protein of the invention is mixed with a potential binding partner or an extract or fraction of a cell under conditions that allow the association of potential binding partners with the protein of the invention. After mixing, peptides, polypeptides, proteins or other molecules that have become associated with a protein of the invention are separated from the mixture. The binding partner bound to the protein of the invention can then be removed and further analyzed. To identify and isolate a binding partner, the entire protein, for instance the entire SKP2 or SKP2-like protein can be used. Alternatively, a fragment of the protein can be used, such as the SKP-1 interacting domain.

As used herein, a cellular extract refers to a preparation or fraction which is made from a lysed or disrupted cell. The preferred source of cellular extracts will be cells derived from human tissue, for instance, malignant tissue. Alternatively, cellular extracts may be prepared from any source of malignant tissue or available cell lines.

A variety of methods can be used to obtain an extract of a cell. Cells can be disrupted using either physical or chemical disruption methods. Examples of physical disruption methods include, but are not limited to, sonication and mechanical shearing. Examples of chemical lysis methods include, but are not limited to, detergent lysis and enzyme lysis. A skilled artisan can readily adapt methods for preparing cellular extracts in order to obtain extracts for use in the present methods. Once an extract of a cell is prepared, the extract is mixed with the protein of the invention under conditions in which association of the protein with the binding partner can occur. A variety of conditions can be used, the most preferred being conditions that closely resemble conditions found in the cytoplasm of a human cell. Features such as osmolarity, pH, temperature, and the concentration of cellular extract used, can be varied to optimize the association of the protein with the binding partner.

After mixing under appropriate conditions, the bound complex is separated from the mixture. A variety of techniques can be utilized to separate the mixture. For example, antibodies specific to a protein of the invention can be used to immunoprecipitate the binding partner complex. Alternatively, standard chemical separation techniques such as chromatography and density-sediment centrifugation can be used.

After removal of non-associated cellular constituents found in the extract, the binding partner can be dissociated from the complex using conventional methods. For example, dissociation can be accomplished by altering the salt concentration or pH of the mixture.

To aid in separating associated binding partner pairs from the mixed extract, the protein of the invention can be immobilized on a solid support. For example, the protein can be attached to a nitrocellulose matrix or acrylic beads. Attachment of the protein to a solid support aids in separating peptide-binding partner pairs from other constituents found in the extract. The identified binding partners can be either a single protein or a complex made up of two or more proteins. Alternatively, binding partners may be identified using the Alkaline Phosphatase fusion assay according to the procedures of Flanagan & Vanderhaeghen, (1998) Annu. Rev. Neurosci. 21, 309-345 or Takahashi et al., (1999) Cell 99, 59-69; the Far- Western assay according to the procedures of Takayama et al, (1997) Methods Mol. Biol. 69, 171-184 or Sauder et al., J. Gen. Virol. (1996) 77, 991-996 or identified through the use of epitope tagged proteins or GST fusion proteins.

Alternatively, the nucleic acid molecules of the invention can be used in a yeast two-hybrid system. The yeast two-hybrid system has been used to identify other protein partner pairs and can readily be adapted to employ the nucleic acid molecules herein described (see Stratagene Hybrizap^® two-hybrid system). G. Methods to Identify Agents that Modulate Expression

Another embodiment of the present invention provides methods for identifying agents that modulate the expression of a nucleic acid encoding the SKP2 protein, or of a nucleic acid encoding the SKP2 or SKP2-like protein such as a protein. Such assays may utilize any available means of monitoring for changes in the expression level of the nucleic acids of the invention. As used herein, an agent is said to modulate the expression of a nucleic acid, for instance a nucleic acid encoding the protein having the sequence of SKP2, SKP2-like proteins, SKPl, CUL-1, or any F-box containing protein such as a ZF protein, if it is capable of up- or down-regulating expression of the nucleic acid in a cell. In one assay format, cell lines that contain reporter gene fusions between the open reading frame of SKP2 or a SKP2-like protein and any assayable fusion partner may be prepared. Numerous assayable fusion partners are known and readily available including the firefly luciferase gene and the gene encoding chloramphenicol acetyltransferase (Alam et al, (1990) Anal. Biochem. 188, 245-254). Cell lines containing the reporter gene fusions are then exposed to the agent to be tested under appropriate conditions and time. Differential expression of the reporter gene between samples exposed to the agent and control samples identifies agents which modulate the expression of a nucleic acid encoding an SKP2, SKP2-like or ZF protein.

Additional assay formats may be used to monitor the ability of the agent to modulate the expression of a nucleic acid encoding a SKP-2 or SKP2-like protein. For instance, mRNA expression may be monitored directly by hybridization to the nucleic acids of the invention. Cell lines are exposed to the agent to be tested under appropriate conditions and time and total RNA or mRNA is isolated by standard procedures such those disclosed in Sambrook et al, (1989) Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory Press.

Probes to detect differences in RNA expression levels between cells exposed to the agent and control cells may be prepared from the nucleic acids of the invention. It is preferable, but not necessary, to design probes which hybridize only with target nucleic acids under conditions of high stringency. Only highly complementary nucleic acid hybrids form under conditions of high stringency. Accordingly, the stringency of the assay conditions determines the amount of complementarity which should exist between two nucleic acid strands in order to form a hybrid. Stringency should be chosen to maximize the difference in stability between the probe:target hybrid and potential probe:non-target hybrids.

Probes may be designed from the nucleic acids of the invention through methods known in the art. For instance, the G+C content of the probe and the probe length can affect probe binding to its target sequence. Methods to optimize probe specificity are commonly available in Sambrook et al, (1989) Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory Press or Ausubel et al, (1995) Current Protocols in Molecular Biology, Greene Publishing. Hybridization conditions are modified using known methods, such as those described by Sambrook et al, (1989) and Ausubel et al, (1995) as required for each probe. Hybridization of total cellular RNA or RNA enriched for polyA+ RNA can be accomplished in any available format. For instance, total cellular RNA or RNA enriched for polyA+ RNA can be affixed to a solid support and the solid support exposed to at least one probe comprising at least one, or part of one of the sequences of the invention under conditions in which the probe will specifically hybridize. Alternatively, nucleic acid fragments comprising at least one, or part of one of the sequences of the invention can be affixed to a solid support, such as a silicon based wafer or a porous glass wafer. The wafer can then be exposed to total cellular RNA or polyA+ RNA from a sample under conditions in which the affixed sequences will specifically hybridize. Such wafers and hybridization methods are widely available, for example, those disclosed by Beattie, (WO9511755). By examining for the ability of a given probe to specifically hybridize to a RNA sample from an untreated cell population and from a cell population exposed to the agent, agents which up or down regulate the expression of a nucleic acid encoding the SKP2 protein are identified.

Hybridization for qualitative and quantitative analysis of mRNA may also be carried out by using a RNase Protection Assay (i.e., RPA, see Ma et al, Methods (1996) 10, 273-238). Briefly, an expression vehicle comprising cDNA encoding the gene product and a phage specific DNA dependent RNA polymerase promoter (e.g., T7, T3 or SP6 RNA polymerase) is linearized at the 3' end of the cDNA molecule, downstream from the phage promoter, wherein such a linearized molecule is subsequently used as a template for synthesis of a labeled antisense transcript of the cDNA by in vitro transcription. The labeled transcript is then hybridized to a mixture of isolated RNA (i.e., total or fractionated mRNA) by incubation at 45°C overnight in a buffer comprising 80% formamide, 40 mM Pipes, pH 6.4, 0.4 M NaCl and 1 mM EDTA. The resulting hybrids are then digested in a buffer comprising 40 μg/ml ribonuclease A and 2 μg/ml nbonuclease. After deactivation and extraction of extraneous proteins, the samples are loaded onto urea-polyacrylamide gels for analysis.

In another assay format, agents which effect the expression of the instant gene products, cells or cell lines would first be identified which express said gene products physiologically. Cells and cell lines so identified would be expected to comprise the necessary cellular machinery such that the fidelity of modulation of the transcriptional apparatus is maintained with regard to exogenous contact of agent with appropriate surface transduction mechanisms and the cytosolic cascades. Further, such cells or cell lines would be transduced or transfected with an expression vehicle (e.g., a plasmid or viral vector) construct comprising an operable non-translated 5 '-promoter containing end of the structural gene encoding the instant gene products fused to one or more antigenic fragments, which are peculiar to the instant gene products, wherein said fragments are under the transcriptional control of said promoter and are expressed as polypeptides whose molecular weight can be distinguished from the naturally occurring polypeptides or may further comprise an immunologically distinct tag. Such a process is well known in the art (see, Sambrook et al, (1989) Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory Press).

Cells or cell lines transduced or transfected as outlined above would then be contacted with agents under appropriate conditions; for example, the agent comprises a pharmaceutically acceptable excipient and is contacted with cells in an aqueous physiological buffer such as phosphate buffered saline (PBS) at physiological pH, Eagles balanced salt solution (BSS) at physiological pH, PBS or BSS comprising serum or conditioned media comprising PBS or BSS and serum incubated at 37°C. Said conditions may be modulated as deemed necessary by one of skill in the art. Subsequent to contacting the cells with the agent, said cells will be disrupted and the polypeptides of the disruptate are fractionated such that a polypeptide fraction is pooled and contacted with an antibody to be further processed by immunological assay (e.g., ELISA, immunoprecipitation or Western blot). The pool of proteins isolated from the "agent contacted" sample will be compared with a control sample where only the excipient is contacted with the cells and an increase or decrease in the immunologically generated signal from the "agent contacted" sample compared to the control will be used to distinguish the effectiveness of the agent.

H. Methods to Identify Agents that Modulate Activity

Another embodiment of the present invention provides methods for identifying agents that modulate at least one activity of a protein of the invention such as SKP2 or SKP2-like proteins. Such methods or assays may utilize any means of monitoring or detecting the desired activity.

The present invention includes methods of screening for compounds which deactivate, or act as antagonists of SKP2 or SKP2-like protein expression. Such compounds may be useful in the modulation of pathological conditions associated with alterations in SKP2, SKP2-like or p27 protein levels.

In one format, the relative amounts of a SKP2 protein between a cell population that has been exposed to the agent to be tested compared to an un-exposed control cell population may be assayed. In this format, probes such as specific antibodies are used to monitor the differential expression of the protein in the different cell populations. Cell lines or populations are exposed to the agent to be tested under appropriate conditions and time. Cellular lysates may be prepared from the exposed cell line or population and a control, unexposed cell line or population. The cellular lysates are then analyzed with the probe.

Antibody probes are prepared by immunizing suitable mammalian hosts in appropriate immunization protocols using the SKP2 or SKP2-like proteins if they are of sufficient length, or if desired, or if required to enhance immunogenicity, conjugated to suitable carriers. Methods for preparing immunogenic conjugates with carriers such as BSA, KLH, or other carrier proteins are well known in the art. In some circumstances, direct conjugation using, for example, carbodiimide reagents may be effective; in other instances linking reagents such as those supplied by Pierce Chemical Co. may be desirable to provide accessibility to the hapten. The hapten peptides can be extended at either the amino or carboxy terminus with a cysteine residue or interspersed with cysteine residues, for example, to facilitate linking to a carrier. Administration of the immunogens is conducted generally by injection over a suitable time period and with use of suitable adjuvants, as is generally understood in the art. During the immunization schedule, titers of antibodies are taken to determine adequacy of antibody formation.

While the polyclonal antisera produced in this way may be satisfactory for some applications, for pharmaceutical compositions, use of monoclonal preparations is preferred. Immortalized cell lines which secrete the desired monoclonal antibodies may be prepared using the standard method of Kohler & Milstein, (1992) Biotechnology 24, 524- 526 or modifications which effect immortalization of lymphocytes or spleen cells, as is generally known. The immortalized cell lines secreting the desired antibodies are screened by immunoassay in which the antigen is the peptide hapten, polypeptide or protein. When the appropriate immortalized cell culture secreting the desired antibody is identified, the cells can be cultured either in vitro or by production in ascites fluid. The desired monoclonal antibodies may be recovered from the culture supernatant or from the ascites supernatant. Fragments of the monoclonals or the polyclonal antisera which contain the immunologically significant portion can be used as antagonists, as well as the intact antibodies. Use of immunologically reactive fragments, such as the Fab, Fab' of F(ab')₂ fragments is often preferable, especially in a therapeutic context, as these fragments are generally less immunogenic than the whole immunoglobulin.

The antibodies or fragments may also be produced, using current technology, by recombinant means. Antibody regions that bind specifically to the desired regions of the protein can also be produced in the context of chimeras with multiple species origin.

Antibody regions that bind specifically to the desired regions of the protein can also be produced in the context of chimeras with multiple species origin, for instance, humanized antibodies. The antibody can therefore be a humanized antibody or human a antibody, as described in U. S. Patent No. 5,585,089 or Riechmann et al, (1988) Nature 332, 323-327.

Agents that are assayed in the above method can be randomly selected or rationally selected or designed. As used herein, an agent is said to be randomly selected when the agent is chosen randomly without considering the specific sequences involved in the association of the a protein of the invention alone or with its associated substrates, binding partners, etc. An example of randomly selected agents is the use a chemical library or a peptide combinatorial library, or a growth broth of an organism.

As used herein, an agent is said to be rationally selected or designed when the agent is chosen on a non-random basis which takes into account the sequence of the target site or its conformation in connection with the agent's action. Agents can be rationally selected or rationally designed by utilizing the peptide sequences that make up these sites. For example, a rationally selected peptide agent can be a peptide whose amino acid sequence is identical to the SKPl or SKP2 interaction domain on a autologous or heterologous target protein which interacts with the SKP2 protein or its targets.

The agents of the present invention can be, as examples, peptides, small molecules, vitamin derivatives, as well as carbohydrates. A skilled artisan can readily recognize that there is no limit as to the structural nature of the agents of the present invention.

The peptide agents of the invention can be prepared using standard solid phase (or solution phase) peptide synthesis methods, as is known in the art. In addition, the DNA encoding these peptides may be synthesized using commercially available oligonucleotide synthesis instrumentation and produced recombinantly using standard recombinant production systems. The production using solid phase peptide synthesis is necessitated if non-gene-encoded amino acids are to be included. Another class of agents of the present invention are antibodies immunoreactive with critical positions of proteins of the invention. For example, antibodies which specifically interact with the SKPl interacting domain, SKP2 interacting domain or the SKP2 C-terminal motif. Antibody agents are obtained by immunization of suitable mammalian subjects with peptides, containing as antigenic regions, those portions of the protein intended to be targeted by the antibodies.

I. High Throughput Assays

The power of high throughput screening is utilized to the search for new compounds which are capable of interacting with the SKP2 or SKP-2 like proteins. For general information on high-throughput screening (see Devlin, (1998) High Throughput Screening, Marcel Dekker; U.S. Patent No. 5,763,263). High throughput assays utilize one or more different assay techniques.

Immunodiagnostics and Immunoassays. These are a group of techniques used for the measurement of specific biochemical substances, commonly at low concentrations in complex mixtures such as biological fluids, that depend upon the specificity and high affinity shown by suitably prepared and selected antibodies for their complementary antigens. A substance to be measures must, of necessity, be antigenic -either an immunogenic macromolecule or a haptenic small molecule. To each sample a known, limited amount of specific antibody is added and the fraction of the antigen combining with it, often expressed as the bound: free ratio, is estimated, using as indicator a form of the antigen labeled with radioisotope (radioimmunoassay), fluorescent molecule (fluoroimmunoassay), stable free radical (spin immunoassay), enzyme (enzyme immunoassay), or other readily distinguishable label.

Antibodies can be labeled in various ways, including: enzyme-linked immunosorbent assay (ELISA); radioimmuno assay (RIA); fluorescent immunoassay

(FIA); chemiluminescent immunoassay (CLIA); and labeling the antibody with colloidal gold particles (immunogold).

Common assay formats include the sandwhich assay, competitive or competition assay, latex agglutination assay, homogeneous assay, microtitre plate format and the microparticle-based assay.

Enzyme-linked immunosorbent assay (ELISA). ELISA is an immunochemical technique that avoids the hazards of radiochemicals and the expense of fluorescence detection systems. Instead, the assay uses enzymes as indicators. ELISA is a form of quantitative immunoassay based on the use of antibodies (or antigens) that are linked to an insoluble carrier surface, which is then used to "capture" the relevant antigen (or antibody) in the test solution. The antigen-antibody complex is then detected by measuring the activity of an appropriate enzyme that had previously been covalently attached to the antigen (or antibody).

For information on ELISA techniques, see, for example, Crowther, (1995) ELISA - Theory and Practice (Methods in Molecular Biology), Humana Press; Challacombe &

Kemeny, (1998) ELISA and Other Solid Phase Immunoassays - Theoretical and Practical Aspects, John Wiley; Kemeny, (1991) A Practical Guide to ELISA, Pergamon Press; Ishikawa, (1991) Ultrasensitive and Rapid Enzyme Immunoassay (Laboratory Techniques in Biochemistry and Molecular Biology) Elsevier.

Colorimetric Assays for Enzymes. Colorimetry is any method of quantitative chemical analysis in which the concentration or amount of a compound is determined by comparing the color produced by the reaction of a reagent with both standard and test amounts of the compound, often using a colorimeter. A colorimeter is a device for measuring color intensity or differences in color intensity, either visually or photoelectrically. Standard colorimetric assays of beta-galactosidase enzymatic activity are well known to those skilled in the art (see, for example, Norton et al, (1985) Mol. Cell. Biol. 5, 281-290). A colorimetric assay can be performed on whole cell lysates using O-nitrophenyl-beta-D-galactopyranoside (ONPG, Sigma) as the substrate in a standard colorimetric beta-galactosidase assay (Sambrook et al, (1989) Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory Press. Automated colorimetric assays are also available for the detection of beta-galactosidase activity, as described in U.S. Patent No. 5,733,720.

Immunofluorescence Assays. Immunofluorescence or immunofluorescence microscopy is a technique in which an antigen or antibody is made fluorescent by conjugation to a fluorescent dye and then allowed to react with the complementary antibody or antigen in a tissue section or smear. The location of the antigen or antibody can then be determined by observing the fluorescence by microscopy under ultraviolet light.

For general information on immunofluorescent techniques, see, for example, Knapp et al, (1978) Immunofluorescence and Related Staining Techniques, Elsevier; Allan, (1999) Protein Localization by Fluorescent Microscopy - A Practical Approach (The Practical Approach Series) Oxford University Press; Caul, (1993) Immunofluorescence Antigen Detection Techniques in Diagnostic Microbiology, Cambridge University Press. For detailed explanations of immunofluorescent techniques applicable to the present invention, see U.S. Patent Nos. 5,912,176; 5,869,264; 5,866,319; 5,861,259. J. Pharmaceutical preparations

The invention also includes pharmaceutical compositions comprising the compounds of the invention together with a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences. Mack Publishing Company, 1995. In addition to the pharmacologically active agent, the compositions of the present invention may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically for delivery to the site of action. Suitable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form, for example, water-soluble salts. In addition, suspensions of the active compounds as appropriate oily injection suspensions may be administered. Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension include, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran. Optionally, the suspension may also contain stabilizers. Liposomes can also be used to encapsulate the agent for delivery into the cell.

The pharmaceutical formulation for systemic administration according to the invention may be formulated for enteral, parenteral or topical administration. Indeed, all three types of formulations may be used simultaneously to achieve systemic administration of the active ingredient.

Suitable formulations for oral administration include hard or soft gelatin capsules, pills, tablets, including coated tablets, elixirs, suspensions, syrups or inhalations and controlled release forms thereof. The agents of the present invention can be administered via parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, or buccal routes. Alternatively, or concurrently, administration may be by the oral route or directly to the lungs. The dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.

The compounds used in the method of treatment of this invention may be administered systemically or topically, depending on such considerations as the condition to be treated, need for site-specific treatment, quantity of drug to be administered and similar considerations.

Topical administration may be used. Any common topical formation such as a solution, suspension, gel, ointment or salve and the like may be employed. Preparation of such topical formulations are well described in the art of pharmaceutical formulations as exemplified, for example, by Remington's Pharmaceutical Sciences. For topical application, these compounds could also be administered as a powder or spray, particularly in aerosol form. The active ingredient may be administered in pharmaceutical compositions adapted for systemic administration. As is known, if a drug is to be administered systemically, it may be confected as a powder, pill, tablet or the like or as a syrup or elixir for oral administration. For intravenous, intraperitoneal or intra-lesional administration, the compound will be prepared as a solution or suspension capable of being administered by injection. In certain cases, it may be useful to formulate these compounds in suppository form or as an extended release formulation for deposit under the skin or intramuscular injection. In a preferred embodiment, the compounds of this invention may be administered by inhalation. For inhalation therapy the compound may be in a solution useful for administration by metered dose inhalers or in a form suitable for a dry powder inhaler.

An effective amount is that amount which will modulate the activity or alter the level of a target protein. A given effective amount will vary from condition to condition and in certain instances may vary with the severity of the condition being treated and the patient's susceptibility to treatment. Accordingly, a given effective amount will be best determined at the time and place through routine experimentation. However, it is anticipated that in the treatment of a tumor in accordance with the present invention, a formulation containing between 0.001 and 5 percent by weight, preferably about 0.01 to 1 percent, will usually constitute a therapeutically effective amount. When administered systemically, an amount between 0.01 and 100 mg per kg body weight per day, but preferably about 0.1 to 10 mg/kg, will effect a therapeutic result in most instances.

In practicing the methods of this invention, the compounds of this invention may be used alone or in combination, or in combination with other therapeutic or diagnostic agents. In certain preferred embodiments, the compounds of this invention may be coadministered along with other compounds typically prescribed for these conditions according to generally accepted medical practice. The compounds of this invention can be utilized in vivo, ordinarily in mammals, preferably in humans.

In still another embodiment, the compounds of the invention may be coupled to chemical moieties, including proteins that alter the functions or regulation of target proteins for therapeutic benefit. These proteins may include in combination other inhibitors of cytokines and growth factors that may offer additional therapeutic benefit in the treatment of tumors. In addition, the molecules of the invention may also be conjugated through phosphorylation to biotinylate, thioate, acetylate, iodinate using any of the cross-linking reagents well known in the art.

K. Transgenic Animals

The term "animal" as used herein includes all vertebrate animals, except humans. It also includes an individual animal in all stages of development, including embryonic and fetal stages. A "transgenic animal" is an animal containing one or more cells bearing genetic information received, directly or indirectly, by deliberate genetic manipulation at a subcellular level, such as by microinjection or infection with recombinant virus. This introduced DNA molecule may be integrated within a chromosome, or it may be extra-chromosomally replicating DNA. The term "germ cell-line transgenic animal" refers to a transgenic animal in which the genetic information was introduced into a germ line cell, thereby conferring the ability to transfer the information to offspring. If such offspring in fact possess some or all of that information, then they, too, are transgenic animals. Transgenic animals containing mutant, knock-out, modified genes or gene constructs to over-express or conditionally express a gene corresponding to the cDNA sequence of SEQ ID NO: 66 or related sequences are encompassed in the invention.

The information may be foreign to the species of animal to which the recipient belongs, foreign only to the particular individual recipient, or genetic information already possessed by the recipient. In the last case, the introduced gene may be differently expressed compared to the native endogenous gene. The genes may be obtained by isolating them from genomic sources, by preparation of cDNA from isolated RNA templates, by directed synthesis, or by some combination thereof.

To be expressed, a gene should be operably linked to a regulatory region. Regulatory regions, such as promoters, may be used to increase, decrease, regulate or designate to certain tissues or to certain stages of development the expression of a gene. The promoter need not be a naturally occurring promoter. The "transgenic non-human animals" of the invention are produced by introducing "transgenes" into the germline of the non-human animal. The methods enabling the introduction of DNA into cells are generally available and well-known in the art. Different methods of introducing transgenes could be used. Generally, the zygote is the best target for microinjection. In the mouse, the male pronucleus reaches the size of approximately twenty microns in diameter, which allows reproducible injection of one to two picoliters of DNA solution. The use of zygotes as a target for gene transfer has a major advantage. In most cases, the injected DNA will be incorporated into the host gene before the first cleavage (Brinster et al, (1985) Proc. Natl. Acad. Sci. USA 82, 4438-4442.). Consequently, nearly all cells of the transgenic non-human animal will carry the incorporated transgene. Generally, this will also result in the efficient transmission of the transgene to offspring of the founder since 50% of the germ cells will harbor the transgene. Microinjection of zygotes is a preferred method for incorporating transgenes in practicing the invention.

Retroviral infection can also be used to introduce a transgene into a non-human animal. The developing non-human embryo can be cultured in vitro to the blastocyst stage. During this time, blastomeres may be targets for retroviral infection. Efficient infection of the blastomeres is obtained by enzymatic treatment to remove the zona pellucida. The viral vector system used to introduce the transgene is typically a replication-defective retrovirus carrying the transgene (Jahner et al, (1985) Proc. Natl. Acad. Sci. USA 82, 6927-6931; Van der Putten et al, (1985) Proc. Natl. Acad. Sci. USA 82, 6148-6152). Transfection is easily and efficiently obtained by culturing the blastomeres on a monolayer of virus-producing cells (Van der Putten et al, (1985) Proc. Natl. Acad. Sci. USA 82, 6148-6152; Stewart et al, (1987) EMBO J. 6, 383-388). Alternatively, infection can be performed at a later stage. Virus or virus-producing cells can be injected into the blastocoele (Jahner et al, (1982) Nature 298, 623-628). Most of the founder animals will be mosaic for the transgene since incorporation occurs only in a subset of the cells which formed the transgenic non-human animal. Furthermore, the founder animal may contain retroviral insertions of the transgene at a variety of positions in the genome; these generally segregate in the offspring. In addition, it is also possible to introduce transgenes into the germ line, albeit with low efficiency, by intrauterine retroviral infection of the midgestation embryo (Jahner et al, (1982) Nature 298, 623-628).

A third type of target cell for transgene introduction is the embryonal stem cell (ES). ES cells are obtained from pre-implantation embryos cultured in vitro (Evans et al, (1981) Nature 292, 154-156; Bradley et al, (1984) Nature 309, 255-256; Gossler et al, (1986) Proc. Natl. Acad. Sci. USA 83, 9065-9069). Transgenes can be efficiently introduced into ES cells by DNA transfection or by retrovirus-mediated transduction. The resulting transformed ES cells can thereafter be combined with blastocysts from a non-human animal. The ES cells colonize the embryo and contribute to the germ line of the resulting chimeric animal.

The methods for evaluating the presence of the introduced DNA as well as its expression are readily available and well-known in the art. Such methods include, but are not limited to DNA (Southern) hybridization to detect the exogenous DNA, polymerase chain reaction (PCR), polyacrylamide gel electrophoresis (PAGE) and Western blots to detect DNA, RNA and protein. The methods include immunological and histochemical techniques to detect expression of a gene.

As used herein, a "transgene" is a DNA sequence introduced into the germline of a non-human animal by way of human intervention such as by way of the Examples described below. The nucleic acid sequence of the transgene, in this case a form of SEQ ID NO: 66, may be integrated either at a locus of a genome where that particular nucleic acid sequence is not otherwise normally found or at the normal locus for the transgene. The transgene may consist of nucleic acid sequences derived from the genome of the same species or of a different species than the species of the target animal.

As discussed above, a "vector" is any means for the transfer of a nucleic acid into a host cell. Preferred vectors are plasmids and viral vectors, such as retroviruses. Viral vectors may be used to produce a transgenic animal according to the invention. Preferably, the viral vectors are replication defective, that is, they are unable to replicate autonomously in the target cell. In general, the genome of the replication defective viral vectors which are used within the scope of the present invention lack at least one region which is necessary for the replication of the virus in the infected cell. These regions can either be eliminated (in whole or in part), or be rendered non-functional by any technique known to a person skilled in the art. These techniques include the total removal, substitution (by other sequences, in particular by the inserted nucleic acid), partial deletion or addition of one or more bases to an essential (for replication) region. Such techniques may be performed in vitro (on the isolated DNA) or in situ, using the techniques of genetic manipulation or by treatment with muiagenic agents.

Preferably, the replication defective virus retains the sequences of its genome which are necessary for encapsidating the viral particles. The retroviruses are integrating viruses which infect dividing cells. The retrovirus genome includes two LTRs, an encapsidation sequence and three coding regions (gag, pol and env). The construction of recombinant retroviral vectors has been described (see, for example, Bernstein et al, (1985) Genet. Eng. 7, 235; McCormick, (1985) Biotechnol. 3, 689-691). In recombinant retroviral vectors, the gag, pol and env genes are generally deleted, in whole or in part, and replaced with a heterologous nucleic acid sequence of interest. These vectors can be constructed from different types of retrovirus, such as, HIV, MoMuLV (murine Moloney leukemia virus), MSV (murine Moloney sarcoma virus), HaSV (Harvey sarcoma virus); SNV (spleen necrosis virus); RSV (Rous sarcoma virus) and Friend virus.

In general, in order to construct recombinant retroviruses containing a nucleic acid sequence, a plasmid is constructed which contains the LTRs, the encapsidation sequence and the coding sequence. This construct is used to transfect a packaging cell line, which cell line is able to supply in trans the retroviral functions which are deficient in the plasmid. In general, the packaging cell lines are thus able to express the gag, pol and env genes. Such packaging cell lines have been described in the prior art, in particular the cell line PA317 (U.S. Patent No. 4,861,719); the PsiCRIP cell line (WO9002806) and the GP+envAm-12 cell line (WO8907150). In addition, the recombinant retroviral vectors can contain modifications within the LTRs for suppressing transcriptional activity as well as extensive encapsidation sequences which may include a part of the gag gene (Bender et al, (1987) J. Virol. 61, 1639-1646). Recombinant retroviral vectors are purified by standard techniques known to those having ordinary skill in the art.

In one aspect the nucleic acid encodes antisense RNA molecules. In this embodiment, the nucleic acid is operably linked to suitable regulatory regions (discussed above) enabling expression of the nucleic acid sequence, and is introduced into a cell utilizing, preferably, recombinant vector constructs, which will express the antisense nucleic acid once the vector is introduced into the cell. Examples of suitable vectors includes plasmids, adenoviruses, adeno-associated viruses (see, for example, U.S. Patent No. 4,797,368, U.S. Patent No. 5,139,941), retroviruses (see above), and herpes viruses. For delivery of a therapeutic gene the vector is preferably an adeno-associated virus. Adenoviruses are eukaryotic DNA viruses that can be modified to efficiently deliver a nucleic acid of the invention to a variety of cell types. Various serotypes of adenovirus exist. Of these serotypes, oreference is given, within the scope of the present invention, to using type two or type five human adenoviruses (Ad 2 or Ad 5) or adenoviruses of animal origin (see WO9426914). Those adenoviruses of animal origin which can be used within the scope of the present invention include adenoviruses of canine, bovine, murine, ovine, porcine, avian, and simian origin.

The replication defective recombinant adenoviruses according to the invention can be prepared by any technique known to the person skilled in the art. In particular, they can be prepared by homologous recombination between an adenovirus and a plasmid which carries, inter alia, the DNA sequence of interest. The homologous recombination is effected following cotransfection of the said adenovirus and plasmid into an appropriate cell line. The cell line which is employed should preferably (i) be transformable by the said elements, and (ii) contain the sequences which are able to complement the part of the genome of the replication defective adenovirus, preferably in integrated form in order to avoid the risks of recombination. Recombinant adenoviruses are recovered and purified using standard molecular biological techniques, which are well known to one of ordinary skill in the art.

A number of recombinant or transgenic mice have been produced, including those which express an activated oncogene sequence (U.S. Patent No. 4,736,866); express Simian SV 40 T-antigen (U.S. Patent No. 5,728,915); lack the expression of interferon regulatory factor 1 (IRF-1) (U.S. Patent No. 5,731,490); exhibit dopaminergic dysfunction (U.S. Patent No. 5,723,719); express at least one human gene which participates in blood pressure control (U.S. Patent No. 5,731,489); display greater similarity to the conditions existing in naturally occurring Alzheimer's disease (U.S. Patent No. 5,720,936); have a reduced capacity to mediate cellular adhesion (U.S. Patent No. 5,602,307); possess a bovine growth hormone gene (Clutter et al, (1996) Genetics 143, 1753-1760) or are capable of generating a fully human antibody response (Zou et al, (1993) Science 262, 1271-1274). While mice and rats remain the animals of choice for most transgenic experimentation, in some instances it is preferable or even necessary to use alternative animal species. Transgenic procedures have been successfully utilized in a variety of non- murine animals, including sheep, goats, chickens, hamsters, rabbits, cows and guinea pigs (see Aigner et al, (1999) Biochem. Biophys. Res. Commun. 257, 843-850; Castro et al, (1999) Genet. Anal. 15, 179-187; Brink et al., (2000) Theriogenology 53, 139-148;

Colman, (1999) Genet. Anal. 15, 167-173; Eyestone, (1999) Theriogenology 51, 509-517; Baguisi et al., (1999) Nat. Biotechnol. 17, 456-461 ; Prather et al, (1999) Theriogenology 51, 487-498; Pain et al., (1999) Cells Tissues Organs 165, 212-219; Fernandez et al., (1999) Indian J. Exp. Biol. 37, 1085-1092; U.S. Patent Nos. 5,908,969; 5,792,902; 5,892,070; 6,025,540).

The practice of the present invention will employ the conventional terms and techniques of molecular biology, pharmacology, immunology and biochemistry that are within the ordinary skill of those in the art. For example, see Sambrook et al, (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press. Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. The following working examples therefore, specifically point out preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

EXAMPLES

Example 1 - Recombinant proteins, fusion proteins and protein tags

Cyclin E, p27, SKPl, and SKP2 were each cloned into pVL1392 (PharMingen) vector as glutathione-S-transferase (GST) fusion proteins. In addition to GST fusion proteins, SKP2 or SKP2-like proteins could be fused with a protein interaction domain such as Max, which binds to c-Myc, to target c-Myc for ubiquitination and degradation in cells. Human CUL-1, SKP2 and SKPl cDNA were also cloned directly into baculovirus pVL1392 or pVL1393 expression vectors. The construction of these baculoviruses was accomplished as previously described (Zhang et al, (1995) Cell 82, 915-925). The baculoviruses for CDK2 and GST-cyclin A were also constructed as previously described (Zhang et al, (1995) Cell 82, 915-925). The cDNA clone encoding human El ubiquitin was cloned into the baculovirus expression vector, pAcSG-His-NT (PharMingen), as a histidineό tagged protein. In addition, SKP2 or SKP2-like proteins could be tagged with a protein interaction domain such as Max, which binds to c-Myc, to target c-Myc for ubiquitination and degradation in cells.

The El protein was expressed in the baculovirus expression system and purified by ubiquitin affinity chromatography (Yu et al, (1998) Proc. Natl. Acad. Sci. USA 95, 11324-11329). The purification was monitored by protein staining and the El activity was assayed by covalent conjugation of biotinylated ubiquitin (Pagano et al, (1995) Science 269, 682-685). For ³⁵S- labeled p27, SF9 cells were infected with baculoviruses encoding GST-p27. Forty hours post-infection, cells were labeled with ³⁵S-methionine for three hours as described (Zhang et al, (1995) Cell 82, 915-925). The labeled GST-p27 protein was isolated by glutathione Sepharose beads and the p27 portion was released from the beads by thrombin treatment for thirty minutes at room temperature (Calbiochem) (Guan et al, (1991) Anal. Biochem. 192, 262-267). Thrombin was subsequently inactivated by one mM phenylmethyl-sulfonyl fluoride (PMSF). The purified p27 is monitored by autoradiography and quantified by protein staining and Western-blot analysis.

To assemble cyclin E/CDK2, GST-cyclin E and CDK2 baculoviruses were individually expressed in SF9 cells. The lysates were prepared in hypotonic buffer (20 mM Hepes, pH 7.2, 5 mM KC1, 1.5 mM MgCb, 0.5 mM DTT). The lysates containing GST-cyclin E and CDK2 were mixed and incubated in the presence of 10 mM ATP at 30 °C for one hour to assemble the active cyclin E/CDK2 kinase. The kinase was then affinity purified using the glutathione beads and quantified by protein staining and Western blot. The activity of purified kinase was monitored by the histone HI assay (Zhang et al, (1995) Cell 82, 915-925). To produce SCF^SKP2 complex, baculoviruses encoding GST-SKP1 and CUL-1, in the presence or absence of baculoviruses encoding SKP2, were co-infected into insect SF9 cells and were affinity purified using a glutathione Sepharose column. The successful assembly of the complex was monitored and quantified by protein staining and Western-blot analysis (Figure 15). The in vitro translated proteins were produced and labeled with ³⁵S-methionine in TNT rabbit reticulocyte lysates according to the manufacturer's instructions (Promega).

The human CDC34 cDNA clone was cloned into pGEXKG as a GST fusion protein and expressed in bacteria BL21. GST-CDC34 was isolated by glutathione column and the GST portion was removed by thrombin. The CDC34 protein was further purified with a MonoQ column and monitored by protein staining. The methyl ubiquitin and ubiquitin aldehyde were commercially obtained (BostonBiochem).

Anti-p27 (sc-528) antibodies were purchased commercially (Santa Cruz Biotechnology). Rabbit anti-cyclin E, SKP2, and CDK2 polyclonal antibodies and anti- HA epitope tag monoclonal antibody (12CA5) were described previously (Zhang et al, (1995) Cell 82, 915-925; Xiong et al, (1993) Nature 366, 701-714). For some experiments, a monoclonal anti-human cyclin E antibody (HE12) and a polyclonal anti- mouse cyclin E antibody (M20) were used (Santa Cruz). The anti-T7-tag monoclonal antibody was obtained from Novagen. Immunoprecipitation and Western-blot analyses were performed as described previously (Zhang et al, (1995) Cell 82, 915-925). For direct Western-blotting, cells were lysed directly in 0.1% SDS, and viscosity was reduced by passing the lysates through a 22-gauge needle. Approximately 40 μg of proteins were loaded directly onto an SDS-polyacrylamide gel for Western-blot analysis. Identical results were obtained from direct Western-blot analyses as from immunoprecipitation followed by Western-blot analyses.

Example 2 - Phosphorylation-dependent p27 degradation

Selective p27 degradation in cell free systems has been reported previously in synchronized S-phase extracts but not in GI cell extracts (Nguyen et al, (1999) Mol. Cell. Biol. 19, 1190-1201; Brandeis & Hunt (1995) EMBO J. 15, 5280-5289). To determine the proteins that control p27 stability, cytosolic extracts from asynchronized and exponentially growing HeLa cells were prepared.

HeLa cells were grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum at 37 °C. For extract preparation, suspension HeLa cells were grown to 0.5-1 x 10⁶ cells/ml (log-phase) and extracts were prepared as previously described (Brandeis & Hunt (1996) EMBO J. 15, 5280-5289). Cell pellets were washed twice with phosphate-buffered saline (PBS) and then with hypotonic buffer. The cells were re-suspended in two volumes of hypotonic buffer. They were lysed by Dounce homogenization using a loose pestle. The cytosolic extracts were prepared by centrifugation at 15,000 rpm using a Sorvall SS34 rotor. Aliquots of the extracts were immediately frozen in liquid nitrogen and stored at -80 °C. For a typical degradation reaction of p27, 200 μg cytosolic extract was used in a total volume of 50 μl, with no greater than 20% dilution of the extract. The reaction mixture also contained 2 mM ATP, 20 mM creatine phosphate, 50 μg/ml creatine kinase, 20 mM Hepes, pH7.2, 1 mM DTT, and 10 mM MgCk. The reactions were initiated by adding ⁵S-labeled p27 (0.25-0.5 μg) and cyclin E/CDK2 (1-3 μg) and incubation at 30 °C for one to three hours. The amount of cyclin E/CDK2 required for p27 degradation was titrated batchwise for different extract preparations to determine the necessary threshold level of cyclin E/CDK2. The requirement of cyclin E/CDK2 is also dependent on the amount of exogenously added p27, reflecting the fact that p27 serves both as an inhibitor and a substrate for the kinase. Substantial amounts of endogenous p27 were present in the extract which was also degraded by addition of cyclin E/CDK2. The reactions were stopped by adding 0.1 % SDS, followed by one ml of lysis buffer and in the presence of protease inhibitors (5 μg/ml of leupeptide, soybean trypsin inhibitor and aprotinin plus 100 mM benzamidine). The reaction products were immunoprecipitated using p27 antibodies, fractionated in SDS-PAGE, and visualized by autoradiography. Degradation of endogenous p27 in the extracts was monitored by directly loading onto an SDS-PAGE in the SDS sample buffer, followed by Western-blotting with p27 antibodies.

Recombinant ³⁵S-labeled p27 was not degraded when incubated with this cytosolic extract indicating that p27 was quite stable. Addition of an active cyclin E/CDK2 kinase to the extract led to the rapid degradation of p27 (Figures 1A, IB). The requirement of cyclin E/CDK2 for p27 destruction was due to its ability to phosphorylate p27 at threonine 187, which has been shown to trigger p27 degradation (Sheaff et al, (1997) Genes Dev. 11, 1464-78; Vlach et al, (1997) EMBO J. 16, 5334-44). Conversion of threonine 187 to glycine (T187G) stabilized p27 in the extract and confirmed the requirement for phosphorylation of threonine 187 (Figure 1A). In the presence of cyclin E/CDK2, a fraction of p27 slightly shifted its electrophoretic mobility which was sensitive to phosphatase (Figure IC), indicating that these proteins are phosphorylated forms of p27.

A fraction of p27 was converted into multiple and high molecular weight species in the presence of cyclin E/CDK2 (Figure IC), which are insensitive to phosphatase treatment. Addition of specific inhibitors of 26S proteosome, such as MG132, stabilized p27 and resulted in accumulation of both the phosphorylated and the high molecular weight forms of p27 (Figure IC). Modified ubiquitins such as methyl ubiquitin and ubiquitin aldehyde also caused accumulation of p27 ladders (Figure 2) due to their ability to interfere with the degradation rate or inhibition of deubiquitination of highly polyubiquitinated proteins (Hershko et al, (1987) Proc. Natl. Acad. Sci. USA 84, 1829-1833). These observations indicate that the high molecular weight species of p27 are the poly-ubiquitinated. This in vitro system therefore faithfully recapitulated the ubiquitin-dependent p27 degradation in a cyclin E/CDK2-dependent process which requires phosphorylation of the threonine 187 residue on p27.

Example 3 - Alteration of p27 levels bv depletion of SKP SKP2 & CUL-1 Using the in vitro p27 degradation system, the potential involvement of candidate ubiquitin E3 ligases, the SCF complexes (SKPl, CDC53/Cullins, F-box proteins), for p27 degradation was examined. The SCF complexes represent a conserved family of protein complexes that target phosphorylated proteins for ubiquitin-dependent proteolysis (Patton et a , (1998) Trends Genet. 14, 236-243; Maniatis, (1999) Gene Dev. 13, 505-510). CUL-1 was examined first to determine whether a human CDC53 homologue is necessary for p27 degradation.

For depletion of CUL-1, SKPl or SKP2 proteins from HeLa extracts, four mg of affinity purified CUL-1, SKPl or SKP2 antibodies (Zhang et al, (1995) Cell 82, 915-925) or IgG were coupled to one ml protein A-Sepharose column. Five to ten ml of HeLa extracts were used to pass through the antibody-protein A column three times at 4°C. The flow-through fractions from the columns were collected and examined for the efficiency of depletion using Western blot analysis. These fractions were then used as depleted extracts. For restoration of p27 degradation activity in SKP2 depleted extract, two μg of purified SKP2, SCF^{S P2} or SC (no SKP2) complexes were added to SKP2 depleted extracts and the degradation of p27 was monitored as described above. Depletion of CUL-1 abolished the ability of the extracts to degrade p27 while parallel mock depletion using purified IgG from the pre-immune serum had no effect (Figure 3 A). Western-blot analysis confirmed that CUL-1 was removed from the extracts by the column (Figure 2D). Specific immuno-depletion of another component of the SCF complex, SKPl, also resulted in the inhibition of p27 degradation which led to an increase in p27 levels (Figures 3B, 3C).

The requirement for CUL-1 and SKPl for p27 degradation implies that an F-box protein is involved. The F-box protein is a component in the SCF complexes that interacts directly with the phosphorylated substrates and thus defines the substrate specificity for ubiquitination (Maniatis, (1999) Genes Dev. 13, 505-510; Skowyra et al, (1997) Cell 91 , 209-219; Winston et al, (1999) Genes Dev. 13, 270-83). To identify the F-box protein(s) that specifically bind to p27, ³⁵S-labeled HeLa cell extracts were incubated with GST-p27 either with or without prior phosphorylation by cyclin E/CDK2. Examination of the labeled proteins specifically associated with the phosphorylated GST-p27 beads revealed the presence of a 45 kDa protein, which is similar to the molecular weight of the F-box protein SKP2. To determine whether SKP2 is involved in p27 degradation, HeLa extracts were subjected to immuno-depletion with an affinity purified SKP2 antibody column. Removal of SKP2 by immunodepletion of SKP2 from the extract resulted in the inhibition of p27 degradation activity in the extract (Figure 3C, 3D). This data in combination with the SKPl depletion experiments indicates that depletion of SKP proteins results in modulation of SKP activity which can increase expression of p27.

Example 4 - SKP2 binds to phosphorylated p27

To directly examine the specific binding of SKP2 to the phosphorylated form of p27, a pair of peptides corresponding to the carboxy-terminal end of p27 (amino acids

175-198) was synthesized. Threonine 187 was phosphorylated in the first peptide but not in the second peptide (Figure 4A). The peptides were each coupled to SulfoLink agarose beads which were then used as affinity resins for binding analysis of F-box proteins. These peptides were initially tested to determine if they could interact with several known F-box proteins, including SKP2, β-TrCP and MD6, as well as a number of unpublished F-box proteins identified through EST database search, in vitro translated and ³⁵S-labeled F-box proteins were incubated with the p27 peptide beads. Analysis of the F-box proteins associated with p27 peptide beads revealed a specific interaction between SKP2 and the phosphorylated threonine 187 p27 peptide (Figure 4B). No significant interactions were observed if the non-phosphorylated form of the peptide was used. Specific associations between the p27 phosphopeptide and other available F-box proteins were not detected (Figure 4B, data not shown). These data indicate that SKP2 can interact selectively and specifically with the p27 phosphopeptide.

To determine whether endogenous SKP2 in the HeLa extract can also interact with p27 phosphopeptide, the peptide beads were incubated with the extracts. The peptides, containing either the carboxy-terminal end of p27 (amino acids 175-198), CSDGSPNAGSVEQTPKKPGLRRRQT, and phosphopeptides

CSDGSPNAGSVEQ*TPKKPGLRRRQT (*T denotes phosphorylated threonine 187 of p27) (SEQ ID NO: 1) were synthesized using the peptide synthesis facility at the Yale University School of Medicine. The phosphorylated threonine 187 (*T) and the non-phosphorylated forms of the p27 carboxy-terminal peptides were conjugated to SulfoLink beads (Pierce) through the cysteine residue added at the amino-terminus of the peptides according to manufacturer's instruction (Pierce). For coupling reactions, 0.5 mg of peptides were conjugated onto two ml of Sulfolink beads for thirty minutes and the residue sites on the beads was blocked by 20 mM cysteine for two hours at room temperature. The beads were washed extensively first with PBS followed by hypotonic buffer and stored at 4°C. For F-box protein binding assays, 10 μl of the in vitro translated F-box proteins, including SKP2, β-TrCP, and MD6, were mixed 20 μl peptide beads in 250 μl of lysis buffer containing protease inhibitors (5 μg/ml leupeptide, trysin inhibitor, aprotinin, and 100 mM benzamidine) and 100 mM NaF. Binding assays were performed at 4 °C for one hour with agitation. The beads were washed with detergent buffer for four times and the proteins associated with the beads were analyzed. A similar procedure was used for the extract binding except 100-400 μg of HeLa extracts were used as the source of SCF complexes, replacing the in vitro translated F-box proteins.

Western-blot analysis detected a strong and specific interaction between endogenous SKP2 and the p27 phosphopeptide (Figure 4C). The p27 peptide without threonine 187 phosphorylation did not significantly interact with SKP2. As a control for SKP2 binding, binding of β-TrCP to the p27 peptides was also measured. β-TrCP is an F-box protein that binds and targets phosphorylated β-catenin and IB for ubiquitin-dependent degradation (Maniatis, (1999) Genes Dev. 13, 505-510). The data indicated that although β-TrCP was also present in the extract, no interactions between β- TrCP and the p27 phosphopeptide were detected in these assays (Figure 4C). These studies confirm that endogenous SKP2 in the HeLa extract specifically recognizes and binds to the phosphorylated form of p27. In addition to SKP2 binding only to the phosphorylated form p27 peptide, it also binds only to the phosphorylated form of cyclin E peptide SPLPSGLL*TPPQSGKKQSSGPEMA (amino acids 372-395 where *T denotes phosphorylated threonine 187 of p27) (SEQ ID NO: 4). SKP2 can therefore be inhibited by a phosphopeptide other than p27 phosphopeptide.

Previous studies indicated that SKP2 interacts with SKPl and CUL-1 in vivo (Yu et al, (1998) Proc. Natl. Acad. Sci. USA 95, 11324-11329; Lisztwan et al, (1998) EMBO J. 17, 368-83; Lyapinaet al, (1998) Proc. Natl. Acad. Sci. USA 95, 7451-7456; Michel et al, (1998) Cell Growth Differ. 9, 435-449). SKP2 interactions with SKPl and CUL-1 were also observed in the HeLa extracts (Figure 4D). It was necessary to confirm that SKPl and CUL-1 could interact directly with the p27 since depletion of either of these proteins from the extracts also abolished p27 degradation. Confirmation of such a direct interaction would eliminate the possibility that other indirect mechanisms were responsible for the increase in p27 following removal of SKP 1 or CUL- 1. Using the peptide bead pull-down assays, specific interactions of SKPl or CUL-1 with the phosphorylated threonine p27 peptide beads were detected in contrast to the non-phosphorylated peptide where no such interaction was detected (Figure 4C). To determine whether SKP2 mediates SKPl and CUL-1 binding to p27 phosphopeptide, SKPl or CUL-1 binding in SKP2 depleted extracts was examined. Depletion of SKP2 from the extract significantly reduced the binding of SKPl (Figure 4E) or CUL-1 (data not shown) to the p27 phosphopeptide beads, although the total levels of SKPl and CUL-1 in the extract were not substantially altered by SKP2 depletion (Figure 4E and data not shown). These studies indicate that SKP2 is the SCF component that binds to the phosphorylated threonine 187 of p27. Upon SKP2 binding to phosphorylated p27, SKP2 associates with SKPl and CUL-1 and targets p27 for ubiquitin-dependent degradation by the 26S proteasome. Modulation of the activity of SKP2 can therefore increase the levels of p27.

Example 5 - SKP2-dependent degradation of p27 The effect of addition of the SCF^SKP2 complex to the SKP2 depleted extract was investigated to determine if restoration of SKP2 is sufficient to restore p27 degradation activity. Recombinant SCF^SKP2 complexes were expressed, assembled using the baculovirus expression system and purified. When the recombinant SCF^SKP2 complex was added back into the SKP2 depleted extract, restoration of p27 degradation was observed (Figure 5 A). Restoration of p27 degradation was dependent on the presence of SKP2 in the complex because complexes assembled in the absence of SKP2 could not rescue the SKP2 deficiency in the extract. Addition of purified SKP2 alone could partially rescue p27 degradation in the SKP2 depleted extract (data not shown) but the assembled SCF^SKP2 complex consistently produced better restoration, indicating that the SCF^SKP2 complex itself is required for p27 degradation. When SKP2 expression is under the control of a tetracycline-inducible promotor in HeLa cells, removal of tetracycline results in expression of SKP2. Induction of SKP2 resulted in approximately a significant decrease in total cellular p27. Since SKP2 only targets the Thrl87-phosphorylated p27 for degradation, the down-regulation of p27 by expressing SKP2 indicates that SKP2 is rate limiting where sufficient CUL-1 and SKP-2 are present.

Example 6 - SKP2-dependent ubiquitination of p27 p27 ubiquitination was also assayed directly using the recombinant SCF^SKP2 complex. In a purified system containing the recombinant SCF^{S1 P2} complex, cyclin E/CDK2, ubiquitin activation enzyme El, and ATP, a fraction of p27 was converted into multiple high molecular weight species (Figure 5B). The formation of high molecular weight p27 was dependent on the presence of ubiquitin and CDC34, a conserved E2 conjugating enzyme that is implicated in SCF-mediated ubiquitination (King et al, (1996) Science 274, 1652-1659; Plon et al, (1993) Proc. Natl. Acad. Sci. USA 90, 10484-10488). The E2 conjugating enzyme for SCF^SKP2 is probably a human CDC34 homolog. These data suggest that the SCF^SKP2 complex can ubiquitinate p27 in the presence of El and E2. However, p27 ubiquitination using the purified proteins was not very efficient. It is possible that SCF^SKP2 may require additional modifications or activities for efficient p27 ubiquitination.

Example 7 - SKP2 binds to phosphorylated cyclin E

N-acetyl-L-leucinyl-L-leucinal-L-norleucinal (LLNL) and hydroxyurea (HU) were purchased from Sigma. The cyclin E carboxy peptides (residues 371-394) CASPLPSGLLTPPQSGKKQSSGPEM containing either the Thr380-phosphorylated (TP- CP) or non-phosphorylated (TP-C) forms were synthesized and coupled to Sulfo-Link agarose beads (Pierce) as described previously (Tsvetkov et al, (1999) Curr. Biol. 9, 661- 664) (TP: corresponding to Thr380 and Pro381 in cyclin E). Cyclin E mutant peptides, TA-CP (CASPLPSGLLTAPQSGKKQSSGPEM) (SEQ ID NO: 5), SP-C and SP-CP (CASPLPSGLLSPPQSGKKQSSGPEM) (SEQ ID NO: 6), were synthesized accordingly. A cysteine residue was added to the amino-terminal end of these peptides to facilitate coupling to the beads. The cyclin E cDNA was tagged by the T7-epitope tag at its amino- terminus in pCGT, and its expression was under CMV promoter control. The SKP2 dominant negative mutant (SKP2DN) lacking the F-box was constructed as described (Carrano et al, (1999) Nat. Cell. Biol. 1 , 193-199). Both the wild-type and mutant SKP2 were cloned into the retrovirus vector pBabe. The full-length cDNA clones of FBL-2, -5, - 6 and -8 were commercially purchased (Research Genetics) and were sequenced for confirmation.

To identify the F-box protein(s) that might bind to the phosphorylated Thr380 in cyclin E, a pair of peptides that correspond to the carboxy-terminal end of cyclin E that includes the critical Thr380 (Figure 6A) were synthesized. One peptide (TP-CP) contained the phosphorylated Thr380, and the other had a nonphosphorylated Thr380 (TP- C). Each of these peptides was immobilized onto agarose beads, which were then used to determine the binding of various F-box proteins. The F-box proteins were in vitro translated and ³⁵S-methionine-labeled. They were used directly to test for binding to TP- CP or TP-C beads. Using this assay, it was determined that SKP2 selectively interacted with the cyclin E phosphopeptide TP-CP, while no detectable interactions were observed between SKP2 and the nonphosphorylated cyclin E peptide TP-C. The interaction between the phosphorylated cyclin E peptide and SKP2 is specific, since it was not possible to detect the binding of cyclin E TP-CP to other F-box proteins, including various FBLs (Winston et al, (1999) Curr. Biol. 9, 1180-1182; Cenciarelli et al, (1999) Curr. Biol. 9, 1177-9), which bears close homologies to SKP2 as well as the more distantly related β-TrCP and MD6 (Figure 6B and data not shown).

SKP2 normally forms a complex with SKPl and CUL-1 (Tsvetkov et al, (1999) Curr. Biol. 9, 661-664). To determine whether the SCF^SKP2 complex binds specifically to the phoshorylated cyclin E peptide, a cytosolic HeLa cell extract was used as the source of SCF complexes (Figure 6C). Thus, SKP2, SKPl, and CUL-1 all interact specifically with the cyclin E phosphopeptide TP-CP but not with the nonphosphorylated cognate peptide TP-C (Figure 6C). To rule out the possibility that SKP2 nonspecifically binds to phosphorylated peptides, a number of mutant peptide derivatives were synthesized in which either Thr380 in cyclin E was converted into serine or phosphoserine (SP-C or SP- CP) or Pro381 was converted into alanine but with Thr380 remaining phosphorylated (TA- CP). Binding assays indicate that SKP2 did not interact with the mutant SP-CP and TA- CP phosphopeptides. Similar results were obtained in a peptide competition experiment in which increasing amounts of either TP-CP, TA-CP, or SP-CP phosphopeptides were used as competitors for the association between SKP2 and the TP-CP beads (Figure 6D).

Example 8 - Phosphorylation-dependent cyclin E degradation SKP2 expression is periodic in a cell-cycle-dependent manner, with a peak level in the S phase (Zhang et al, (1995) Cell 82, 915-925). Recent evidence suggests that SKP2 is a limiting component of the SCF^{S P2} complex for S phase entry and for the degradation of p27 (Sutterluty et al, (1999) Nat. Cell Biol. 1, 207-214; Tsvetkov et al, (1999) Curr. Biol. 9, 661-664; Carrano et al, (1999) Nat. Cell. Biol. 1, 193-199; Zhang et al, (1995) Cell 82, 915-925). To determine whether cyclin E is a target for ubiquitination by SKP2, the levels of T7-epitope-tagged cyclin E were examined after its transfection into HeLa cells in the presence or absence of SKP2. SKP2 expression caused a substantial reduction in the levels of co-expressed cyclin E. This effect is dependent on the Thr380 residue in cyclin E. When Thr380 was converted into glycine (T380G), which could not be phosphorylated, the mutant cyclin E was much more resistant to SKP2 (Figure 7 A).

Pulse-and-chase experiments indicated that SKP2 significantly shortened the half-life of the cyclin E protein (Figure 8A).

In addition, expression of SKP2 induced the formation of high-molecular- weight ladders of cyclin E (Figure 7B-E) in both 293 and mouse embryonic fibroblast cells. The SKP2-dependent formation of high-molecular-weight ladders of cyclin E was mostly abolished if Thr380 of cyclin E was mutated into glycine (T380G). To determine whether the high-molecular-weight species were polyubiquitinated forms of cyclin E, the effect of expressing an HA-tagged ubiquitin (HAUb) on cyclin E was examined. Expression of HAUb also led to the accumulation of the high-molecular-weight forms of cyclin E similar to the ones induced by SKP2 (Figure 7C). Immunoprecipitation with anti-HA epitope antibody followed by Western-blotting with T7-tagged cyclin E revealed that the high- molecular-weight species of cyclin E were polyubiquitinated forms of cyclin E (Figure 7D).

Furthermore, expression of SKP2 greatly promoted high levels of incorporation of HAUb into cyclin E, as compared with that of HAUb alone (Figure 7D). These observations indicate that expression of SKP2 is sufficient to cause the polyubiquitination of cyclin E in vivo. In addition, the effect of T380G mutation in cyclin E on the polyubiquitination of cyclin E indicates that ubiquitination is dependent on the presence of Thr380. However, in the absence of Thr380, a weaker but detectable level of cyclin E ubiquitination was observed (Figure 7B). This ubiquitination was also promoted by SKP2. Although the phosphorylated Thr380 provides a major binding site for SKP2, there exists additional minor sites in cyclin E that can be used for SKP2 binding and cyclin E ubiquitination.

Example 9 - SKP2-dependent ubiquitination of cyclin E independent of p27 The F-box proteins usually interact directly with their phosphorylated substrates.

To determine the potential association of SKP2 with full-length cyclin E, T7-tagged wild- type or the T380G mutant form of cyclin E was expressed in the presence or in the absence of N-acetyl-L-leucinyl-L-leucinal-L-norleucinal (LLNL), a specific inhibitor of the 26S proteasome in vivo (Figure 8B). Immunoprecipitation followed by Western-blotting indicated that both un-ubiquitinated and ubiquitinated forms of cyclin E were associated with SKP2 (Figure 8B). The cyclin E T380G mutant was also found to be associated with SKP2 (Fig. 3B) but to a lesser extent. This is consistent with the earlier observation (Figure 7B) that the phosphorylated Thr380 is a major site for SKP2 binding but that there are additional minor sites in cyclin E for SKP2 binding and ubiquitination. Since cyclin E-SKP2 interaction (Figure 8B) and ubiquitination (Figure 7E) occurred in p27-/- mouse embryonic cells, this indicates that cyclin E ubiquitination (Figure 7E and 8B) and its interaction with SKP2 (Figure 8B) are independent of p27.

Although cyclin E ubiquitination is independent of p27, in the presence of co- expressed CDK inhibitor p27, cyclin E degradation was inhibited even in the presence of SKP2 (Figure 8C). This observation indicates that p27 might inhibit cyclin E autophosphorylation on Thr380, leading to resistance to SKP2-mediated ubiquitin- dependent degradation of cyclin E. The effect of p27 is not to be due to a competition between p27 and cyclin E for SKP2 binding, since a non-phosphorylated mutant form of p27 in which the critical Thrl87 was converted into glycine (T187G) did not bind. This data is consistent with the previous report that p27 inhibits the Thr380-dependent cyclin E degradation (Clurman et al, (1996) Genes Devel. 10, 1979-1990). Expression of SKP2 also affects the endogenous cyclin E level. When SKP2 was expressed in cells using recombinant retrovirus delivery system, a significant decrease in endogenous cyclin E levels was observed (Figure 9A and B). As observed before, ectopic expression of SKP2 also led to the reduction of p27 levels. The possibility that cyclin E down-regulation is due to a secondary effect of SKP2 on the S phase was eliminated since SKP2 caused the decrease of cyclin E even in cells that were synchronized in the S phase by hydroxyurea (Figure 7B). Conversely, expression of a dominant-negative SKP2 (DN) that is defective in F-box, a binding site for SKPl, caused the accumulation of endogenous cyclin E (Figure 8C and D). Such an effect on the endogenous cyclin E is independent of p27, since SKP2DN-mediated elevation of cyclin E could occur in p27-/- mouse embryonic fibroblasts (Figure 8D). This observation is consistent with our finding that SKP2-mediated-ubiquitination of cyclin E occurs in p27-/- mouse embryonic fibroblasts (Figure 7E). This data indicates that SKP2-mediated cyclin E ubiquitination is p27- independent. Applicants have identified SKP2 as an F-box protein that mediates ubiquitin- dependent degradation of cyclin E. SKP2 is an F-box protein that is expressed in late GI, S, and G2 phases, playing a role in S phase of the cell cycle (Zhang et al, (1995) Cell 82, 915-925). SCF^SKP2 binds and targets the CDK inhibitor p27 for ubiquitin-dependent degradation. In addition, SKP2 also interacts with cyclin E and plays a role in the ubiquitin-dependent degradation of cyclin E. This SKP2-mediated cyclin E ubiquitination and degradation is mostly dependent on the presence of Thr380 in cyclin E (Figure 14), although weak cyclin E ubiquitination in the absence of Thr380 was also promoted by SKP2 in vivo.

Applicants have also identified that SKP2 performs a dual function during the Gl/S transition. It is required for the ubiquitin-dependent degradation of p27 in late GI (Sutterluty et al, (1999) Nat. Cell. Biol. 1, 207-14; Tsvetkov et al, (1999) Curr. Biol. 9, 661-664; Carrano et al, (1999) Nat. Cell. Biol. 1, 193-199). The degradation of p27 by SCF^SKP2 activates cyclin E/CDK2 and promotes entry into the S-phase (Sutterluty et al, (1999) Nat. Cell. Biol. 1, 207-14; Coats et al, (1996) Science 272, 877-880). Once cells are in the S phase, cyclin E is degraded which may be required for terminating the S-phase initiation events, allowing the cells to progress from the S phase into the G2 phase (Clurman et al, (1996) Genes Dev. 10, 1979-1990; Won et al, (1996) EMBO J. 15, 4182- 4193).

Applicants have determined that a number of phosphorylation dependent and ubiquitin-dependent degradation events occur during the Gl/S transition, which are temporally regulated. The expression of SKP2 in the late GI and S phases leads to assembly of the SCF^SKP2 complex. Previous reports suggest that the phosphorylation status of p27 and cyclin E could be temporally separated. p27 phosphorylation on the critical Thrl87 has been shown to occur in the late GI phase and p27 ubiquitination has been reported to require its binding to the cyclin E/CDK2 complex (Montagnoli et al, (1999) Genes Dev. 13, 1181-1189). The phosphorylation of Thrl 87 in p27 triggers the binding of SKP2, leading to the subsequent ubiquitin-dependent degradation of p27.

It has been shown that binding of p27 to cyclin E/CDK complexes inhibits the activity of cyclin E/CDK2 and cyclin E degradation (Clurman et al, (1996) Genes Dev. 10, 1979-1990). The binding of p27 therefore prevents phosphorylation on Thr380 in cyclin E or there is a competition between p27 and cyclin E for the binding of SKP2. p27 binding can also cause a conformational change in cyclin E so that Thr380 in cyclin E is not exposed for phosphorylation or SKP2 binding. Applicants have determined that SKP2 binds to the p27 phosphopeptide with higher affinity than that of cyclin E peptide (data not shown). Thus the affinities between SKP2 and p27 or cyclin E may also affect the ubiquitination rate of p27 and cyclin E by SKP2. Once p27 is degraded, the cyclin

E/CDK2 kinase activity is activated, leading to the S-phase entry. Activation of cyclin E also leads to its autophosphorylation in Thr380 (Clurman et al, (1996) Genes Dev. 10, 1979-1990; Won & Reed, (1996) EMBO J. 15, 4182-4193). The phosphorylation of Thr380 promotes the SKP2 binding which in turn results in the ubiquitin-dependent degradation of cyclin E.

The efficiency of the ubiquitination reaction by the SCF complexes is very high. Based on the in vitro and in vivo p27 and cyclin E degradation using SKP2, the reaction efficiency can be 80-90% or even higher to 100% (Figure 14). This is a low estimation, since SKP2 only binds to phosphorylated substrates, the complete reaction is thus dependent on the extent of the substrate phosphorylation, which in turn relies on activities of kinases and phosphatase that regulate the levels of substrate phosphorylation in vivo or in the cell extracts. Conversely, using the p27 phosphopeptide, it is possible to deplete almost all SKP2 in the cell extract. This indicates that SKP2 can bind to its substrates with very high affinity.

Example 10 - SKP2 Fusion Proteins Capable of Altering Substrate Specificity

The substrate-specificity of SCF complexes can be altered if the substrate-binding domains of the F-box protein such as LRR in SKP2 or WD repeats in β-TRCP are replaced by other protein-protein interaction motifs. As a first test for such a possibility, a hybrid protein that contains the amino-terminus of β-TRCP up to its F-box motif was created (residues 1-204, the F-box is located between residues 148-191). However, the substrate-targeting domain of the WD repeats is replaced by the LRR region of SKP2 (residues 169-435, the F-box is between residues 112-151) (Figure 10). Such a fusion creates a hybrid protein (β-TRCP.N/SKP2.C) that contains the F-box region of β-TRCP and the SKP2 substrate-binding domain (leucine-rich repeats or LRR). This hybrid β- TRCP.N/SKP2.C protein would be expected to have an altered substrate specificity.

Instead of normally targeting β-catenin and IκB by β-TRCP, the hybrid protein should target SKP2-specific substrates, such as cyclin E or p27, for ubiquitination and degradation. As expected, when this fusion protein is introduced into 293 cells, it is fully active to ubiquitinate cyclin E for polyubiquitination in the same way as SKP2 (Figure 11). Thus, swapping the domain of F-box proteins can alter the substrate specificity of F- box proteins.

By fusing the amino-terminus of β-TRCP and the carboxy terminus of SKP2, a fully active hybrid β-TRCP .N/SKP2.C protein was produced to target cyclin E for ubiquitination. The results from the β-TRCP.N/SKP2.C hybrid protein suggest that alteration of specificity of the F-box proteins can be made. However, it could not be ruled out that the LRR region of SKP2 contains a motif that is also required for SKPl binding or the SCF ubiquitination activity.

Sequence comparison (Figure 13) has revealed the presence of a relatively conserved motif at the carboxy-terminal region of SKP2 (residues 321-374) and β-TRCP (residues 429-497). This motif is also present in the yeast F-box CDC4 carboxy-terminus (residues 388-463). For convenience this domain will be designated SCM for SKP2 C- terminal motif. The role of this motif is to mediate the intearction between the F-box proteins and SKPl or other components of the SCF complexes. This possibility is based on our finding that von Hippel-Lindau disease protein ("NHL"), a human tumor suppressor protein that binds to a SKPl -like protein, elongin C/SIII in a putative SCF-like ubiquitin E3 ligase CUL-2/elongin B/C complex, also has this domain (residues 146-195) (Stebbins et al, (1999) Science 284, 455-461). Fusing the F-box, a protein interaction domain, such as Max or the MDM2 amino-terminus, and this conserved SCM domain, should improve the ubiquitination of targeted protein by various hybrid proteins.

The VHL binds to CUL-2 and Elongin C (also called SIIIC) (Pause et al, (1997) Proc. Νatl. Acad. Sci. USA 94, 2156-2161). Human CUL-2 is a close homologue of CUL-1 while Elongin C/SIIIC shares substantial homology with SKPl (Pause et al, (1997) Proc. Νatl. Acad. Sci. USA 94, 2156-2161 ; Kipreos et al, (1996) Cell 85, 829- 839). The formation of the VHL/CUL-2-Elongin C complex (Duan et al, (1995) Science 269, 1402-1406; Kibel et al, (1995) Science 269, 1444-1446), with additional components such as Elongin B (also called SIIIB, a ubiquitin-like protein) and Rbxl (Kamura et al, (1999) Science 284, 657-661; Duan et al, (1995) Science 269, 1402-1406; Kibel et al, (1995) Science 269, 1444-1446), has been suggested to contain an SCF-like ubiquitin ligase activity (Pause et al, (1997) Proc. Νatl. Acad. Sci. USA 94, 2156-2161). Immuno- purified SCF complexes from both yeast (Seol et al, (1999) Genes Dev. 13, 1614-1626) and human cells can ubiquitinate proteins associated with SCF complexes if they are co- incubated with ubiquitin, ATP, CDC34 E2 conjugating enzyme and El (Figure 14). Similar ubiquitination activity has been found to associate with the purified VHL/CUL- 2/Elonin C/Elongin B complex (Lisztwan et al, (1999) Genes Dev. 13, 1822-1833). Since it was determined that VHL shares certain homology with the SCM of SKP2 in the α- domain, it is likely VHL/CUL-2/Elongin C/Elongin B is an SCF-like E3 ubiquitin ligase that uses VHL as a substrate targeting subunit. Under such circumstances, the protein- knockout technique proposed for SKP2 or other F-box proteins can also be applied to the use of VHL. Thus it is expected that if one fuses a protein interaction domain with VHL, the VHL fusion protein should act to ubiquitinate the target protein through the interaction between the protein interaction domain and the target. CUL-1 and CUL-2 belong to the cullin family (Kipreos et al, (1996) Cell 85, 829- 839), which so far contains several additional members such as CUL-3 (Singer et al, (1999) Genes Dev. 13, 2375-2387; Michel & Xiong, (1998) Cell Growth Differ. 9, 435- 449), CUL-4A and 4B (Kipreos et al, (1996) Cell 85, 829-839; Chen et al, (1998) Cancer Res. 58, 3677-3683), vasopressin-activated calcium-mobilizing receptor- 1 (Stankovic et al, (1997) Genomics 40, 267-276), and anaphase-promoting complex 2 (APC2) (Stankovic et al, (1997) Genomics 40, 267-276). Based on the homology between CUL-1 and other members of cullin family, it is expected that these cullin family members should act as ubiquitin E3 ligases. In addition, if similar fusion proteins for the substrate- targeting components of these cullin family members are constructed, it is possible to alter the substrate specificity of these ubiquitin E3 ligases in the same design as proposed for that of SCF complexes.

Example 11 - F-box antagonist peptides block the SKPl /F-box protein interaction The F-box region is a peptide motif composed of 40-50 amino acids that is present in a variety of otherwise unrelated proteins (Winston et al, (1999) Curr. Biol. 9, 1180- 1182; Cenciarelli et al, (1999) Curr. Biol. 9, 1177-1179). The F-box region is required for the SKPl interaction for the assembly of the SCF complex (SKPl, CUL-1, F-box proteins) (Zhang et al, (1995) Cell 82, 915-925; Bai et al, (1996) Cell 86, 263-274). Since F-box proteins regulate many important proteins such as β-catenin, IκB, p27, cyclin E that are involved in tumorigensis, signal transduction, cell cycle regulation, and development (Maniatis, (1999) Genes Dev. 13, 505-510; Koepp et al, (1999) Cell 97, 431-434; Sidow et al, (1999) Nat. Genet. 23, 104-107; Kawakami et al, (2000) Curr. Biol. 10, 463-466), it is anticipated that modulation of the various SCF complexes would provide a means to control and alter the biological consequences that involve the SCF activity. One way to interfere the SCF activity to alter the developmental, cell cycle, tumorigenic, or signaling pathways is to use the peptides or peptide analogues derived from the F-box region and use them as an antagonist peptide for SCF activities.

The method can be used for targeted protein knockout for genetic and biochemical analysis in cells and animals. It will help to elucidate the normal functions of a target protein in cells and animal or in human by creating deficient mutants of targeted protein. It can also be used to correct the diseases by altering the level of the disease protein or its antagonists. It can be used for testing the function and regulation of the targeted protein in diseases, drug sensitivity, development, cell growth and differentiation, programmed cell death, behavior, gene expression patterns, and learning and memory. The method can also be used for detecting protein-protein or peptide-protein interaction by fusing SKP2 or F-box proteins with a protein or peptide that bind to a target protein. Ubiquitination of the target protein can be used as the means of detection.

Example 12 - SKP2-Like Proteins SKP2-like proteins are proteins that contain a SKPl interacting domain that is homologous to the SKP2 sequence LPDELLLGIFSCLCLPELLKVSGVCKRWYRL ASDESLWQTLDL (SEQ ID NO: 2) (amino acids 112-154) (Zhang et al, (1995) Cell 82, 915-925; Bai et al, (1996) Cell 86, 263-274; Patton et al, (1998) Trends Genet. 14, 236- 243; Skowyra et al, (1997) Cell 91, 209-219; Yu et al, (1998) Proc Natl Acad Sci USA. 95, 11324-11329; Winston et al, (1999) Genes Dev. 13, 270-283; Winston et al, (1999) Curr. Biol. 9, 1180-1182; Cenciarelli et al, (1999) Curr. Biol. 9, 1177-1179). The SKPl interacting domain is the region on the SKP2 protein that interacts with the SKPl protein. This region is also called the F-box for SKPl binding (Bai et al, (1996) Cell 86, 263-274). The SKPl interacting domain is present in a variety of proteins from yeast to human, including: ( 1 ) Xenopus b-TrCP which has the sequence LPARGLDHIAENILSYLDAKSL CSAELVCKEWYRV TSDGMLWKKL (SEQ ID NO: 3) (amino acids 135-157): (2) human b-TrCP (amino acids 148-192), which is identical to SEQ ID NO: 3 (Bai et al, (1996) Cell 86, 263-274; Winston et al, (1999) Genes Dev. 13, 270-283; Spevak et al, (1993) Mol. Cell. Biol. 13, 4953-4966); and (3) some yeast proteins such as CDC4 and GRR1 (Bai et al, (1996) Cell 86, 263-274; Skowyra et al, (1997) Cell 91, 209-219).

These proteins can replace SKP2 to form a complex with SKPl and CUL-1 or their yeast homologues SKPl or CDC53. Like SKP2, they bind to phosphorylated proteins and target them for ubiquitination and degradation. More than ten human SKP2-like proteins have been identified and obtained through ESTdatabase (Figure 12) (Winston et al, (1999) Curr. Biol. 9, 1180-1182; Cenciarelli et al, (1999) Curr. Biol. 9, 1177-1179. See also, for example, SEQ ID NO: 26-61. Although the present invention has been described in detail with reference to examples above, it is understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims. All cited patents and publications referred to in this application are herein incorporated by reference in their entirety.

Claims

We claim,

1. A method of altering the level of polypeptide in a cell comprising altering the amount of one or more of the proteins selected from the group consisting of SKPl, SKP2, SKP2-like protein and CUL- 1.

2. The method of claim 1 wherein the polypeptide is phosphorylated.

3. The method of claim 1 wherein the SKP-2 like protein is selected from the group consisting of ZFl (SEQ ID NO: 27), ZF3 (SEQ ID NO: 29), ZF4 (SEQ ID NO: 31), ZF5 (SEQ ID NO: 33), ZF6 (SEQ ID NO: 35), ZF7 (SEQ ID NO: 37), ZF8 (SEQ ID NO: 39), ZF9 (SEQ ID NO: 41), ZFl 1 (SEQ ID NO: 43), ZF13 (SEQ ID NO: 45), ZF16 (SEQ ID NO: 47), ZF18 (SEQ ID NO: 49), ZF19 (SEQ ID NO: 51), ZF20 (SEQ ID NO: 53), ZF23 (SEQ ID NO: 55), ZF24 (SEQ ID NO: 57), ZF25 (SEQ ID NO: 59) and ZF26 (SEQ ID NO: 61).

4. The method of claim 1 wherein the polypeptide is p27 (SEQ ID NO: 65).

5. The method of claim 1 wherein the polypeptide is selected from the group consisting of cyclin E (SEQ ID NO: 63), Max (SEQ ID NO: 9), Mad (SEQ ID NO: 11 ), c- Myc (SEQ ID NO: 13), MDM2 (SEQ ID NO: 15), p53 (SEQ ID NO: 17), Bax (SEQ ID NO: 19), Bad (SEQ ID NO: 21) and Bcl-2 (SEQ ID NO: 23).

6. The method of claim 1 wherein the level of polypeptide is increased by decreasing the amount of SKP2.

7. The method of claim 1 wherein the level of polypeptide is reduced by increasing the amount of SKP2.

8. A method of altering the level of SKP2 comprising altering the amount of p27 polypeptide which is available for binding with SKP2.

9. A method of modulating the activity of SKP2 comprising contacting SKP2 with a peptide comprising a SKP2 interaction domain which is available for binding with SKP2.

10. The method of claim 9 wherein the peptide is phosphorylated.

11. The method of claim 10 wherein the SKP2 interaction domain is derived from p27.

12. The method of claim 10 wherein the SKP2 interaction domain is derived from cyclin E.

13. The method of claim 9 wherein the peptide comprises any one of the amino acid sequences of SEQ ID NO: 1, 2, 3, 4, 5, or 6.

14. A method of treating a tumor in a mammal comprising altering the level of SKP protein in the cells of said tumor.

15. The method of claim 14 wherein the SKP protein is SKP2 or allelic variants thereof.

16. A method of detecting a tumor in a mammal wherein the level of SKP2 is used as a diagnostic indicator to determine the progression of said tumor.

17. A method of detecting a tumor in a mammal wherein the level of SKP2 is used as a prognostic indicator to determine the progression of said tumor.

18. A method of monitoring the treatment of a tumor in a mammal wherein the level of SKP2 is used as a diagnostic indicator to monitor the success of a said treatment.

19. A method of monitoring the treatment of a tumor in a mammal wherein the level of SKP2 is used as a prognostic indicator to monitor the success of a said treatment.

20. A method of testing an agent for the ability to modulate an interaction between SKP2 and a target protein wherein the method comprises:

(a) fusing SKP2 with a target protein interaction domain to produce a SKP2 fusion protein;

(b) contacting the agent, the SKP2 fusion protein and the target protein; and

(c) determining whether the interaction of the SKP2 fusion protein with the target protein has been modulated by the agent.

21. A method of altering the level of a target protein in a cell comprising inserting a heterologous target protein interaction domain into SKP2 or a SKP2-like protein to produce a fusion protein, and contacting the fusion protein with the target protein.

22. The method of claim 21 wherein the SKP-2 like protein is selected from the group consisting of ZFl (SEQ ID NO: 27), ZF3 (SEQ ID NO: 29), ZF4 (SEQ ID NO: 31), ZF5 (SEQ ID NO: 33), ZF6 (SEQ ID NO: 35), ZF7 (SEQ ID NO: 37), ZF8 (SEQ ID NO: 39), ZF9 (SEQ ID NO: 41), ZFl 1 (SEQ ID NO: 43), ZF13 (SEQ ID NO: 45), ZF16 (SEQ ID NO: 47), ZF18 (SEQ ID NO: 49), ZF19 (SEQ ID NO: 51), ZF20 (SEQ ID NO: 53),

ZF23 (SEQ ID NO: 55), ZF24 (SEQ ID NO: 57), ZF25 (SEQ ID NO: 59) and ZF26 (SEQ ID NO: 61).

23. A method of altering the level of a target protein in a cell comprising expressing a cDNA coding for a SKP2 fusion protein comprising a SKP2 protein fused with a target protein interaction domain which is specific for the target protein.

24. A method of ubiquitinating a target protein in a cell comprising fusing a target protein interaction domain with SKP2, and permitting the SKP2 fusion protein to contact with the target protein.

25. The method of either claim 23 or 24 wherein the target protein is selected from the group consisting of p27 (SEQ ID NO: 65), cyclin E (SEQ ID NO: 63), Max (SEQ ID NO: 9), Mad (SEQ ID NO: 11), c-Myc (SEQ ID NO: 13), MDM2 (SEQ ID NO: 15), p53 (SEQ ID NO: 17), Bax (SEQ ID NO: 19), Bad (SEQ ID NO: 21) and Bcl-2 (SEQ ID NO: 23).

26. A method of modulating protein ubiquitination in a cell comprising altering the amount of SKP2 which is available to facilitate protein ubiquitination.

27. A fusion protein comprising a first protein comprising at least one SKP2 C- terminal motif (SCM) capable of interacting with SKPl and forming a complex with CUL-1 and a second protein which is capable of interacting with a heterologous target protein.

28. The fusion protein of claim 27 wherein the fusion protein contains only one

SCM capable of interacting with SKPl.

29. The fusion protein of claim 27 wherein the SCM is selected from any one of the following proteins selected from the group consisting of SKP2 (SEQ ID NO: 67), ZFl (SEQ ID NO: 27), ZF3 (SEQ ID NO: 29), ZF4 (SEQ ID NO: 31), ZF5 (SEQ ID NO: 33), ZF6 (SEQ ID NO: 35), ZF7 (SEQ ID NO: 37), ZF8 (SEQ ID NO: 39), ZF9 (SEQ ID NO: 41), ZFl 1 (SEQ ID NO: 43), ZF13 (SEQ ID NO: 45), ZFl6 (SEQ ID NO: 47), ZFl8 (SEQ ID NO: 49), ZF19 (SEQ ID NO: 51), ZF20 (SEQ ID NO: 53), ZF23 (SEQ ID NO: 55), ZF24 (SEQ ID NO: 57), ZF25 (SEQ ID NO: 59) and ZF26 (SEQ ID NO: 61).