AU701733B2 - GDU, a novel signalling protein - Google Patents

Description

WO 96/34951 PCT/AU96/00258 1 GDU, A Novel Signalling Protein The present invention relates to a previously unidentified erbB receptor target designated GDU. The present invention relates to a polynucleotide encoding GDU and to methods of detecting the presence of

GDU.

Many intracellular targets for receptor tyrosine kinases (RTKs) contain one or more src homology (SH)2 domains. These are conserved, non-catalytic domains of approximately 100 amino acids which bind to short peptide sequences containing phosphotyrosine (Cohen et al, Cell 80, 237- 248, 1995). Since receptor autophosphorylation on specific tyrosine residues follows RTK activation, SH2 domains mediate receptor-substrate, as well as other protein-protein interactions, during signal transduction. SH2 domains contain not only a pocket lined with basic residues which binds the phosphotyrosine but also an additional binding pocket or groove which interacts with amino acids C-terminal to this residue, this determining the specificity of the interaction. The particular autophosphorylation sites present on a given RTK therefore define the SH2 domain-containing signalling proteins that it can recruit and hence, to a large extent, the signalling specificity of the receptor. SH2 domains are often accompanied in signalling proteins by two other conserved protein modules; SH3 domains, which bind to proline-rich peptide ligands, and pleckstrin-homology

(PH)

domains. The function of the latter remains ill-defined, and both protein and phospholipid ligands have been described.

SH2 domain-containing proteins can be divided into two classes (Schlessinger and Ullrich Neuron, 9,383-301 1992); Class I, which also possess a catalytic function e.g. phospholipase C-yl (PLC-yl) and the GTPase activating protein for Ras (Ras-GAP), and Class II, which contain only noncatalytic protein modules and are thought to function as adaptors, linking separate catalytic subunits to receptors or other signalling proteins e.g. Grb2.

The tissue expression of particular SH2 domain-containing proteins varies from ubiquitous, e.g. Grb2, which performs a fundamental role in linking tyrosine kinases to Ras signalling, to relatively restricted e.g. Grb7, which is mainly expressed in the liver and kidney (Margolis et al Proc. Natl. Acad.

Sci. USA, 89, 8894-8898, 1992). Presumably the latter protein performs relatively specialised signalling functions. The CORT (cloning of receptor targets) technique, in which cDNA expression libraries are screened with the WO 96/34951 PCT/AU96/00258 2 tyrosine phosphorylated C-terminus of the EGF receptor represents a powerful methodology for the identification and characterisation of novel, SH2 domain-containing, receptor substrates (Skolnik et al Cell 65, 83-90, 1991).

Members of the erbB family of RTKs and their ligands are implicated both in normal mammary gland development and the growth and progression of human breast cancer. Furthermore, marked alterations in the expression or activity of several SH2 domain-containing proteins have been observed in human breast cancers or breast cancer-derived cell lines, suggesting that this represents an additional level at which RTK signalling may be modulated in this disease (Daly, Breast Cancer Res Treat, 34, 85-92, 1995). We therefore chose normal human mammary epithelial cells as a basis for a CORT screening program and hence identification of novel, and relatively tissue specific, erbB receptor targets.

Screening of a HMEC 184 XEXlox cDNA library isolated 1 Ras-GAP, 2 Grb2 cDNAs and a cDNA encoding a novel SH2 domain-containing protein. This protein, designated GDU or Grbl4 (the designations "GDU" and "Grbl4" are used interchangeably herein), is related both in molecular architecture and sequence homology to Grb7 and GrblO, previously identified erbB receptor targets. These three proteins also share significant sequence homology, over an approximately 300 amino acid region encompassing the PH domain, with the C. elegans gene F10E9.6. The latter gene has recently been shown to encode a protein (mig 10) critical for longitudinal neuronal migration in C. elegans; members of the Grb7 gene family, including GDU, may therefore be involved in the regulation of cell migration in higher organisms.

Analysis of GDU gene expression in normal breast epithelial cells and a large series of human breast cancer cell lines revealed that expression was limited predominantly to normal breast cells and the more highly differentiated, estrogen receptor positive, breast cancer cell lines. Also, GDU mRNA was overexpressed in the DU-145 prostate carcinoma cell line relative to the normal prostate and two other prostate cancer cell lines. GDU may therefore serve as a prognostic indicator and/or a tumour marker in both breast and prostate cancer. Furthermore, since altered expression of GDU may contribute to the abnormal proliferation, invasion and/or migration of cancer cells, GDU signal transduction may provide a novel therapeutic target in human cancer. Finally, since GDU is involved in downstream signalling initiated by the platelet derived growth factor receptor (PDGFR), it may provide a target in diseases or conditions in which PDGF plays a regulatory role e.g. wound healing, fibrotic conditions, atherosclerosis.

In a first aspect the present invention consists in a polynucleotide encoding GDU, the polynucleotide having a sequence which encodes a polypeptide having an amino acid sequence as shown in Figure 2 or a sequence which hybridises thereto under stringent conditions.

As used herein, stringent conditions are those that employ low ionic strength and high temperatures for washing, for example, 0.015 M NaC1/0.0015 M sodium citrate/0.1% NaDodSO 4 at 50°C; employ during hybridisation a denaturing agent such as formamide, for example, (vol/vol) formamide with 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaC1, 75 mM sodium citrate at 42 0 C; or employ 50% formamide, 5 x SSC (0.75 M NaC1, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 g/ml), 0.1% SDS and 10% dextran sulfate at 42 0 C in 0.2 x SSC and 0.1% SDS.

1 20 In a preferred embodiment of the present invention the polynucleotide has a sequence as shown in Figure 2.

In a second aspect the present invention consists in a polypeptide, the polypeptide having an amino acid sequence as shown in Figure 2.

In a third aspect the present invention consists in an antibody raised 25 against the polypeptide of the second aspect of the present invention.

The antibody may be monoclonal or polyclonal, however, it is presently preferred that the antibody is a monoclonal antibody.

In a fourth aspect, the present invention consists in an oligonucleotide probe of at least 12 nucleotides, the oligonucleotide probe having a sequence such that the probe selectively hybridises to the polynucleotide of the first aspect of the present invention under stringent conditions.

In a preferred embodiment of this aspect of the present invention the oligonucleotide is labelled. In a further preferred embodiment of the present invention the oligonucleotide is of at least 18 nucleotides.

In a fifth aspect the present invention consists in method of detecting the presence of GDU in a sample, the method comprising reacting the sample with an antibody of the second aspect of the present invention or a oligonucleotide probe of the fourth aspect of the present invention and detecting the binding of the antibody or the probe.

In order that the nature of the present invention may be more clearly understood preferred forms thereof will now be described with reference to the following examples and Figures in which:- Figure 1 shows a schematic representation of Grbl4 structure with a restriction map for the Grbl4 cDNA and the cDNA clones used to derive the Grbl4 sequence aligned underneath. The initial clone isolated by CORT screening was designated clone 1. Two other clones and were o

U

WO 96/34951 PCT/AU96/00258 4 isolated from the 184 cell line library by screening using clone 1 as a probe.

The Grbl4 cDNA sequence was completed using two clones L5 and L6, isolated from a human liver cDNA library. Abbreviations are as follows: A; Apa I; Av; Avr II, X; Xho I; E; Eco RI. The numbers refer to distance in bp.

Figure 2 shows the nucleotide and amino acid sequence of Grbl4.

The PH domain is underlined and the SH2 domain indicated by bold type.

The translation termination codon is shown by an asterisk in the amino acid sequence. Numbers refer to distances in bp.

Figure 3 shows the sequence homology between Grbl4, Grb7, GrblO and F10E9.6. As alignment of the amino acid sequences of Grb14, mouse Grb7, mouse GrblO and C. elegans F10E9.6 was obtained using the computer programs Clustal W and SeqVu. Identical residues are boxed. A highly conserved proline-rich motif is indicated by the dotted underline, the PH domain by the broken underline and the SH2 domain by the bold underline.

Only the central region of F10E9.6 exhibiting homology with the Grb7 family is shown. Amino acid residues for each protein are numbered (from the initiation methionine) on the right.

Screening of a Normal Breast Epithelial Cell cDNA Library by the CORT Technique CORT screening of two cDNA libraries prepared from normal breast epithelial cells led to the isolation of recombinants which exhibited differential binding to the phosphorylated EGFR C-terminus. Upon excision of the corresponding pEXlox plasmids and sequencing of the DNA inserts, two recombinants which bound very strongly were identified as Grb2 cDNA clones (Lowenstein et al 1992, Cell 70, 431-442, 1992), and a clone exhibiting moderate binding corresponded to ras-GAP (Trahey et al. Science 242, 1696- 1700, 1988 The final clone, designated GDU, bound only weakly to the EGFR. A database search with the corresponding cDNA sequence did not detect an exact match but revealed significant sequence homology with the SH2 domain-containing protein Grb7 (Margolis et al PNAS 89, 8894- 8898,1992). The cDNA (GDU Clone 1 in Figure 1) encoded a short stretch of amino acids followed by a C-terminal SH2 domain; homology to Grb7 was apparent over this entire open reading frame.

WO 96/34951 PCT/AU96/00258 Characterisation of GDU In order to obtain the full length cDNA sequence for GDU, two cDNA library screens were performed. In the first, the cDNA insert from Clone 1 was used to screen the breast cDNA library. Screening of 5 x 105 recombinants isolated 2 cDNAs, designated 1-1 and 1-2, of 1.6 and 1.4kb, respectively (Figure In the second, a 213bp EcoRI-Xho I restriction fragment derived from 1-1 (Figure 1) was used to screen a human liver cDNA library. Screening of 1 x 106 recombinants isolated 2 cDNAs, designated and L6, of 1.3 and 1.7kb, respectively (Figure Clones 1-1, 1-2, L5 and L6 were sequenced in their entirety on both strands to obtain the cDNA sequence shown in Figure 2. The 2.4kb of DNA sequence derived from these overlapping clones corresponds closely to the size of the three most abundant mRNA species detected upon Northern blot analysis.

Analysis of the cDNA sequence identified an open reading frame of 540 amino acids. The initiation codon is preceded by an in-frame termination codon and is surrounded by a consensus sequence for strong translational initiation. The encoded protein is similar both in molecular architecture and amino acid sequence to Grb7 (Margolis et al, Proc. Natl.

Acad. Sci. USA 89, 8894-8898, 1992) and the recently identified GrblO (Ooi et al, Oncogene 10, 1621-1630, 1995), consisting of a N-terminal region containing at least one proline-rich motif, a central region which exhibits significant homology to the putative C. elegans protein F10E9.6 (Stein et al EMBO J, 13, 1331-1340, 1994) and which also encompasses a PH domain, and a C-terminal SH2 domain. An alignment of the amino acid sequences of GDU, Grb7, GrblO and F10E9.6 is shown in Figure 3.

GDU is similar in size to Grb7, GrblO possessing a more extended N-terminus. The N-terminal region exhibits low sequence homology between GDU, Grb7 and GrblO apart from a highly conserved amino acid motif PS/AIPNPFPEL. Also of note is the presence of two clusters of basic residues which flank this motif. Overall the N-terminal region of GDU displays a lower proline content than that of Grb7 and GrblO (GDU amino acids 1-110; 11% proline, GrblO amino acids 1-113; 15%, Grb7 amino acids 1-103; 23%).

GDU, Grb7 and GrblO share a central, conserved region of approximately 320 amino acids which exhibits significant homology to a domain found in the C. elegans protein F10E9.6. Over this region, GDU WO 96/34951 PCT/AU96/00258 6 bears 48, 55 and 28% amino acid identify respectively with Grb7, GrblO and F10E9.6 (Figure The core of this region is provided by a PH domain (Figures 1, 2 and over which GDU exhibits 56, 61 and 35% amino acid identity, respectively, with Grb7, GrblO and F10E9.6. However, as noted by Ooi et al, (Oncogene 10, 1621-1630, 1995) another region of particularly marked homology spanning approximately 100 amino acids exists aminoterminal to the PH domain (Figure 3).

The most highly conserved region amongst Grb7 family members is the SH2 domain (Figure The GDU SH2 domain displays 67 and 74% amino acid identity, respectively, with the corresponding domain in Grb7 and Grbl0.

Northern Blot Analysis of GDU Gene Expression The tissue specificity of GDU gene expression was investigated by hybridizing Northern blots of poly A RNA isolated from a variety of human tissues to a GDU specific cDNA probe. GDU gene expression was highest in the testis, ovary, heart, liver, skeletal muscle, kidney and pancreas.

Moderate expression was detected in the small intestine, colon, peripheral blood leukocytes, brain and placenta, whilst expression in the spleen, thymus, prostate and lung was low or undetectable. Several mRNA transcripts were detected which displayed tissue-specific variation in their relative abundance. The three most prominent transcripts were approximately 2.3, 2.4 and 2.5kb. Often co-expressed with one or two of these transcripts was a transcript of approximately 9.5kb. In the ovary a still larger transcript of undetermined size was also expressed.

Since the Grbl4 cDNA was originally isolated from a cDNA library prepared from normal human breast epithelial cells, we were interested in determining the expression profile of Grbl4 mRNA in a panel of human breast cancer cell lines. Upon Northern blot analysis of total RNA isolated from 3 normal human breast epithelial cell strains and 19 human breast cancer cell lines, Grbl4 gene expression could be detected in HMEC 184 and HMEC-219-4 cells, 6/7 ER+ human breast cancer cell lines and 2/12 ER- cell lines (Table Thus Grbl4 gene expression appears largely restricted to normal breast epithelial and ER+ breast cancer cells. Differential expression of Grbl4 was also observed amongst human prostate cancer cell lines.

Although Grbl4 mRNA expression was undetectable in the normal prostate, WO 96/34951 PCT/AU96/00258 7 low expression could be detected in the PC3 and LnCaP prostate cancer cell lines and high expression in the DU145 line (Table 1).

Origin Cell Line Expression Normal human breast epithelial Human breast cancer, ER+ Human breast cancer, ER- Human prostate cancer Human epidermoid carcinoma Human embryonic kidney HMEC 184 HMEC-219-4 HMEC-1001-7 T-47D ZR-75-1 MCF-7 BT-483 MDA-MB-134 MDA-MB-361 BT-474 MDA-MB-330 MDA-MB-468 SK-BR-3 BT-549 H3578T DU-4475 MDA-MB-157 MDA-MB-175 MDA-MB-231 MDA-MB-436 MDA-MB-453 PC3 LnCaP DU-145 A431 HEK 293 Table 1. Expression of Grbl4 mRNA in different human cell lines. Total cellular RNA was extracted from the indicated cell lines and subjected to Northern blot analysis using a Grbl4 cDNA probe. The relative expression levels of Grbl4 mRNA were then scored on a scale from (low) to (high). undetectable expression.

WO 96/34951 PCT/AU96/00258 8 Expression of Grbl4 Protein In order to characterize the Grbl4 protein a polyclonal antiserum was raised against a GST-Grbl4 SH2 domain fusion protein. Following affinity purification, this antiserum was used to Western blot cell lysates derived from cell lines in which Grbl4 mRNA was either expressed at high levels (DU145 and HEK 293) or was undetectable (A431 and SK-BR-3) (Table The antiserum recognized a protein of approximately 58kDa in DU145 cells, whilst in HEK 293 cells a tight doublet of this mobility was detected. These bands were not observed upon Western blotting with preimmune serum or in the cell lines which do not express Grbl4 mRNA. This estimated size of Grbl4 upon SDS-PAGE is in accordance with the predicted size of the translation product of the Grbl4 cDNA Since DU145 cells overexpress Grbl4 mRNA relative to the two other prostate carcinoma cell lines examined (Table we investigated whether this was accompanied by an upregulation of Grbl4 protein expression.

Upon Western blot analysis, Grbl4 was clearly detectable in DU145, but not PC3 or LnCaP, cell lysates, indicating that Grbl4 protein is overexpressed in this cell line.

Phosphorylation of Grbl4 In order to characterize further the role of Grbl4 in receptor tyrosine kinase signalling, the phosphorylation state of Grbl4 was investigated before and after growth factor stimulation. Since the anti-Grbl4 antiserum 264 did not immunoprecipitate Grbl4 under either native or denaturing conditions, we utilized an expression construct (pRcCMVFIag) which tagged Grbl4 with the 8 amino acid Flag epitope at the C-terminus. This construct was stably transfected into HEK 293 cells, leading to the isolation of stable clones of cells expressing an epitope-tagged Grbl4 which could be immunoprecipitated with the M2 anti-Flag monoclonal antibody and Western blotted with either this antibody or anti-Grbl4 antiserum 264.

Immunoprecipitation of Grbl4 from serum starved cells which were metabolically labelled with 32 P-orthophosphate demonstrated that Grbl4 was phosphorylated in this basal state. Phosphoamino acid analysis of the isolated protein demonstrated that phosphorylation was on serine residues.

WO 96/34951 PCT/AU96/00258 9 Treatment of the cells with EGF did not significantly increase this level of phosphorylation, although activation of native EGFRs could be demonstrated by anti-phosphotyrosine blotting of the cell lysates. However, stimulation with PGDF BB resulted in an approximately 1.5 fold increase within 5 min of administration, and transient transfection of a cDNA encoding P-PDGFRs into the cells further amplified this response to approximately 2-fold. The small increase in phosphorylation which occurred when this construct was present in the absence of PGDF BB was presumably due to the constitutive activation of RTKs often observed with this system. Phosphoamino acid analysis demonstrated that the PDGFinduced increases in Grbl4 phosphorylation also occurred on serine residues.

As will be recognised by persons skilled in this field the present inventors have identified a novel signalling molecule which they have designated GDU or Grbl4. GDU has the potential to be used as a prognostic indicator/tumour marker in both breast and prostate cancer. In addition, as GDU may influence invasive/ metastatic behaviour it may also serve as a marker of invasive/metastatic disease in these and other cancers. Finally, the involvement of GDU in signalling by the PDGFR suggests that it may represent a therapeutic target in diseases or conditions in which PDGF plays a regulatory role.

Signalling via GDU could be targeted by competitive peptides or dominant negative mutants, or restored by gene therapy. The information provided herein will clearly assist in the rational design of a GDU antagonist.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Claims (7)

1. A polynucleotide encoding GDU, the polynucleotide having a sequence which encodes a polypeptide having an amino acid sequence as shown in Figure 2 or a sequence which hybridises thereto under stringent conditions.

2. A polynucleotide as claimed in claim 1 in which the polynucleotide has a sequence as shown in Figure 2.

3. A GDU polypeptide, the polypeptide having an amino acid sequence as shown in Figure 2.

4. An antibody raised against the polypeptide as claimed in claim 3. An antibody as claimed in claim 4 in which the antibody is a monoclonal antibody.

6. An oligonucleotide probe of at least 12 nucleotides, the oligonucleotide probe having a sequence such that the probe selectively hybridises to the 15 polynucleotide as claimed in claim 1 under stringent conditions.

7. An oligonucleotide probe as claimed in claim 6 in which the "I oligonucleotide probe is labelled.

8. An oligonucleotide probe as claimed in claim 6 or claim 7 in which the oligonucleotide is of at least 18 nucleotides. 20 9. A method of detecting the presence of GDU in a sample, the method comprising reacting the sample with an antibody as claimed in claim 4 or I claim 5 or an oligonucleotide probe as claimed in any one of claims 6 to 8 and detecting the binding of the antibody or the probe. Dated this fourteenth day of December 1998. GARVAN INSTITUTE OF MEDICAL RESEARCH Patent Attorneys for the Applicant: F B RICE CO