©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Functional Interaction between c-Src and Its Mitotic Target, Sam 68 (*)

Stephen J. Taylor (1), Mordechai Anafi (2), Tony Pawson (2), David Shalloway (1)(§)

From the (1) Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853 and the (2) Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The c-Src tyrosine kinase phosphorylates and binds to a 68-kDa RNA-binding protein in mitotic cells. We have examined the mechanism and functional consequence of the interaction of c-Src with this protein, Sam 68 (Src associated in mitosis, 68 kDa). In whole cell homogenates, Sam 68 was the predominant substrate and binding partner of overexpressed c-Src. Mitotic, tyrosine-phosphorylated Sam 68 bound selectively to recombinant SH2 domains with significantly different affinities (c-Src Ras GTPase activating protein > p85 (amino-terminal) > Grb2 p85 (COOH-terminal)). In vitro translated Sam 68 also bound selectively to recombinant SH3 domains, with the highest affinity for the Src and p85 SH3 domains. SH3 binding was inhibited by specific Sam 68 peptides. In vitro translated Sam 68 bound directly to immobilized poly(U), and this was inhibited by binding of Src and p85 SH3 domains to Sam 68. The results suggest that the selection of Sam 68 as a mitotic target by c-Src is the result of highly specific interaction with SH2 and SH3 domains and that this interaction may modulate the RNA binding activity of Sam 68.


INTRODUCTION

The c-Src tyrosine kinase is activated in mitotic cells as a consequence of dephosphorylation of its regulatory phosphotyrosine residue, Tyr-527 in chicken c-Src (1, 2, 3) . Mitotic dephosphorylation of Tyr-527 is accompanied by an increased ability of the c-Src SH2 domain, which is otherwise occupied by phosphorylated Tyr-527 in an intramolecular interaction, to bind to tyrosine-phosphorylated ligands (4) . Mitotic substrates of c-Src might therefore be phosphorylated by activated c-Src and bind to the disengaged SH2 domain. We and Fumagalli and colleagues (5, 6) found such a potential mitotic target of c-Src, which we term Sam (Src associated in mitosis) 68. Sam 68() is tyrosine phosphorylated in mitotic cells expressing wild type and mutationally activated c-Src and forms a stable complex with Src by interactions with both the SH3 and SH2 domains of Src. Peptides derived from purified Sam 68 revealed that it was closely related to the product of a gene, previously cloned as encoding GAP-associated p62 (7) . However, important differences in immunoreactivity, protein and nucleic acid binding, and electrophoretic mobility have distinguished the 62-kDa tyrosine-phosphorylated protein associated with Ras GAP from Sam 68 (5, 6, 8, 9, and this report). Sam 68 was able to bind to RNA homopolymers, specifically poly(U), in vitro (5) . This is consistent with the primary structure of the p62 clone, which contains a KH domain (10, 11) and a region of similarity to the RGG box (12) , both of which are predictive of RNA-binding proteins.

Intracellular signal transduction involves the transient or stable formation of highly specific protein-protein interactions, which are mediated by protein modules, paradigms of which are SH2 and SH3 domains (13) . SH2 domains bind to phosphotyrosine (PTyr)-containing peptides in a sequence-specific manner and mediate the recruitment of SH2-containing target proteins to tyrosine-phosphorylated proteins such as activated growth factor receptors. Phosphopeptide binding to SH2 domains can also regulate enzymatic activity. For instance, intramolecular binding of phosphorylated Tyr-527 to the c-Src SH2 domain represses its tyrosine kinase activity (4, 14, 15, 16) . SH3 domains bind to sequences containing multiple proline residues, with a minimal consensus of P XXP (17, 18) . They also participate in signaling complex formation and can also modulate enzymatic activity. For example, phosphoinositide 3-kinase is activated by Src SH3 domain binding to its p85 subunit (19) . The SH3 domain of c-Src is also required for the repression of Src activity by PTyr-527-SH2 domain interaction (20, 21, 22) .

We have set out to determine the basis of the selectivity with which c-Src phosphorylates and binds to its potential mitotic target, Sam 68. We have found that Src competitively selects Sam 68 as the predominant substrate and binding partner in cell homogenates and that this can be explained by the specificity with which Sam 68 binds to the Src SH3 and SH2 domains. We have also found that SH3 domain binding to Sam 68 decreases its ability to bind to poly(U) in vitro, suggesting that Src-Sam 68 interaction may have functional consequences in vivo.


EXPERIMENTAL PROCEDURES

Plasmids and Recombinant Proteins

Plasmid pGEM-p68 h was created by inserting the human p62 (7) coding region from p62R2 (supplied by P. Polakis, Onyx Pharmaceuticals) into the EcoRI site of pGEM-3Z. Construction of cDNAs encoding GST fusion proteins with c-Src SH3 (23) , c-Src SH2 (24) , v-Src SH3, v-Src SH3 W118R and p85 SH3 (25) , Abl SH3 (26) , Grb2 (C) SH3, Fgr SH3, spectrin SH3, and PLC SH3 (27) , p85 (N,C) SH2 and GAP (N) SH2 (28) , and Grb2 SH2 (29) have been described. Human Crk (N) SH3 (residues 133-184) was constructed by cloning the relevant fragment produced by polymerase chain reaction into the BamHI- EcoRI sites of pGEX 2T. GST fusion proteins were purified from isopropyl-1-thio--D-galactopyranoside-induced Escherichia coli lysates with glutathione-Sepharose beads. Purified fusion proteins were eluted from the beads with 5 mM glutathione, 50 mM Tris-Cl, pH 8, 100 mM NaCl. Glutathione was removed by gel filtration on desalting columns, and the protein concentrations of purified, soluble fusion proteins were determined.

Cell Lysis, Cell Lysate Manipulations, Immunoprecipitation, and Immunoblotting

Cell lines overexpressing wild type c-Src (NIH(pMcsrc/foc)B), partially activated c-Src(RF) (NIH(pR148416/pSV2neo/cos)E), and fully activated c-Src (F527) (NIH(pcsrc527/foc/ep)B1) have been described (5) . Homogenization of cells and incubation of homogenates in the presence of 5 mM EDTA was as described (30) , except that cells were collected by trypsinization rather than scraping, and subcellular fractionation was not performed. After incubation for 45 min at 37 °C, homogenates (400 µl) were incubated for 15 min at room temperature in the presence or absence of 10 mM MgCl(5 mM free Mg)/1 mM ATP. Homogenates were solubilized by addition of an equal volume of 2 concentrated 3T3 lysis buffer (5) . Cell lysis, immunoprecipitations, and immunoblotting were as previously described (5) . An anti-Sam 68 rabbit antiserum was generated against peptide C corresponding to the 11 carboxyl-terminal residues of the human p62 sequence (7) conjugated to keyhole limpet hemeocyanin via an amino-terminal cysteine residue. Polyclonal anti-GAP antibodies and monoclonal anti-PTyr antibody (4G10) were from UBI.

For affinity precipitation of proteins with GST-SH2 domains, cells were lysed in 3T3 lysis buffer containing 1% SDS and then diluted 4-fold with lysis buffer (without SDS), and DNA was sheared by passage through a 22-gauge needle. Lysates were cleared by centrifugation in an SM24 rotor at 15,000 rpm for 20 min and precleared by incubating with GST-saturated glutathione beads. Precleared lysate (200 µg) was incubated with 10 µg of GST or GST-SH2 domain for 40 min on ice. Complexes were adsorbed to glutathione beads (20 µl) for 40 min, the beads were washed three times with 3T3 lysis buffer containing 0.1% SDS, and eluted proteins were immunoblotted and probed with anti-PTyr or anti-Sam 68 antibodies.

In Vitro Binding Assays

[S]Methionine-labeled Sam 68 was produced by in vitro transcription of pGEM-p68 h with SP6 DNA polymerase (Promega) and translation of the resulting mRNA in a rabbit reticulocyte lysate system (Promega). [S]Methionine metabolically labeled Sam 68 was immunoprecipitated from lysates of NIH 3T3 cells, which had been labeled for 12-16 h with [S]methionine/cysteine (ICN; 50 µCi/ml) in methionine-depleted medium. Immunoprecipitation or affinity precipitation of in vitro translated Sam 68 was performed in TLB (25 mM HEPES, pH 7.5, 150 mM NaCl, 2 mM EDTA, 0.5% Nonidet P-40, 0.1% SDS, 10 µg/ml leupeptin, and 10 µg/ml aprotinin). Prior to immunoprecipitation, lysates were precleared by incubation with pre-immune serum and protein A-Sepharose beads. Before precipitation with GST fusion proteins, lysates were precleared by incubation with GST-saturated glutathione-Sepharose beads. Precipitations were performed by incubating lysates with antiserum (10 µl/lane) or soluble GST-fusion protein in the presence or absence of competing peptides for 45 min at 4 °C, followed by incubation with protein A-Sepharose or glutathione-Sepharose beads for 30 min. Precipitates were washed 3-4 times with TLB. Precipitation with poly(U) beads (Pharmacia Biotech Inc; AGPOLY(U)) was for 10-15 min at 4 °C. Poly(U) precipitates were washed 3-4 times with TLB containing 1 mg/ml heparin. Precipitates were eluted with SDS-polyacrylamide gel electrophoresis sample buffer and subjected to SDS-polyacrylamide gel electrophoresis (9% acrylamide) and fluorography. To confirm precipitation of equal amounts of GST fusion proteins, gels were Coomassie Blue stained.

Peptides used in these studies were synthesized by Chiron Mimotopes and were biotinylated at the amino-terminal residue. Peptides were modeled on proline-rich potential SH3-binding motifs from human Sam 68 (7) , mouse 3BP1 (26) , and bovine p85 (31) . Peptide 1, QTPSRQPPLPH (Sam 68 residues 32-42); peptide 2, ATQPPPLLPPSAT (Sam 68 residues 60-72); peptide 3, RGRGAAPPPPPVPRGRG (Sam 68 residues 289-306); peptide 4, VTRGVPPPPTVRGA (Sam 68 residues 329-342); peptide 5, RIPLPPPPAPE (Sam 68 residues 354-365); peptide 3BP1, RAPTMPPPLPPVPPQP (3BP1 residues 265-280); peptide p85, SPPTPKPRPPRPLPV (p85 residues 83-97); peptide C, CGAYREHPYGRY (Sam 68 residues 433-443).


RESULTS AND DISCUSSION

To examine the specificity with which c-Src phosphorylates and associates with Sam 68 in a physiological context, homogenates of NIH 3T3-derived c-Src overexpressor cells were incubated in the presence of EDTA to inhibit endogenous kinase activity, allowing global dephosphorylation of cellular proteins by endogenous phosphatases. Reincubation of EDTA-treated homogenates with Mg/ATP resulted in the tyrosine phosphorylation of two predominant proteins with molecular masses of 60 and 68 kDa, revealed by anti-PTyr immunoblotting (Fig. 1, lane 5). The 60-kDa phosphorylated protein comigrated with c-Src, which was the predominant PTyr-containing protein in untreated lysates ( lanes 3 and 5). The 68-kDa phosphoprotein comigrated with tyrosine-phosphorylated Sam 68 in lysates from mitotic c-Src(RF) overexpressor cells ( lanes 2 and 5). Immunoprecipitation of c-Src from detergent-solubilized homogenates demonstrated that tyrosine-phosphorylated Sam 68 co-immunoprecipitated with c-Src after ATP treatment or with c-Src(RF) from mitotic lysates ( lanes 6-9). No other coprecipitating PTyr-containing proteins were detected. The identity of the coprecipitating tyrosine-phosphorylated protein as Sam 68 was confirmed by probing a duplicate blot with a commercial anti-Sam 68 antibody (data not shown). Therefore, Sam 68 appears to be the preferred substrate and binding partner of dephosphorylated, activated c-Src in total cellular homogenates, as in mitotic cells in vivo. This implies that Sam 68 possesses highly specific recognition motifs for phosphorylation by and for binding to c-Src and/or that high affinity interaction of Sam 68 phosphotyrosine residues with the c-Src SH2 domain protects Sam 68 from tyrosine dephosphorylation.


Figure 1: Src preferentially selects Sam 68 as a substrate and binding partner in a cell-free system. Unsynchronized ( U) or mitotic ( M) c-Src(RF) expressor cells were directly lysed in lysis buffer. Unsynchronized wild type ( WT) c-Src overexpressor cells were homogenized, incubated in the presence of EDTA, and then incubated in the presence (+) or absence () of Mg/ATP and solubilized with lysis buffer. Lysates ( lanes 1-5) or anti-Src immunoprecipitates ( lanes 6-9) were immunoblotted and probed with anti-PTyr antibodies. Bound antibodies were detected by incubation with I-protein A followed by autoradiography. The positions of molecular mass markers (in kDa), Sam 68 (p68), and Src are indicated.



To further study Sam 68-Src interactions, we generated a polyclonal antiserum against a peptide based on the Sam 68 carboxyl-terminal as predicted from the p62 cDNA sequence (see below). This antiserum immunoprecipitated a tyrosine-phosphorylated, 68-kDa protein from Src(F527)-transformed NIH 3T3 cells (Fig. 2, lane 3) and recognized an anti-PTyr and GST-SH3 domain-precipitable 68-kDa protein on Western blots ( lanes 1, 3, and 6). The same antibody did not detect any proteins in Western-blotted anti-Ras GAP immunoprecipitates, although a coprecipitating 62-66-kDa protein (GAP-associated p62), as well as p190, were clearly detected with an anti-PTyr antibody ( lanes 4). These results are consistent with those of Ogawa et al. (8) who concluded that Sam 68 and GAP-associated p62 are distinct proteins. However, we cannot exclude the possibility that differences in immunoreactivity, SH3 binding, electrophoretic mobility, and GAP binding in vivo are due to differential post-translational or post-transcriptional modifications of the same gene product.


Figure 2: Sam 68 is distinguishable from GAP-associated p62. Lysates from unsynchronized c-Src(F527) overexpressor cells were immunoprecipitated with pre-immune ( lanes 2), anti-Sam 68 ( lanes 3), or anti-GAP ( lanes 4) sera or affinity precipitated with GST ( lanes 5) or GST-Src SH3 ( lanes 6). Whole cell lysates ( WCL) ( lanes 1) and precipitates were immunoblotted, and duplicate blots were probed with anti-PTyr or anti-Sam 68 antibodies. Detection was by chemiluminescence.



To analyze the specificity of binding of mitotic, tyrosine-phosphorylated Sam 68 to individual SH2 domains, lysates from unsynchronized or mitotic c-Src(RF) cells were incubated with equal amounts of the SH2 domains of c-Src, Grb2, Ras GAP (amino-terminal), and the p85 subunit of phosphoinositide 3-kinase (amino- and COOH-terminals), and the precipitated complexes were immunoblotted and probed with anti-PTyr or anti-p68 antibodies (Fig. 3). Lysates from c-Src(F527) cells were also probed to ensure that each SH2 domain was capable of binding tyrosine-phosphorylated proteins. As expected, Sam 68 was precipitated by the Src SH2 domain from mitotic but not from unsynchronized Src(RF) or Src(F527) transformed cells ( lanes 7-9). Mitotic, tyrosine-phosphorylated Sam 68 also bound to other SH2 domains with apparent affinity Src Ras GAP (N) > p85 (N) > Grb 2, with little detectable binding to the p85 (C) SH2 domain ( lanes 8, 11, 14, 17, and 20). The ability of mitotic, tyrosine-phosphorylated Sam 68 to bind selectively to other SH2 domains and the presence of multiple potential tyrosine phosphorylation sites in Sam 68 (7) suggests that other SH2 domain-containing proteins may be recruited to the Src-Sam 68 complex in mitotic cells. We have, however, been unable to detect association of Sam 68 with either Ras GAP or p85 in vivo (Fig. 2).()


Figure 3: Tyrosine-phosphorylated Sam 68 from mitotic cells selectively binds recombinant SH2 domains. Lysates from unsynchronized ( U) or mitotic ( M) c-Src(RF) cells or unsynchronized c-Src(F527) cells ( F) were affinity precipitated with 10 µg of GST ( lanes 4-6) or GST-Src SH2 ( lanes 7-9), GST-Grb2 SH2 ( lanes 10-12), GST-GAP amino-terminal SH2 ( lanes 13-15), GST-p85 amino-terminal SH2 ( lanes 16-18), or GST-p85 COOH-terminal SH2 ( lanes 19-21). Lysates ( lanes 1-3) and precipitates were immunoblotted, probed with anti-PTyr or anti-Sam 68, and detected by chemiluminescence.



In vitro transcription and translation of the human p62 cDNA produced an [S]methionine-labeled protein of slightly faster electrophoretic mobility than Sam 68 immunoprecipitated from [S]methionine metabolically labeled NIH 3T3 cells (Fig. 4, lanes 1 and 3). This may reflect post-translational modification of Sam 68 in fibroblasts. Transcription and translation of the human p62 cDNA produced a protein that was specifically immunoprecipitated by anti-Sam 68 ( lanes 4 and 5) and affinity precipitated by GST-Src SH3 ( lane 7), confirming that Sam 68 is the product of the p62 cDNA. We have previously shown that Sam 68 is precipitated from NIH 3T3 lysates by poly(U) beads (5) . To determine whether Sam 68 binds directly to poly(U), this experiment was repeated with in vitro translated Sam 68. Specific precipitation of Sam 68 with poly(U) beads was observed. This was competed by soluble poly(U) but not by poly(C) ( lanes 10-12), demonstrating direct Sam 68-poly(U) interaction. Interestingly, Sam 68 was efficiently precipitated by poly(I)-poly(C) beads (Fig. 7, lane 11), suggesting that it may be able to bind to double-stranded RNA.


Figure 4: Sam 68 is the product of the p62 cDNA and binds poly(U) directly. Sam 68 was immunoprecipitated from [S]methionine metabolically labeled NIH 3T3 cells in the presence or absence of peptide C (40 µM) ( lanes 1 and 2). In vitro translated [S]methionine-labeled product of the human p62 cDNA was immunoprecipitated with anti-Sam 68 with or without 50 µM peptide C ( lanes 4 and 5), affinity precipitated with GST ( lane 6) or GST-Src SH3 with or without peptides C or 3 ( lanes 7-9), or affinity precipitated with poly(U) beads with or without soluble poly(U) or poly(C) (1 mg/ml) ( lanes 10-12) or with poly(U) beads pretreated with micrococcal nuclease ( lane 13). Lane 3 contains the input translation product (20% of amount used for precipitations).




Figure 7: SH3 domains inhibit binding of poly(U) to Sam 68. In vitro translated Sam 68 was precipitated with poly(U) beads ( lanes 2-10), poly(I)-poly(C) beads ( lane 11), nuclease-treated poly(U) beads ( lane 12), or glutathione beads ( lane 13) in the presence or absence of 10 µg (unless otherwise indicated) of the indicated GST fusion proteins. The precipitation in lane 6 was performed in the presence of 250 µM peptide 3. Lane 1 contains 20% of input translation product.



The specificity of the interaction between Sam 68 and SH3 domains was examined by affinity precipitating in vitro translated Sam 68 with different GST-SH3 domain fusion proteins (Fig. 5). Sam 68 was bound most effectively by the SH3 domains of c-Src and v-Src, but appreciable binding was also observed with the p85 SH3 domain ( lanes 3-6, 14, and 16). The SH3 domains of Crk (amino-terminal), Abl, and PLC bound Sam 68 to lesser extents, while no binding was detectable to the SH3 domains of Fgr, spectrin, and Grb2 (COOH-terminal). Binding to the v-Src SH3 domain was abolished by point mutation of Trp-118, which is conserved in SH3 domains. Similar results were obtained by precipitating Sam 68 from NIH 3T3 lysates with different GST-SH3 domains (data not shown).


Figure 5: Sam 68 selectively binds recombinant SH3 domains. In vitro translated Sam 68 was precipitated with GST ( lane 2) or the indicated GST-SH3 fusion proteins ( lanes 3-16). 5 µg of fusion protein was used for precipitation except where indicated (0.5 µg; lanes 3, 5, and 7). Lanes 1 and 17 contain 20% of input translation product.



To define the SH3 binding sites within Sam 68, peptides corresponding to proline-rich, potential SH3 binding sites in Sam 68 were synthesized. The ability of these peptides to inhibit Sam 68 binding to the SH3 domains of Src or p85 was assessed (Fig. 6). Peptide 3, corresponding to residues 289-306 in Sam 68, completely inhibited Sam 68 binding to Src SH3 ( lane 6). The same concentrations of peptides corresponding to residues 354-365 of Sam 68 (peptide 5) and 265-280 of 3BP1 partially competed with Sam 68 for Src SH3 binding ( lanes 8 and 9). In contrast, peptide 3 only partially competed for binding of Sam 68 to p85 SH3. Peptide 4 from Sam 68 and residues 89-97 of p85 also partially blocked binding ( lanes 14, 15, and 18). We conclude that Src SH3 binds primarily to a region within residues 289-306 of Sam 68, while the p85 SH3 domain binds to this and at least one other site with apparently lower affinity. In similar experiments, the Ab1 SH3 domain exhibited a binding profile similar to that of the Src SH3 domain, while the Crk (N) and PLC SH3 binding profiles resembled that of p85 (data not shown). The results suggest that Sam 68 interacts in vivo with the Src SH3 domain in the region of residues 289-306 but that other SH3 binding sites in Sam 68 may mediate interaction with other SH3 domain-containing proteins, such as p85, and possibly other c-Src molecules.


Figure 6: Proline-rich peptides differentially inhibit Sam 68 binding to Src and p85 SH3 domains. In vitro translated Sam 68 was precipitated with 10 µg of GST ( lane 2), GST-Src SH3 ( lanes 3-10), or GST-p85 SH3 ( lanes 11-18) in the presence or absence of 100 µM of the indicated peptides. Lane 1 contains 20% of input translation product.



To gain insight into the functional consequences of the Src SH3-Sam 68 interaction, we examined whether Src SH3 was able to modify the RNA binding activity of Sam 68 in vitro (Fig. 7). Affinity precipitation of in vitro translated Sam 68 by poly(U) beads was blocked in a concentration-dependent manner by the Src SH3 domain ( lanes 2-5). This inhibition was blocked by Sam 68 peptide 3 ( lane 6), consistent with the binding data above. The ability of other SH3 domains to inhibit poly(U) binding correlated with their ability to bind to Sam 68 ( lanes 7-10). Coomassie Blue staining of precipitates verified that SH3 domains did not directly bind to the poly(U) beads (data not shown), indicating that binding of SH3 domains to Sam 68 blocks its ability to bind poly(U). It will be important to determine whether SH3 domains and poly(U) compete for overlapping binding sites or whether SH3 binding causes a conformation change in Sam 68 that decreases its intrinsic poly(U) binding activity.

Taken together, the results suggest that the selectivity with which Src associates with and phosphorylates Sam 68 in mitotic cells may stem from the high specificity of binding of Sam 68 sequences to the Src SH3 and SH2 domains. As a working model, we suggest that binding of Sam 68 to the Src SH3 domain may enable phosphorylation of a tyrosine residue, which then binds to c-Src SH2. Subsequent tyrosine phosphorylation of bound Sam 68 may create SH2 binding sites for other SH2-containing proteins, while those SH3 binding sites not engaged in binding to Src may recruit SH3-containing proteins to the Src-Sam 68 complex. In this way, Src-Sam 68 could direct the subcellular localization or modulate the activity of signaling proteins during the mitotic transition. This model of Sam 68 acting as a Src-elicited SH2/SH3 docking protein is consistent with recent studies by Richard et al. (32) who found that tyrosine-phosphorylated Sam 68 co-immunoprecipitated with Grb2 from lysates of HeLa cells cotransfected with Sam 68, Fyn, and Grb2-encoding plasmids and with Grb2 and phospholipase C from v-Src-transformed NIH 3T3 cells.

The ability of Src SH3 to inhibit binding of poly(U) by Sam 68 raises the possibility that interaction of c-Src with Sam 68 modifies, either by decreasing binding ability or changing specificity, Sam 68-RNA interactions. The consequences of such modulations in mitotic cells might be to suppress processing/translation of inappropriate ( e.g. G1/S-specific) messages or to prepare Sam 68 for interaction with G1-specific messages after mitosis. Since the Src SH3 domain also binds to ribonucleoprotein K (5, 33) , it will be interesting to determine whether this interaction also results in a modulation of RNA binding ability.

It is clear from the results presented here and elsewhere that Sam 68 is the product of the cDNA reported to encode GAP-associated p62. Antibodies raised against peptides or recombinant protein fragments derived from the reported p62 cDNA sequence specifically recognize Sam 68 ( Fig. 2and Refs. 5, 8, and 32), and transcription/translation of this clone in vitro and in vivo produces a protein with all of the characteristics of Sam 68 ( Fig. 3and Refs. 9 and 32). However, it is also clear that Sam 68 and GAP-associated p62 differ significantly in many respects. Unlike Sam 68, GAP-associated p62 does not bind to the Src SH3 domain ( Fig. 2and Ref. 6) or to poly(U) beads (8) , does not exhibit increased tyrosine phosphorylation in mitosis (Ref. 6),() and does not react with anti-Sam 68 antibodies (including commercially available anti-p62 antibodies) ( Fig. 2and Ref. 8). Furthermore, Sam 68 does not appear to associate with GAP in vivo ( Fig. 2 and Ref. 8). In light of these data and until the exact nature of GAP-associated p62 is ascertained, we suggest that Sam 68 and GAP-associated p62 be regarded as distinct proteins and be referred to accordingly.


FOOTNOTES

*
This research was supported by National Institutes of Health Grant CA 32317 (to D. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed. Tel.: 607-254-4896; Fax: 607-255-2428.

The abbreviations used are: Sam 68, Src associated in mitosis (68 kDa); GAP, GTPase-activating protein; GST, glutathione S-transferase; PTyr, phosphotyrosine.

S. J. Taylor and D. Shalloway, unpublished observations.

S. J. Taylor and D. Shalloway, unpublished results.


ACKNOWLEDGEMENTS

We thank Paul Polakis for the human Sam 68 cDNA, Piera Cicchetti for bacteria expressing GST-Abl SH3, Stéphane Richard for communication of unpublished results, Shubha Bagrodia, Douglas Laird, and Ross Resnick for discussions, Eden Heinemann for preparation of figures, and Paula Slocum for preparation of the manuscript.


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