The Human p62 cDNA Encodes Sam68 and Not the RasGAP-Associated p62 Protein

and Not the RasGAP-Associated either anti-HA or anti-P antibodies. Conversely, a 68 kDa p62 Protein protein was detected in anti-HA and anti-P but not anti-GAP immunoprecipitates reprobed with anti-HA (Figure 1A, bottom). The anti-HA antibodies also immunoprecip-itated a Tyr-phosphorylated protein of approximately 68 The p62 protein is tyrosine (Tyr) phosphorylated in cells expressing activated Tyr kinases (Ellis et al., 1990) and kDa from mitotic but not asynchronous extracts (Figure associates with RasGAP in a phosphotyrosine (pTyr)-1B, top). In addition, anti-C antibodies (see Figure 2A) and SH2 domain–dependent fashion (Marengere and recognized two proteins of approximately 68 kDa that Pawson, 1992; Moran et al., 1990). The putative p62 were Tyr phosphorylated only in mitotic cells. (Presum-protein was purified from v-Src-transformed murine ably, the larger protein corresponded to the product fibroblasts using anti-pTyr antibodies and oligo-of the epitope-tagged hump62 cDNA, and the smaller nucleopeptide probes based on peptide sequences ob-protein corresponded to endogenous Sam68.) When the tained to isolate a human cDNA, hump62 (GenBank ac-same blot was reprobed with anti-HA antibodies, only cession number M88108; Wong et al., 1992). We and a 68 kDa protein was detected in anti-HA and anti-C others (Fumagalli et al., 1994; Taylor and Shalloway, immunoprecipitates from both asynchronous and mi-1994; Weng et al., 1994) recently identified Sam68 totic cells (Figure 1B, bottom). Together these results (Courtneidge and Fumagalli, 1994), a mitotic substrate of demonstrate that the product of the hump62 cDNA has Src. Sequence analysis of peptides from murine Sam68 a molecular mass of 68 kDa and the properties of Sam68 showed that it was highly homologous to Hump62. Yet (see also Taylor et al., 1995). Sam68 and p62 have distinct properties: p62 is not asso-We reported previously that a commercial antibody ciated with Src in interphase or mitosis; conversely, against the central portion of the predicted Hump62 Sam68 does not associate with RasGAP and is not Tyr protein recognized both Sam68 and a 62 kDa protein, phosphorylated in interphase cells (Fumagalli et al., leading us to conclude that Sam68 and p62 were anti-1994). Here we show that the hump62 cDNA encodes genically related (Fumagalli et al., 1994). However, we Sam68, but not p62, and further that Sam68 and p62 were unable to detect p62 with other batches of this are unlikely to be related proteins. antibody (data not shown), consistent with reports from We used antibodies generated to regions of Hump62 All four antibodies we generated …