Oncogene (1998) 17, 3073 ± 3082 ã 1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00 http://www.stockton-press.co.uk/onc Gads is a novel SH2 and SH3 domain-containing adaptor protein that binds to tyrosine-phosphorylated Shc Stanley K Liu and C Jane McGlade Department of Medical Biophysics, Ontario Cancer Institute, AMGEN Institute, University of Toronto, 620 University Avenue, Suite 706, Toronto, Ontario, Canada, M5G 2C1 Shc proteins are important substrates of receptor and human tumor cells with known tyrosine kinase gene cytoplasmic tyrosine kinases that couple activated alterations (Pelicci et al., 1995). Following tyrosine- receptors to downstream signaling enzymes. Phosphor- phosphorylation, Shc associates with the SH2 and ylation of Shc tyrosine residues 239 and 317 leads to SH3 domain-containing adaptor protein, Grb2 (Low- recruitment of the Grb2-Sos complex, thus linking Shc enstein et al., 1992; Rozakis-Adcock et al., 1992). This phosphorylation to Ras activation. We have used interaction is mediated by the phosphorylated tyrosine phosphorylated peptides corresponding to the regions residues 239 and 317 of Shc which are both found spanning tyrosine 239/240 and 317 of Shc in an within a sequence conforming to a high anity expression library screen to identify additional down- binding site for the Grb2 SH2 domain (Salcini et stream targets of Shc. Here we report the identi®cation al., 1994; van der Geer et al., 1996; Gotoh et al., of Gads, a novel adaptor protein most similar to Grb2 1996; Harmer and DeFranco, 1997). The Grb2 SH3 and Grap that contains amino and carboxy terminal domains mediate its stable association with the Ras SH3 domains ¯anking a central SH2 domain and a 120 guanine nucleotide exchange factor, Sos, and mem- amino acid unique region. Gads is most highly expressed brane localization of the Grb2-Sos complex results in in the thymus and spleen of adult animals and in human activation of Ras (Li et al., 1993; Buday and leukemic cell lines. The binding speci®city of the Gads Downward, 1993; Chardin et al., 1993; Egan et al., SH2 domain is similar to Grb2 and mediates the 1993; Rozakis-Adcock et al., 1993; Aronheim et al., interaction of Gads with Shc, Bcr-Abl and c-kit. Gads 1994). Thus, it has been proposed that Shc is involved does not interact with Sos, Cbl or Sam68, although the in coupling the activation of cell surface receptors to isolated carboxy terminal Gads SH3 domain is able to Ras activation (Rozakis-Adcock et al., 1992). Phos- bind these molecules in vitro. Our results suggest that the phorylation of Y239/Y240 may also lead to c-myc unique structure of Gads regulates its interaction with induction and suppression of apoptosis in BaF3 cells downstream SH3 domain-binding proteins and that Gads in a Ras-independent manner, suggesting that Shc has may function to couple tyrosine-phosphorylated proteins other functions (Gotoh et al., 1996). In addition, Shc such as Shc, Bcr-Abl and activated receptor tyrosine phosphopeptides containing residues Y239/Y240 bind kinases to downstream eectors distinct from Sos and to a variety of as yet unidenti®ed phosphoproteins Ras (van der Geer et al., 1996). Together, these data suggest that Shc may participate in diverse signal Keywords: adaptor protein; SH2 domain; SH3 domain; transduction pathways by interacting with multiple Shc; Bcr-Abl cytoplasmic signaling molecules through binding of SH2 domain-containing proteins to phosphorylated residues Y239, Y240 and Y317. To elucidate novel signaling pathways downstream Introduction of Shc, we sought to identify molecules that associated speci®cally with the phosphorylated Y239/Y240 or Ubiquitously expressed Shc proteins play a role in Y317 residues of Shc. In this report, we describe the coupling growth factor receptor activation to intracel- characterization of a novel protein, Gads (Grb2- lular signaling pathways and are major targets of related adaptor downstream of Shc) which possesses tyrosine-phosphorylation following activation of re- amino and carboxy terminal SH3 domains ¯anking a ceptor tyrosine kinases (Pelicci et al., 1992; reviewed central SH2 domain and a unique region rich in in Bon®ni et al., 1996), and receptors that lack glutamine and proline residues. Gads shares the intrinsic tyrosine kinase activity including G-protein greatest identity with Grb2 and Grap suggesting its coupled receptors (van Biesen et al., 1995; Touhara et role as an adaptor molecule (Lowenstein et al., 1992; al., 1995; Chen et al., 1996); ligation of integrins Clark et al., 1992; Suen et al., 1993; Feng et al., 1996; (Wary et al., 1996); and in cells expressing activated Trub et al., 1997). The SH2 domain-binding speci®city Src (McGlade et al., 1992; Dilworth et al., 1994), Sea of Gads appears to be similar to that of Grb2 and (Crowe et al., 1994) or Lck (Baldari et al., 1995). In mediates the interaction of Gads with tyrosine- addition, Shc is constitutively phosphorylated in phosphorylated Shc, Bcr-Abl and c-kit. In the context of the full-length molecule, the speci®city of the Gads SH3 domains is distinct from Grb2, suggesting that Gads may function to couple tyrosine-phosphorylated Correspondence: CJ McGlade proteins to downstream eectors distinct from Sos and Received 29 May 1998; revised 31 July 1998; accepted 31 July 1998 Ras. Gads, a novel adaptor binds to tyrosine-phosphorylated Shc SK Liu and CJ McGlade 3074 Results a Cloning and expression of Gads (Grb2-related adaptor downstream of Shc) To identify proteins interacting with tyrosine-phos- phorylated Shc, a mixture of biotinylated phosphopep- tides corresponding to the known Shc tyrosine- phosphorylation sites (Y239, Y240 and Y317) were used to screen a 16-day mouse embryo expression library. Partial cDNAs encoding several known and novel SH2 domain-containing proteins were isolated (unpublished results). One of these partial cDNAs was found to contain an open-reading frame encoding an SH2 domain, a unique region rich in glutamine and proline residues, and a carboxy terminal SH3 domain; this partial cDNA was most closely related to the adaptor protein Grb2 (Lowenstein et al., 1992). We b have termed this protein Gads, for Grb2-related adaptor downstream of Shc. Comparison of the Gads nucleotide and protein sequence against the Genbank database (Benson et al., 1998) identi®ed a murine EST sequence (accession number AA175718) encoding an SH3 domain and a partial SH2 domain, which overlapped with the 5' coding region of Gads. To obtain the entire predicted open-reading frame of Gads, primers corresponding to the 5' sequence of the murine EST and to the 3' sequence of the partial cDNA isolated in the screen were used to PCR amplify cDNA made from an 11-day mouse embryo. The DNA sequence of the ampli®ed cDNA contains a putative open-reading frame containing 322 amino acids coding for a protein with a predicted molecular weight of Figure 1 Amino acid sequence of Gads and comparison with 36.8 kDa. It contains an amino terminal SH3 domain, Grb2 and Grap. (a) The deduced amino acid sequence of Gads is followed by an SH2 domain, a unique region (aa 150 ± shown. The SH3 domains are underlined and the SH2 domain is 269) and a carboxy terminal SH3 domain (Figure 1a). boxed. The conserved tryptophan residue in the EF1 region is denoted (!). (b) Schematic representations of Grb2, Gads and To con®rm the sequence of the 5' region of Gads, Grap; the double-slash represents the unique region in Gads. The 5' RACE was performed (data not shown). The percentage identities between the individual domains are denoted assembled 1.37 kb cDNA likely represents the full- length cDNA since a stop codon is observed upstream from the putative initiating methionine in both the most tissues examined. The Gads cDNA cloned from murine EST sequence and the 5'RACE product, and mouse embryo likely represents the observed 1.7 kb the size corresponds to the observed message size by messages, as the assembled Gads cDNA is 1.37 kb not Northern blotting (Figure 2). We predict that a human including the poly-A tract. The nature of the 4 kb and gene in the region 22q12 encodes a Gads homologue 7.5 kb messages is unknown, but both the unique (accession number Z82206) because it is 90.6% similar region probe and a probe corresponding to the full- to the mouse protein. The protein sequences of the length Gads detected all three messages (data not SH2 and SH3 domains of Gads are most similar to the shown). adaptor protein Grb2 and the recently identi®ed Grb2- related adaptor protein, Grap (Figure 1b) (Feng et al., Gads binding to Shc is mediated by phosphorylation of 1996; Trub et al., 1997). Based on its similarity to these tyrosine 239 or 317 adaptor proteins, and its apparent lack of a catalytic region as deduced from the sequence, Gads likely To determine the speci®city of Gads interaction with encodes an adaptor protein. the biotinylated Shc phosphopeptides, the puri®ed To examine the expression pattern of Gads, North- phage encoding the partial Gads cDNA were plated, ern blot analyses were performed with poly(A)+RNAs protein expression induced, immobilized onto nitrocel- isolated from embryonic and adult murine tissue, and lulose ®lters and incubated with individual biotinylated adult human tissues. A probe corresponding to the Shc phosphopeptides or control peptides. The unique region of Gads detected two major transcripts pY239Y240,pY239pY240, and pY317 peptides bound to the of 4 kb and 1.7 kb primarily in adult mouse spleen and immobilized Gads, whereas neither Y239pY240 peptides testis and in day 11 ± 17 mouse embryos (Figure 2a). nor the unphosphorylated peptides bound (Figure 3a). Identical-sized transcripts were detected in human This indicates that the Gads SH2 domain can thymus and peripheral blood leukocytes (Figure 2b).