(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 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 are important substrates of receptor and human tumor cells with known tyrosine kinase 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 anity 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 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 e€ectors 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 e€ectors 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). speci®cally bind to phosphorylated Y239 or Y317 Examination of human cancer cell lines revealed high residues, which are within Grb2 SH2 consensus binding levels of Gads expression in K562 and MOLT-4 cells sites (pYXNX; where X is any amino acid), and does (Figure 2b). A minor message was detected at 7.5 kb in not bind to phosphorylated Y240, which is not within Gads, a novel adaptor binds to tyrosine-phosphorylated Shc SK Liu and CJ McGlade 3075 a to a phenylalanine (Figure 3b). GST-Gads bound to the FLAG-tagged Shc mutants when either the Y239 or Y317 residues were phosphorylated, but did not bind to the 3F mutant con®rming that Gads interaction with Shc is mediated by tyrosine-phosphor- ylation at these sites (Figure 3b). In addition, the binding of wild type, F239/Y240 and F317 Shc mutants to Gads appeared to be less ecient than F239/F240 or the F240 mutant, suggesting that phosphorylation at tyrosine 240 may have an inhibitory e€ect on binding. To verify that full-length Gads can associate with endogenous tyrosine-phosphorylated Shc, COS-1 cells were transiently transfected with pcDNA3.1 vector encoding HA-epitope tagged Gads (pcDNA3.1-HA- Gads). Following EGF-stimulation of transfected COS-1 cells, all three Shc isoforms were detected in the anti-HA immunoprecipitate (Figure 3c), indicating that Shc is co-immunoprecipitated with HA-tagged Gads in a growth factor dependent manner. A 40 kDa protein corresponding to HA-tagged Gads was detected in the anti-HA immunoprecipitates, and also b in the anti-Shc immunoprecipitate from EGF-stimu- lated cells, con®rming that HA-tagged Gads co- immunoprecipitates with Shc. Tyrosine-phosphory- lated proteins of 40 kDa, 100 kDa and 175 kDa in addition to the p46, p52, and p66 Shc isoforms are co- immunoprecipitated with HA-Gads following EGF- stimulation (Figure 3c). Subsequent immunoblotting of the membrane with anti-EGF receptor antibodies identi®ed the 175 kDa phosphoprotein as the EGF receptor (data not shown).

Gads possesses a unique SH3 domain-binding speci®city To further identify binding partners for Gads and to compare its binding speci®city to that of Grb2 we used the K562 cell line which expressed high levels of Gads. Immobilized GST and GST-fusion proteins corre- sponding to the Gads SH2, SH3 domains, full-length Gads, or Grb2 were incubated with cell lysate and bound proteins were analysed by immunoblotting with Figure 2 Northern blot analyses of Gads in murine and human anti-phosphotyrosine antibodies (Figure 4). Similar tissues and cancer cell lines. (a) Northern blots of adult murine tissue and developing murine embryonic tissue were probed using sized phosphoproteins precipitated with both Gads a fragment of the Gads cDNA corresponding to the unique and Grb2, although a major phosphoprotein of region. The blots were stripped and reprobed with b-actin cDNA 120 kDa (identi®ed as Cbl) was absent in the full- as a measure of RNA loading. (b) Northern blots of human length Gads complexes. Stripping and immunoblotting cancer cell lines and human tissue were probed using cDNA of the membrane with anti-Shc antibodies identi®ed the corresponding to the unique region of Gads. The blots were stripped and reprobed with b-actin cDNA as a measure of RNA 52 kD phosphoprotein as Shc, and indicated that Shc loading is able to associate with full-length Gads or the isolated Gads SH2 domain in vitro (Figure 4). Another phosphoprotein known to bind to Grb2 in K562 cells a Grb2-consensus binding site. Therefore, Gads and is the 210 kDa Bcr-Abl oncoprotein. Subsequent Grb2 SH2 domains likely have similar binding immunoblotting with anti-Abl antibodies revealed that speci®cities. In addition, a tryptophan residue corre- Bcr-Abl was also bound to full-length Gads, and the sponding to W121 in the EF1 region of the Grb2 SH2 isolated Gads SH2 and carboxy terminal SH3 domains domain, which has been shown to be critical for (Figure 4). binding speci®city (Marengere et al., 1994), is Many of the proteins which bind to the SH3 conserved within the Gads SH2 domain (Figure 1a). domains of Grb2 and Grap have been identi®ed and We also tested the binding of Gads to mutant Shc include Sos, Sam68 and Cbl (Rozakis-Adcock et al., proteins in which each of the tyrosine-phosphorylation 1993; Feng et al., 1996; Trub et al., 1997; Richard et sites was replaced by phenylalanine (Y to F). The al., 1995; Lock et al., 1996; Donovan et al., 1994). FLAG-tagged mutant Shc proteins become tyrosine- Given the similarity of Gads with Grb2 and Grap we phosphorylated when co-transfected with v-src in 293T have examined the interaction of Gads or the isolated cells, with the exception of the 3F mutant in which all SH3 domains of Gads with these SH3 domain-binding three tyrosines (Y239, Y240, Y317) have been changed proteins. Full-length Gads and the isolated amino Gads, a novel adaptor binds to tyrosine-phosphorylated Shc SK Liu and CJ McGlade 3076 terminal SH3 domain of Gads failed to bind to Sos, Gads may couple to SH3 domain-binding proteins that Sam68 or Cbl, whereas the isolated carboxy terminal are di€erent than those that have been found to bind SH3 domain bound to all of these proteins (Figure 4). either Grb2 or Grap. As expected, Grb2 bound to Sos, Sam68 and Cbl. In addition, no association between full-length Gads and Gads binds to Bcr-Abl and c-kit through its SH2 domain Sos was detected in 293T cells transfected with v-src, p815 mastocytoma cells, PDGF stimulated Rat2 Both the isolated carboxy terminal SH3 domain and ®broblasts or anti-CD3 activated Jurkat T cells (data SH2 domain of Gads were observed to bind to Bcr-Abl not shown). These results suggest that the binding of in K562 cell lysate, suggesting that both domains are the Gads carboxy terminal SH3 domain is altered in able to associate with Bcr-Abl. This is similar to the the context of the full-length Gads protein and that reported interaction between Grb2 and Bcr-Abl in

a c

b

Figure 3 Gads associates speci®cally with Shc pY239 or pY317. (a) Puri®ed phage encoding the partial Gads cDNA were plated, protein expression induced, immobilized onto nitrocellulose ®lters and incubated with biotinylated Shc phosphopeptides or control peptides preconjugated to streptavidin-alkaline phosphatase as indicated. Bound peptides were detected by colourimetric reaction with NBT-BCIP. (b) 293T cells were transfected with v-src expression vector alone (7), or in combination with expression vector encoding FLAG-tagged full-length wildtype Shc (wt) or FLAG-tagged full-length Shc containing point mutations of each of the tyrosine- phosphorylation sites (F239F240,F239Y240,Y239F240,F317, 3F). Relative transfection eciency was con®rmed by Western blotting of whole cell lysate (wcl) from the transfected 293T cells with anti-FLAG antibody (upper panel). The tyrosine-phosphorylation status of individual FLAG-tagged Shc mutants was determined by performing anti-FLAG immunoprecipitations of the transfected 293T cell lysate, following by Western blot analysis with anti-phosphotyrosine antibody (middle panel). In vitro binding experiments using puri®ed GST-Gads were performed, and bound FLAG-tagged Shc mutant proteins detected by immunoblotting with anti-FLAG antibody (bottom panel). (c) COS-1 cells were transfected with pcDNA3.1-HA-Gads, unstimulated (7) or stimulated (+) with EGF, and immunoprecipitations performed using a non-speci®c antibody (anti-mouse IgG: RaM), anti-Shc antibody, or anti-HA antibody. Immunoprecipitates and whole cell lysates (wcl) were subjected to Western blotting with anti-phosphotyrosine antibody conjugated to HRP to detect tyrosine-phosphorylated proteins (top panel). The blot was striped, cut lengthwise and separately reprobed with anti- Shc antibody (middle panel) or anti-HA antibody (bottom panel) Gads, a novel adaptor binds to tyrosine-phosphorylated Shc SK Liu and CJ McGlade 3077 a

b

Figure 4 Gads possesses a unique SH3 domain-binding speci®city. Puri®ed, immobilized GST or GST-fusion proteins corresponding to the Gads SH2, amino terminal or carboxy terminal SH3 domains (N-SH3, C-SH3), full-length Gads (Gads), or Grb2 were incubated with K562 cell lysate and the bound proteins analysed by SDS ± PAGE, and immunoblotting with anti-phosphotyrosine antibodies. The blot was subsequently Figure 5 Gads interacts with Bcr-Abl and c-kit via its SH2 stripped and reprobed with anti-Shc, anti-Abl, anti-Sam68, anti domain. (a) Puri®ed, immobilized GST or GST-fusion proteins Sos1, and anti-Cb1 antibodies as described in Materials and corresponding to full-length wildtype Gads (wt) or full-length methods Gads with an inactivating point mutation in the amino terminal SH3 domain (N*/C SH3), in the carboxy terminal SH3 domain (N/C* SH3), in both SH3 domains (N*/C* SH3) or in the SH2 domain (SH2*) were incubated with K562 cell lysate, and bound which both the isolated SH2 and SH3 domains of proteins analysed by immunoblotting with anti-phosphotyrosine Grb2 bound to Bcr-Abl in vitro (Pendergast et al., antibody. As a positive control for phosphotyrosine-containing 1993; Puil et al., 1994). To determine which domain proteins, total cell lysate was also analysed. Bcr-Abl and Shc were identi®ed by stripping the blot and immunoblotting with anti-Abl mediates the interaction with Bcr-Abl in the context of and anti-Shc antibodies, respectively (data not shown). (b) the full-length Gads protein, the SH3 domains or SH2 Puri®ed, immobilized GST or GST-fusion proteins correspond- domain of full-length Gads were inactivated and ing to full-length wildtype Gads (wt) or full-length Gads with an assayed for their ability to precipitate tyrosine- inactivating point mutation in the SH2 domain (SH2*) were incubated with P815 cell lysate, and bound proteins analysed by phosphorylated proteins from K562 cell lysate (Figure immunoblotting with anti-phosphotyrosine antibody. c-kit and 5A). Gads amino terminal and carboxy terminal SH3 Shc were identi®ed by stripping the blot and immunoblotting with domains were inactivated through P47L and P313L anti-c-kit and anti-Shc antibodies, respectively (data not shown) point mutations, respectively, while the SH2 domain was inactivated through an R83K point mutation; mutation of the corresponding amino acids in Grb2 C*, N*/C, or N/C*) still precipitated Bcr-Abl, implying was previously demonstrated to block SH3 and SH2 that the interaction between Gads and Bcr-Abl is domain-mediated interactions (Fukazawa et al., 1995). primarily mediated through the Gads SH2 domain. In The Gads mutant de®cient in SH2 domain-binding addition, mutation of the Gads SH2 domain abrogated (SH2*) did not precipitate the 210 kDa phosphopro- association with Shc, as identi®ed by stripping the blot tein corresponding to Bcr-Abl, as identi®ed by and Western blotting with anti-Shc antibodies (Figure stripping the blot and Western blotting with anti-Abl 5 and data not shown), con®rming that the SH2 antibodies (Figure 5 and data not shown), while the domain of Gads speci®cally mediates its interaction Gads mutants de®cient in SH3 domain-binding (N*/ with Shc. Interestingly, binding of an unidenti®ed Gads, a novel adaptor binds to tyrosine-phosphorylated Shc SK Liu and CJ McGlade 3078 phosphoprotein of 100 kDa (p100) was lost in Gads blotting with anti-phosphotyrosine antibody (Figure mutants in which the carboxy terminal SH3 domain 7a); for comparison, cell lysate, anti-Shc, and anti-Abl was inactivated (N/C* SH3) suggesting that p100 may immunoprecipitates were also included. Whereas a be a speci®c target of the Gads carboxy terminal SH3 wide range of phosphoproteins were observed to co- domain in vivo. Mutation of the amino terminal SH3 immunoprecipitate with both Shc and Bcr-Abl, only domain did not appear to alter the pro®le of two predominant 210 and 190 kDa phosphoproteins phosphotyrosine-containing proteins bound by Gads. co-immunoprecipitated with Gads. The co-immuno- The proto-oncogene c-kit encodes a receptor precipitating 210 kDa phosphoprotein was identi®ed tyrosine kinase required for the normal development as Bcr-Abl by subsequent immunoblotting with anti- of melanocytes, germ cells and hematopoetic cells in Abl antibodies (data not shown). Bcr-Abl was seen to the mouse (van der Geer, et al., 1994). Both Grb2 and co-immunoprecipitate with Gads, and as previously Grap SH2 domains have been shown to interact with demonstrated, with Grb2 (Figure 7b) (Pendergast et c-kit (Tauchi et al., 1994b; Feng et al., 1996). To al., 1993; Puil et al., 1994). We next examined whether investigate whether Gads also interacts with activated endogenous Gads co-immunoprecipitates with Shc. c-kit we have used the mastocytoma cell line P815 Anti-Gads and anti-Shc immunoprecipitations were which expresses a mutant c-kit protein with a performed using K562 cell lysate and Western blotted constitutively active kinase domain. The full-length for the presence of Gads (Figure 7b). Gads was Gads protein precipitated a tyrosine phosphorylated detected in the anti-Shc immunoprecipitate suggesting protein corresponding to c-kit from p815 cell lysates. that Gads and Shc are associated in vivo. In addition, The Gads SH2 domain mutant (SH2*) did not the anti-Gads immunoprecipitates were also subse- precipitate c-kit con®rming that the interaction is quently immunoblotted with anti-Sos antibodies. In mediated by the SH2 domain (Figure 5B). In keeping with the results of the in vitro binding addition, longer exposures of the blot revealed the experiments, Sos did not co-precipitate with endogen- presence of a 52 kDa phosphoprotein precipitated by ous Gads (data not shown). Together, these experi- Gads but not by the SH2 mutant which was identi®ed as Shc by stripping and immunoblotting with anti-Shc antibodies (data not shown). a

Gads protein expression To analyse endogenous Gads protein expression, polyclonal antisera were generated against two di€erent peptides within the unique region of Gads. The reactivity of pre-immune and immune Gads antisera was tested on Gads protein that was in vitro transcribed and translated from pBS-Gads (Figure 6a). An immunoreactive band of 40 kDa was detected in the anti-Gads immunoprecipitate from the in vitro transcribed and translated Gads cDNA, and absent in pre-immune immunoprecipitates. This indicates that the anity-puri®ed anti-Gads antibodies speci®cally b react with Gads protein in both immunoprecipitations and in Western blotting. Endogenous Gads protein was also detected in murine and human T cell lines (CTLL2 and Jurkat, respectively) and in K562 cells (Figure 6a). Notably, the human Gads protein displays a slightly slower mobility relative to mouse Gads likely due to variation in the proline content of the unique region of the human and mouse proteins (unpublished data). Anti-Gads immunoprecipitations were also performed in a variety of other cell lines including COS-1, 293T, NIH-3T3 and BaF3 cells, and Gads expression subsequently assayed by Western blotting with anti- Gads antibody (Figure 6b). Gads was not detected in any of these cell lines, suggesting that Gads expression is mainly restricted to hematopoietic cells. This is in agreement with the Northern expression analyses which detects Gads expression predominantly in the thymus Figure 6 Western blot analysis of Gads protein expression. (a) In and peripheral blood lymphocytes. vitro transcription and translation reaction of pBS or pBS-Gads utilizing rabbit reticulocyte lysate were performed as outlined in Materials and methods. The reaction mixtures, or lysates from Gads is associated with Shc and Bcr-Abl in K562 cells CTLL2, Jurkat or K562 cells were subjected to immunoprecipita- tion with pre-immune sera or anity-puri®ed anti-Gads antibody, To examine the binding of endogenous Gads to followed by Western blotting with anti-Gads antibody. (b) phosphotyrosine-containing proteins, K562 cell ly- Lysates from various cell lines: COS-1, 293T, NIH-3T3, BaF3, CTLL2, and Jurkat cells, were subjected to immunoprecipitation sates were immunoprecipitated with anti-Gads anti- with anity-puri®ed anti-Gads antibody followed by Western bodies, and associated proteins analysed by Western blotting with anti-Gads antibody Gads, a novel adaptor binds to tyrosine-phosphorylated Shc SK Liu and CJ McGlade 3079 1996; Trub et al., 1997). Given its restricted expression a we postulate that Gads is also involved in signaling processes within T lymphocytes and are currently investigating this possibility. Indeed we have found that Gads is highly expressed in primary mouse lymphocytes and binds to a restricted set of tyrosine- phosphorylated proteins following T cell receptor activation (SK Liu and CJ McGlade, manuscript in prep). Gads speci®cally binds to Shc derived peptides which contain phosphorylated tyrosine residues 239 or 317, and likewise is able to interact in vitro with Shc mutants that retain either of these phosphorylation sites. Both Y239 and Y317 are embedded in amino acid sequences which conform to the Grb2 SH2 domain-binding consensus sequence (pYXNX), thus the Gads SH2 domain likely possesses a binding speci®city similar to that of the Grb2 and Grap SH2 domains (Songyang et al., 1994; Trub et al., 1997). However we have found that in vivo, the pro®le of phosphotyrosine-containing proteins bound to Gads in K562 cells and in activated lymphocytes (unpublished results) is not identical to that detected in anti-Grb2 b immunoprecipitates suggesting that the binding of Gads to its phosphorylated targets is restricted. Similarly, Trub and colleagues have noted that the Grap SH2 domain appears to precipitate two predominant phosphotyrosine-containing proteins, p36/38, from activated Jurket T cells while numerous tyrosine-phosphorylated proteins are observed to precipitate with Grb2 (Trub et al., 1997). Thus the binding of both Gads and Grap appears to be more Figure 7 Gads associates in vivo with Shc and Bcr-Abl. (a) K562 restricted than that of Grb2. However, we cannot rule cell lysates were immunoprecipitated with anti-Gads, anti-Shc, and anti-Abl antibodies, and immunoblotted with anti-phospho- out that the di€erences in the pro®les of phospho- tyrosine antibodies. (b) Anti-Gads, anti-Shc, and RaM (negative tyrosine-containing proteins observed to precipitate control) immunoprecipitations were performed using K562 cell with Gads are a function of restricted SH3 domain- lysates, and the presence of Gads was assessed by Western mediated interactions. blotting with anti-Gads antibody. The presence of Bcr-Abl was We have found that in isolation the carboxy analysed by anti-Gads, anti-Grb2 and anti-mouse IgG immuno- precipitation from K562 cell lysates, followed by Western blotting terminal Gads SH3 domain can interact with Sos, with anti-Abl antibody Cbl, and Sam68 while the amino terminal SH3 domain does not appear to possess any detectable anity for these proteins. Furthermore, full-length Gads does not ments indicate that Gads is associated with Shc and interact with any of these proteins including Sos, in Bcr-Abl in vivo, and identi®es Gads as a potentially vivo or in vitro, suggesting that Gads likely functions to novel component of the Bcr-Abl signaling complex. couple Bcr-Abl, Shc, and perhaps additional tyrosine- phosphorylated proteins to a downstream pathway that is independent of Sos activation of Ras. The di€erence Discussion in the binding speci®cities between the full-length Gads protein and the isolated SH3 domain may be due to In this report, we describe the identi®cation and the presence of the 120 amino acid unique region in the characterization of Gads, a novel SH3-SH2-SH3 full-length molecule which could alter the spatial domain-containing adaptor protein. Gads is structur- orientation of the SH3 domains of Gads and ally similar to the Grb2 and Grap adaptor proteins, contribute to their interaction with speci®c targets. suggesting that Gads is a new member of the `Grb2- Alternatively, this region could regulate the interaction family' of adaptor proteins (Lowenstein et al., 1992; of the carboxy terminal SH3 domain with its targets Clark et al., 1992; Suen et al., 1993; Feng et al., 1996; via an intramolecular contact between proline rich Trub et al., 1997). Interestingly, Gads possesses a sequences in the unique region and the carboxy unique region rich in proline and glutamine residues terminal SH3 domain. This mode of regulation could located between its SH2 domain and carboxy terminal be analogous to the recently described low-anity SH3 domain which distinguishes it from both Grb2 intramolecular interaction between the SH3 domain of and Grap. Src and Hck kinases with the `SH2-kinase linker' that Gads is predominantly expressed in hematopoietic contributes to the inactive kinase conformation (Xu et cells. The recently cloned Grap adaptor is also al., 1997; Sicheri et al., 1997). A similar regulatory speci®cally expressed in lymphoid tissue and in various mechanism has been proposed to modulate the activity hematopoietic cell lines, and has been proposed to of the adaptor protein Crk-II (Ana® et al., 1996). function downstream of the T cell receptor (Feng et al., Finally, the unique region could mediate the interac- Gads, a novel adaptor binds to tyrosine-phosphorylated Shc SK Liu and CJ McGlade 3080 tion of Gads with another protein which could cloned in-frame into the pGEX-2TK vector (Pharmacia, sterically hinder the binding of SH3 domain target Uppsala, Sweden) or pBluescript (pBS) vector (Stratagene, proteins. Experiments are underway to assess the LaJolla,CA,USA).TheaminoterminalSH3(aa1±65), e€ects of the unique region of Gads in protein SH2 (aa 48 ± 152), and carboxy terminal SH3 (aa 257 ± 322) complex formation and function. regions were ampli®ed from Gads full-length cDNA and cloned into the pGEX-2TK vector. To generate HA-tagged The oncogenic 210 kDa Bcr-Abl protein is asso- Gads cDNA, the full-length Gads coding region was ciated with chronic myelogenous leukemia (reviewed in subcloned into pcDNA3.1-HA (a generous gift of Bryan Gotoh and Broxmeyer, 1997). Bcr-Abl mediated Snow, AMGEN Institute, Ontario, Canada). Full-length transformation is thought to rely on activation of the Grb2 cDNA in pGEX-2T and v-src pECE were kind gifts Ras pathway (Sawyers et al., 1995), and evidence from Mike Moran (University of Toronto, Canada) suggests that this involves adaptor proteins such as The full-length mouse Shc cDNA was cloned into the Shc, Grb2 and SHP-2 which act to couple Bcr-Abl to pFLAG-CMV2 vector (Kodak) to generate the wt Shc- Sos (Tauchi et al., 1994a; Pendergast et al., 1993; Puil pFLAG-CMV2 expression vector. Tyrosine to phenylalanine mutations in Shc tyrosine-phosphorylation sites (F F , et al., 1994; Gishizky et al., 1995). Like Grb2, Gads is 239 240 F Y ,Y F ,F ,F F F (3F)) were generated by also associated with Bcr-Abl through its SH2 domain, 239 240 239 240 317 239 240 317 PCR using mutagenic primers and cloned into the pFLAG- however, Gads does not recruit Sos to this complex. In CMV2 vector. PCR-based mutagenesis was employed to addition, we have found that although Grb2 and Gads generate inactivating P47L and P313L mutations in the are expressed at roughly equivalent levels in K562 cells, amino and carboxy terminal SH3 domains of Gads, and an much more Bcr-Abl can be immunprecipitated with inactivating R83K mutation in the SH2 domain of Gads; the Grb2 than Gads. This di€erence suggests that the mutants were cloned into the pGEX-2TK vector. interaction of Gads with tyrosine-phosphorylated proteins is regulated in vivo or that the Gads may be Cell culture bound to targets in addition to Shc and Bcr-Abl that compete for binding or preclude the association of 293T, COS-1, P815 and NIH-3T3 cells were cultured in Dulbecco's Modi®ed Eagle medium (DMEM) supplemen- Gads with Bcr-Abl and Shc in these cells. We propose ted with 10% (v/v) fetal bovine serum (Gibco BRL, that Gads may couple activated receptors and tyrosine- Gathersburg, MD, USA), 200 mML-glutamine, 5 U/ml phosphorylated proteins such as Shc and Bcr-Abl to penicillin C, and 5 mg/ml streptomycin sulfate. K562 and novel downstream signaling pathways. The identifica- Jurkat cells were cultured in RPMI 1640 medium tion of in vivo targets of the Gads SH3 domains, such supplemented as above. BaF3 cells were maintained in as p100, will reveal its functions in tyrosine kinase RPMI 1640 supplemented as above with the addition of mediated signaling pathways. recombinant IL-3 (100 pg/ml) (R&D Systems, Minneapo- lis, MN, USA), while CTLL2 cells were maintained in RPMI 1640 supplemented as above with the addition of recombinant IL-2 (2 U/ml) (Boehringer-Mannheim, Man- Materials and methods nheim, Germany).

Screening of expression libraries with phosphopeptides Preparation of GST-fusion proteins A 16-day mouse embryo phage expression library Recombinant GST-fusion protein were expressed and (Novagen, WI, Madison, USA) was plated at approxi- puri®ed as previously described (Pelicci et al., 1992). mately 46104 p.f.u./150 mm plate and protein expression Puri®ed protein bound to glutathione-sepharose 4B beads induced as per the manufacturer's directions. The (Pharmacia) were resuspended in an equal volume of NP- nitrocellulose ®lters containing immobilized proteins were 40 lysis bufer (50 mM HEPES, pH 7.4, 150 mM NaCl, washed several times in TBST (20 mM Tris-Base pH 7.5, 2mM EDTA, 100 mM ZnCl2, 1% (v/v) Nonidet-P40, 10% 150 mM NaCl, 0.05% (v/v) Tween-20) and blocked (v/v) glycerol) containing complete protease inhibitors overnight in blocking bu€er (5% BSA (w/v), 1 mM (Boehringer-Mannheim) and 1 mM DTT and stored at dithiothreitol (DTT) in TBST). Blocked ®lters were 7808C. Each of the puri®ed GST-fusion proteins was incubated in blocking bu€er, at RT for 3 h with quantitated by SDS ± PAGE followed by Coomassie

biotinylated phosphopeptides (25 pmol/ml): pY239pY240, staining, and comparison with BSA standards.

DHQpYpYNDFPGKEPP; pY239Y240, DHQp-YYNDFPG- KEPP; Y pY , DHQYpYNDFPGKEPP; Y Y ,DH- 239 240 239 240 Transient transfections QYYNDFPGKEPP; pY317, ELFDDSpYVNIQNLD; Y317, ELFDDPSYVNIQNLD (the numbering of the tyrosine 293T or COS-1 cells were grown to approximately 50% residues denotes the position of the amino acid in the con¯uence in 10 cm culture dishes and co-transfected with human p52 Shc protein), that were pre-conjugated to 5 mg of each expression vector using Lipofectin (Gibco- streptavidin-alkaline phosphatase (BioRad) as described in BRL) according to manufacturer's instructions. Trans- Sparks et al. (1996). Filters were washed ®ve times with fected cells were harvesed 48 h post-transfection. TBST and detection performed through a colorimetric EGF-stimulation of transfected COS-1 cells was carried NBT-BCIP reaction. Positive plaques were isolated and out as follows: 16 h following transfection COS-1 cells were two subsequent rounds of screening were performed to starved in 0.5% (v/v) fetal bovine serum in DMEM for 24 h purify the phage. The pExLox plasmids were isolated from and then stimulated with human recombinant EGF (80 nM) the phage according to the manufacturer's directions and Upstate Biotechnology, Lake Placid, NY, U.S.A.) in DMEM the inserts sequenced. at 378 for 5 min.

Plasmids In vitro transcription and translation The Gads full-length cDNA (aa 1 ± 322) was ampli®ed For in vitro transcription and translation reactions, 1 mgof through Polymerase Chain Reaction (PCR) from an 11-day pBSorpBS-Gadswasincubatedwithrabbitreticulocyte Mouse Embryo Marathon-Ready cDNA library (Clontech, lysate and reaction components for 90 min at 308C Palo Alto, CA, USA) using cDNA speci®c primers and according to manufacturer's directions (Promega, Madi- Gads, a novel adaptor binds to tyrosine-phosphorylated Shc SK Liu and CJ McGlade 3081 son, WI, USA). Each 50 ml reaction was then immunopre- blotting) for a minimum of 1 h prior to addition of cipitated with either pre-immune sera or anity-puri®ed antibody overnight at 48C. Membranes were washed three anti-Gads antibody, and Western blotted with anity- timesinTBSTandincubatedatRTfor30minwithan puri®ed anti-Gads antibody as outlined below. appropriate secondary antibody conjugated to HRP. Following incubation with secondary antibodies, mem- branes were washed three times in TBST and developed Antibodies using ECL (Amersham, Buckinghamshire, UK) as per the Polyclonal Gads antisera were generated by immunizing manufacturer's instructions. rabbits with peptides coupled to keyhole limpet hemocya- nin, corresponding to two di€erent sequences within the unique region of Gads: H1-A, GEEIRPSVNRKLSDH or In vitro binding experiments H1-B, LMHRRHTDPVQLQA. Crude antisera were affi- Thelysateoftransfectedcellsor26107 K562 cells were nity-puri®ed over an immobilized peptide (H1-A or H1-B) prepared as described above, and incubated with approxi- column (Pierce). For anti-Gads immunoprecipitations, 2 mg mately 5 mg of GST-fusion protein coupled to glutathione- of anity-puri®ed anti-Gads antibody (H1-B) was used; sepharose 4B beads for 90 min at 48C. The beads were 1 : 250 dilution of anity-puri®ed anti-Gads antibody (H1- washed three times with NP-40 lysis bu€er, resuspended in A) was used for immunoblotting. Anity-puri®ed anti-Shc reducing SDS-Laemmli sample bu€er, the proteins resolved antibodies were prepared and used as previously described on a polyacrylamide gel and transferred to Immobilon-P (Pelicci et al., 1992). Anity-puri®ed anti-HA antibody membrane. Western blotting and detection of immunor- (12CA5) was purchased from Boehringer-Mannheim and reactive bands was carried out as described above. 2 mg was used for immunoprecipitation. For Western blotting of HA-tagged proteins, anti-HA antibody Northern blot analysis (12CA5) conjugated to HRP (Boehringer-Mannheim) was used at a 1 : 1000 dilution. Anti-FLAG M2 antibody Northern blots containing poly (A)+mRNA isolated from (Kodak, Rochester, NY, USA) was used at 2 mg/sample a variety of murine and human tissues and human cancer for immunoprecipitation and a dilution of 1 : 500 used for cell lines were purchased from Clontech. Western blotting. The anti-phosphotyrosine antibody 4G10 A 0.36 kb fragment (unique region) or a 0.99 kb fragment (Upstate Biotech) was used for Western blotting at a of the Gads cDNA was 32P-radiolabeled by random hexamer 1 : 1500 dilution. Anti-cbl, anti-Sam68, anti-c-kit and anti- priming (Pharmacia) and hybridizied with Northern blots at Sos1 antibodies were purchased from Santa Cruz, Santa 106 c.p.m./ml overnight at 428C in a solution of 50% Cruz, CA USA and used for Western blotting at a dilution formamide, 46SSPE. 1% sodium dodecyl sulfate (SDS), of 1 : 250. Anti-Abl antibody was used at a 1 : 100 dilution 0.5% skim milk powder, 10% dextran sulfate and 10 mg/ml for Western blotting (Calbiochem, Bedford, MA, USA). sheared salmon sperm DNA. Blots were washed twice for Anity-puri®ed anti-GST antibody was used for Western 10 min per wash at room temperature in 26SSC, 0.1% SDS, blotting at a dilution of 1 : 5000 and was a kind gift of Susan then twice for 10 min per wash at 558C in 0.16 SSC, 0.1% McCracken (AMGEN Institute). SDS, and exposed to X-OMAT ®lm (Kodak). The blots were also probed with radiolabeled b-actin cDNA (Clontech) as an indicator of RNA loading. Immunoprecipitation and Western blotting Each 10 cm plate of transfected COS-1 or 293T cells or 26107 CTLL2, Jurkat and K562 cells were lysed in 1 ml of PLC lysis bu€er (50 mM HEPES pH 7.5, 150 mM NaC1,

10% (v/v) glycerol, 1% (v/v) Triton X-100, 1.5 mM MgC12, Acknowledgements

1mM EDTA, 10 mM NaPPi, 100 mM NaF, 1 mM Na3VO4) The authors would like to express sincere thanks to Olga containing complete protease inhibitors and the lysate Agah for expert technical assistance, Christian Smith for clari®ed by centrifugation at 13 500 r.p.m. at 48Cfor assistance with the phosphopeptide screen, Mike Thomas 10 min. Clari®ed lysate was incubated with antibodies as of the AMGEN DNA sequencing group (Thousand Oaks, described above and 50 ml of 20% (v/v) protein G- or CA, USA) for excellent DNA sequencing, the peptide protein A-sepharose (Sigma) at 48C with gentle rotation synthesis group at AMGEN (Boulder, CO, USA) for for 90 min. Immune complexes were washed three times in synthesis of the biotinylated Shc phosphopeptides, the 1 ml NP-40 lysis bu€er, bound proteins eluted by boiling DNA technology group at AMGEN (Boulder, CO, USA) for 5 min in reducing SDS-Laemmli sample bu€er and for oligonucleotide synthesis and Michelle French, David resolved by SDS ± PAGE. Proteins were electrophoretically Siderovski and Dwayne Barber for comments on the transferred to Immobilon-P membrane (Millipore, Bed- manuscript. This work was funded by the Medical ford, MA, USA), and incubated in a blocking solution of Research Council (MRC) of Canada. The nucleotide 5% non-fat skim milk powder (w/v) in TBST or in 1% sequence of Gads has been submitted to the GenBank/ BSA (w/v) in TBST (for anti-phosphotyrosine Western EMBL Data Bank with the accession number (AF053405).

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