Analysis of the Binding of the Src Homology 2 Domain of Csk

Proc. Nati. Acad. Sci. USA Vol. 91, pp. 3984-3988, April 1994 Cell Biology Analysis of the binding of the Src homology 2 domain of Csk to tyrosine-phosphorylated proteins in the suppression and mitotic activation of c-Src (focal adhesion kinase/focal adhesion plaque/glutathlone S-transferase fsib protein/paxillin/proteln-trosie kinase) HISATAKA SABE*, AKIKO HATA*, MASATO OKADAt, HACHIRO NAKAGAWAt, AND HIDESABURO HANAFUSA*t *Laboratory of Molecular Oncology, The Rockefeller University, 1230 York Avenue, New York, NY 10021; and tDivision of Protein Metabolism, Institute for Protein Research, Osaka University, 3-2, Yamadaoka, Suita, Osaka 565, Japan Contributed by Hidesaburo Hanafusa, January 4, 1994

ABSTRACT Csk (C-t minal Src kinase), a protein-tyro- and its relation to cell adhesion to the extracellular matrix sine kinase, bearing the Src homology 2 and 3 (SH2 and SH3) (ECM) are discussed. domains, has been in cted in pbospborylaton of c-Src Tyr- 527, rlg in supp of c-Src inase activity. We found MATERIALS AND METHODS that mutations in the SH2 or SH3 domain of Csk, though they did not affect its knase activity, resulted In a loss ofsuppression Cell Culture. Cell culture and cDNA transfection were of c-Src activity in fibrobast. In normal fibroblasts, tyrosine- done as described (4, 9). Mitotic cells were prepared (17) by phosphorylated paxillin and focal adhesi kinase pp125FAK, incubating cells with nocodazole (0.4 ;g/ml) for7.5 hrat 370C which colocalize at focal adhsin plaques, were the major and harvested by shaking from culture dishes. Chicken proteins to which the Csk SH2 domain bound. Loss of binding embryo fibroblasts (CEFs) were prepared and infected with to these proteins by the Csk SH2 mutants correlated with loss of Rous sarcoma virus (RSV) (18) or treated with 200 pM the activity to suppress c-Src. Co t with this observation, vanadate for 12 hr before analysis, to inhibit cellular protein- the leveis of y iphorylation of paxiflin and pp125FAK tyrosine-phosphatases (19). Cell adhesion experiments were were greatly reduced during mikos, whereas the kinase activity performed according to Burridge et al (20) with some mod- of c-Src was elevated. We suggest that the S112 domain is ification. Briefly, cells were trypsinized, washed twice with Ire rd for Csk to spresc-Src, perhaps in combination th Dulbecco's modified Eagle's medium containing soybean the 513 domain, by aring Csk to a particular e trypsin inhibitor (0.5 mg/ml), suspended in culture medium location where c-Src may exist. Our data also indicate that a without serum, and kept in suspension at 37C for 1 hr. Cells certain fatin ofthe Csk and Src family kinas fuxntion at the were then either harvested or plated on culture dishes coated foca adhsion plaques. The activity ofthe c-Src kinase lo d with rat fibronectin (10 pg/ml, GIBCO/BRL) and incubated at the focal ads plaques appears to be regulated by cell at 3TC for 1 hr in the absence of serum. adhesion to the extracellular matrix. Construction of Csk Mutant cDNAs. For the K222M mutant, in which the Lys-222 ATP-binding site (7, 9) was changed to Met, the mutagenized 122-bp Apa I-Fsp I PCR- Csk (C-terminal Src kinase) was first purified as a kinase amplified fragment was substituted for the corresponding which could phosphorylate the C-terminal tyrosine (Tyr-527) segment of pcDNAIcsk (9). For R106K (Arg-106-* Lys) and of c-Src, suppressing c-Src kinase activity (1), and was S108C (Ser-108 -* Cys) mutants, the mutagenized 315-bp subsequently shown to phosphorylate several other Src fam- Msc I-Sma I PCR-amplified fragments were substituted for ily kinases, such as Lyn, Fyn, Yes, and Lck, at their the corresponding segment of pcDNAcsk. The nucleotide C-terminal tyrosine residues in vitro (2, 3). Our previous sequences ofthese three mutants differ from the parental one results (4) and the data from the targeted disruption of csk as follows [the residue number (ref. 9) and the substituted gene loci (5, 6) have further supported the contention that Csk nucleotides]: K222M, (665T, 687T); R106K, (319A, 320A); negatively regulates the kinase activities ofSrc family kinases S108C, (325T, 327T). Deletion mutants were made by digest- in vivo. Csk has Src homology 2 and 3 (SH2 and SH3) ing double-stranded csk cDNA with BAL-31 nuclease (New domains N-terminal to the kinase domain (7-9). Greater England Biolabs) from the Aat II site at nt 104 ofpcDNAIcsk understanding of the role of the SH2 and SH3 domains and then self-ligated by T4 ligase (New England Biolabs). suggests that these domains coordinate intracellular protein- After being cloned, the mutations and the deletions were protein interactions, thus regulating and propagating signals confirmed by sequence analysis. via protein tyrosine phosphorylation (10-16). An assessment In Vitro Kinase Acivity. Wild-type Csk and its mutants of the apparent necessity of these domains of Csk in its were expressed in COS-7 cells by cDNA transfection (4) and suppression of Src kinase activity, and an identification of their kinase activities were measured after immunoprecipi- proteins binding to these domains, may help us to understand tation with a polyclonal antibody raised against a synthetic the mechanism of Csk suppression of c-Src and Src family peptide corresponding to aa 1-13 of chicken Csk, using kinases in vivo. Such understanding of the mechanism may poly(Glu,Tyr) (Sigma) as a substrate (1). Specific activity was help to clarify the function of Src family kinases in vivo. calculated by dividing each activity by the respective amount In this report, we describe the role of the SH2 and SH3 of Csk protein quantified by immunoblotting with the poly- domains of Csk and the properties of the SH2-binding pro- clonal anti-Csk antibody (7), then normalized to the specific teins in the suppression of c-Src kinase activity by Csk. The activity of wild-type Csk, which was assigned a value of 1.0. mechanism of the mitotic activation of c-Src kinase activity Abbreviations: CEF, chicken embryo fibroblast; ECM, extracellular The publication costs ofthis article were defrayed in part by page charge matrix; GST, glutathione S-transferase; RSV, Rous sarcoma virus; payment. This article must therefore be hereby marked "advertisement" SH2, Src homology 2; SH3, Src homology 3. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 3984 Downloaded by guest on October 1, 2021 Cell Biology: Sabe et al. Proc. Natl. Acad. Sci. USA 91 (1994) 3985 The specific activity of immunoprecipitated c-Src from cell crk/c-src) cells], where Csk overexpression has been shown lysates prepared with RIPA buffer (4) was measured with to suppress the c-Src activity and restore the normal mor- acid-denatured enolase substrate and kinase buffer (21) and phology (4) (Table 1). A kinase-negative Csk mutant (K222M) normalized to the specific activity of c-Src in Rev(II) cells did not suppress the transformation or c-Src activity. Dele- transfected with wild-type csk cDNA (4), which was assigned tion mutations [A18-53 and A12-57; numbers represent a value of 1.0. amino acid residues (9)] in the SH3 domain (aa 14-68) or Production and Purification of Recombinant Chicken Csk in point mutations (R106K and S108C) within the SH2 domain a Baculovirus System. The 2.3-kb Kpn I-EcoRI fragment ofcsk consensus sequence (FLVRES) (12) also markedly reduced cDNA from pcDNAIcsk (9) was ligated into a BamHI site of Csk's ability to suppress c-Src activity in vivo, while the pAcYMi vector (22), after the insert fragment and the vector kinase activities of these mutants remained unchanged. were made blunt-ended with T4 DNA polymerase (Promega). There was basically no difference in the amount of Csk The Kpn I site is located 58 bp upstream of the first ATG protein ofeach mutant, as well as wild type, expressed in this codon. Sixty hours after infection, Sf9 cells (Invitrogen) were experiment (data not shown). harvested, solubilized at 5 x 107 cells per ml in a buffer (1), Csk SH2 Domain Binds to Tyrosine-Phosphorylated Paxillin sonicated briefly, and centrifuged at 100,000 x g for 30 min at and ppl25FAK. Analysis ofthe Csk SH3/2-binding proteins in 40C. Recombinant Csk was purified to a homogeneity by v-Crk/c-Src cells with a GST-Csk SH3/2 fusion protein sequentially fractionating the supernatant on Mono Q (Phar- revealed two major tyrosine-phosphorylated proteins above macia), hydroxyapatite (Mitsui Toatsu Chemicals), and hep- background (Fig. la). Since GST-Csk SH3 fusion protein as arin-Sepharose (Pharmacia) basically as described (1). well as the GST protein alone did not show such binding (Fig. Construction and Expression of Glutathione S-Transferase la), this binding to tyrosine-phosphorylated proteins appears (GST)-Csk Fusion Protein. GST-Csk SH3/2 plasmid and its to be mediated mainly by the Csk SH2 domain. The most mutants within the SH2 domain, encoding aa 9-189 (SH3 and abundant protein, 70-75 kDa, was identified as paxillin (30) SH2 domains) of chicken Csk (9), were constructed by with an anti-paxillin antibody (Fig. 2a). Paxillin consists of ligating each 0.54-kb Msc I-Apa I fragment of chicken csk several isoforms (30) primarily due to a multiple tyrosine cDNA (9) of wild type or R106K or S108C mutant into a Sma phosphorylation (H.S. and H.H., unpublished work) and I site of pGEX-3X (Pharmacia) after blunt-ending by T4 most, if not all, of the isoforms bind to the Csk SH2 domain. polymerase. GST-Csk SH3 plasmid was made by ligating the The second protein, =120 kDa, was shown to be ppl25FAK PCR product of the SH3 domain of chicken csk cDNA (9), (24, 28) (Fig. 2a). Equivalent binding proteins were found in synthesized with 5' primer (5'-GCCCGGGTGTATCGC- normal CEFs and mouse NIH 3T3 fibroblasts (Figs. lb, 2a, CAAGTACAAC-3') and 3' primer (5'-CCGAATTCCTCT- and 3) and parental 3Y1 cells (data not shown). The protein TCTGCACGTAGTT-3') into the Sma I/EcoRI site ofpGEX- band of 65 kDa often appeared even with buffer alone (data 3X. GST fusion proteins were expressed in Escherichia coli not shown), perhaps due to an artifact. Anti-Csk antibody and purified with glutathione-Sepharose (Pharmacia). also coimmunoprecipitated paxillin and ppl25FAK from a cell Antibodies. Antibodies against paxillin were purchased from lysate with endogenous Csk (Fig. 2d), supporting in vivo Zymed or a gift from J. Glenney (Transduction Laboratories, binding of Csk with these proteins. With CEFs transformed Kentucky). Antibodies against mitogen-activated protein ki- by RSV or treated with vanadate, many tyrosine-phosphor- nases (MAP kinases) were from Upstate Biotechnology. An- ylated proteins bound the GST-Csk SH3/2 protein, indicat- tibodies against p130 (23), focal adhesion kinase ppl25FAK ing a potential broad-range binding capacity of the Csk SH2 [monoclonal (23) and polyclonal (24) anti-chicken], p120 (23) domain (Fig. 1 c and d). Some of the Csk SH2-binding and p110 (23) were provided by J. T. Parsons; against integrin proteins in RSV-transformed cells were identified as tensin (31 (25) by K. Yamada, against tensin (26) by L. B. Chen, (26), p130 (23), ppl25FAK, pl20 (23, 31), and paxillin (Fig. 2 against Src (Ab327) (27) by J. Brugge, against ppl25FAK a-c). However, tyrosine-phosphorylated forms of EGF re- (polyclonal anti-mouse) (28) by S. Hanks, and against Csk (7) ceptor, integrin ,B1 (25), pllO (23), pp60v-src, pp6CO-src, MAP by M. Okada. Protein G-Sepharose was used for anti- kinases (32), and p36/calpactin did not bind to the Csk SH2 pp125FAK (monoclonal) and anti-p120 immunoprecipitation. domain (Fig. 2b; ref. 4; data not shown). Anti-p130 antibody was precipitated with rabbit anti-mouse Coincident with the loss ofthe c-Src-suppressive activity in IgM coupled with protein A-Sepharose. Immunoprecipitation v-Crk/c-Src cells (Table 1), the binding capacity ofthe S108C with other antibodies was done with protein A-Sepharose. mutant of GST-Csk SH3/2 protein was almost absent (Figs. Immunoblot Analysis. Cell lysates were prepared in 1% 1 and 2). The binding ofthe R106K mutant to both paxillin and Nonidet P-40 (4), and each 200 ug of lysate protein was incubated with 5 ug ofGST fusion protein for 2 hr at 4°C. The Table 1. Biological activity of Csk mutants washed complexes were separated by SDS/8% PAGE, trans- ferred to Immobilon-P (Millipore), and subjected to immu- Vector Reversion activity* Activity Activity noblot analysis (4) using ECL (Amersham). insert Rev(I) Rev(II) of Cskt of c-Srct In Vitro Binding of Csk with Paxillin. Paxillin immunopre- Wild type 43.9 14.7 1.0 1.0 cipitated with anti-paxillin antibody covalently coupled with None 5.7 1.1 3.2 Affi-Gel 10 (Bio-Rad) beads was incubated in the presence or K222M 4.3 1.5 0.0 3.3 absence ofpurified c-Src (29) in kinase buffer (4) with 0.1 mM A18-53 12.3 1.4 1.0 2.7 ATP for 30 min at 30°C. After c-Src was washed from the A12-57 12.0 2.8 1.2 2.5 immunoprecipitate, paxillin retained on the beads was incu- A&36-177 9.0 1.3 <0.1 2.5 bated with purified recombinant chicken Csk for 2 hr at 4°C R106K 20.3 2.6 1.4 2.0 and then washed again extensively. The final washed samples S108C 12.2 2.5 0.9 3.0 were analyzed by immunoblotting. *Morphological reversion activity of each mutant of csk cDNA in 3Y1(v-crk/c-src) cells was measured as described (4). Numbers RESULTS represent mean values of % of colonies showing morphologies reverted to Rev(I) and Rev(II) types after csk cDNA transfection Both SH2 and SH3 Domains of Csk Are Necessary for from three separate experiments. The representative morphology of Suppression of c-Src Kinase Activity. Various mutants of csk Rev(I) and Rev(II) types was described previously (4); Rev(II) cells cDNAs were transfected into rat 3Y1 cells transformed by show more flattened morphology than Rev(I) cells. c-Src activated in the presence of the v-Crk protein [3Yl(v- tSpecific activity as defined in Materials and Methods. Downloaded by guest on October 1, 2021 3986 Cell Biology: Sabe et aL Proc. Natl. Acad. Sci. USA 91 (1994)

a b c d CN Ne U C1 y u N Ye Q- M 0 o n C 00 C., Cm V 0 v-c) I ° ° I ° °I I I:° I- 0 CI V.nw V) LO gm I kDa _- _00= L 200-

97- :.:. s* 69- in _a _ .W-e RS..V - 46- "3 CE_/R_ 30- -gas C d

v-Crk/c-Src CEF C EF/VQ4 FIG. 1. Analysis of Csk SH2-binding tyrosine-phosphorylated proteins. Bacterially expressed GST and GST fusion proteins containing the wild-type Csk SH3 and SH2 domains (SH3/2), SH3 domain (SH3), SH3/2 ofR106K mutant (R106K), and SH3/2 ofS108C mutant (S108C) were immobilized with glutathione-Sepharose and incubated with 200 Mg of lysate of 3Y1(v-crk/c-src) cells (a), normal CEFs (b), RSV-transformed CEFs (c), or vanadate-treated CEFs (d) and analyzed for proteins associated with each GST fusion protein by immunoblotting with anti-phosphotyrosine antibodies. Total cell lysates (50 Mg) were also analyzed in each case (T). ppl25FAK was also significantly reduced (Figs. 1 and 2). The level was not changed (Fig. 3; data not shown); paxillin was residual binding activity of the R106K mutant correlates with not only dephosphorylated but seemed to be almost absent a slightly higher suppressive activity of this mutant in 3Y1(v- (Fig. 3). The level of Csk protein was essentially the same in crk/c-src) cells as compared with the S108C mutant (Table 1). both mitotic and unsynchronized cells (data not shown). Csk Can Bind to Paxdilin Phosphorylated by c-Src in Vitro. Kin Activity of c-Src Is Increased when Tyrosine Phos- Paxillin immunoprecipitated from 3Y1 (Fig. 2e) or CEFs phorylatlon of Paxilhin and pp125FAK Is Decreased. Tyrosine (data not shown) was a good substrate for c-Src kinase. High phosphorylation ofpaxillin and pp125FAK is regulated by cell levels of tyrosine phosphorylation of paxillin in v-Src- adhesion to ECM and is greatly reduced when fibroblasts are transformed 3Y1 and CEF cells also suggest that paxillin is a detached from culture dishes (20, 28, 35). We therefore Src substrate in vivo (ref. 33; data not shown). Csk bound to examined c-Src activity under these conditions. Tyrosine the paxillin phosphorylated by c-Src but not to the nonphos- phosphorylation of both paxillin and pp125FAK in c-Src phorylated form (Fig. 2e). From analysis with Csk mutants, overexpressing 3Yl(c-src) cells was almost undetectable we confirmed that this binding was mediated by the SH2 when cells were kept in suspension, and the specific auto- domain of Csk (data not shown). phosphorylating activity of c-Src kinase increased -2.2-fold Csk SH2-Binding Proteins Are Altered During Mitosis. We while its phosphorylation activity against enolase increased then examined the levels and phosphorylation status of the 1.7-fold. Activity returned to the basal level when cells proteins to which Csk SH2 binds in cells arrested during were once again adhered to fibronectin, where tyrosine mitosis by nocodazole treatment, in which c-Src has shown phosphorylation ofpaxillin and ppl25FAK were also restored to be activated by altered phosphorylation of Tyr-527 (34). (Fig. 4). However, the change in phosphorylation of Tyr-527 The Csk SH2-binding proteins were greatly altered: tyrosine ofc-Src under these conditions was too small to be quantified phosphorylation ofppl25FAK was reduced, while the protein biochemically (data not shown), as described previously (4,

a O co < J O 00 I I * Cb I ) 200- 0 dl _ _ p125 _-- _ --Tensin 97- _ Ad I; :l ~Paxi11 ? Y-Crkc/c-Src CEF CEF/RSV CEF/RSV-~ -Integrir, e C preincubated d with phosphorylation S W -p1 10 SH3 2 S108C by c-Src kD + LOu O uLO 200- N N N 0N4 N I 0 pl25"" aD CL a I CL CL Paxillin -Src 97-.

_. i- Paxillin -MAP2 Kinases CEF RSV Csk CEF RSV FIG. 2. Identification ofthe tyrosine-phosphorylated Csk SH2-binding proteins and their binding to Csk in vivo and in vitro. (a and b) The same membrane filters in Fig. 1 were blotted with antibodies against paxillin and against pp125FAK (polyclonal) (a), or against tensin, integrin ,1, p110, Src, and MAP kinases (b). (c) Since antibodies against p130, pp125FAK (monoclonal), and p120 were unable to be used for immunoblotting, binding ofthese proteins to the Csk SH2 domain was assessed as follows. Cell lysates (same as in Fig. 1) were incubated with glutathione-Sepharose either coupled with GST-Csk SH3/2 wild type (SH3/2) or coupled with GST-Csk SH3/2 of the S108C mutant Csk (S108C) as a control. The resulting supernatants were immunoprecipitated with each of the antibodies. After washing, immunoprecipitates were analyzed by blotting with anti-phosphotyrosine antibody. (d) Cskwas immunoprecipitatedfrom a lysate of3Y cells overexpressingtransfected cskcDNA, by use ofanti-Csk antibody coupled with protein A-Sepharose beads. After washing, proteins retained on beads were separated by SDS/8% PAGE and analyzed with anti-paxillin antibody or anti-ppl25FAK antibody. Preimmune serum was used as a negative control (PI). (e) Paxillin was immunoprecipitated from 3Y1 cells; halfofthe immunoprecipitate was phosphorylated by c-Src in vitro; phosphorylated and unphosphorylated paxillin were then incubated with purified recombinant Csk. After washing, samples were analyzed by immunoblotting with anti-paxillin antibody, anti-phosphotyrosine antibody (aPY) for paxillin phosphorylation, and anti-Csk antibodies. Downloaded by guest on October 1, 2021 Cell Biology: Sabe et al. Proc. Natl. Acad. Sci. USA 91 (1994) 3987 One can consider at least two models for the role of SH2 00 binding in the phosphorylation of c-Src and Src family C/)I to kinases by Csk: (i) the binding of tyrosine-phosphorylated such as and to Csk may activate M L M L L proteins paxillin pp125FAK kDa the Csk kinase activity or (ii) the binding of these proteins 200- may promote the accessibility of Csk to its substrates, activated forms of Src family kinases. The former appears 97- less likely because purified recombinant Csk was almost as 69- aminmoa P active as the purified recombinant v-Src (H.S. and H.H., qomw unpublished work), suggesting that Csk is active in the absence of the SH2-binding proteins. Furthermore, no significant change in the specific activity of Csk was detected 30- -, when Csk was bound to paxillin phosphorylated by c-Src, 200- under conditions comparable to those used in Fig. 2e (data pl 25 not shown). The second model is more plausible. Csk is a 97- soluble protein in nature (1, 7, 9), and only a few percent of

97- Csk is recovered from the membrane fraction in normal cells . .. Paxillin 9 the of Csk SH2 7t 40 a (9). Perhaps anchoring through binding, possibly in combination with the SH3 domain (11, 15), to the membrane fraction or the cytoskeletal structure, where the FIG. 3. Levels of Csk SH2-binding proteins during mitotic phase Src kinases exist (36), might be necessary for Csk to suppress of NIH 3T3 cells. Total cell lysates (T) and proteins bound to the Src kinases. Indeed, the activated forms of the Src and GST-Csk SH3/2 (SH3/2) or GST-Csk SH3/2 of S108C mutant Fyn kinases can bind to pp125FAKthrough their SH2 domains (S108C) were prepared from unsynchronized (L) or mitotic phase (M) (37,44), and c-Src can also bind to paxillin via its SH3 domain NIH 3T3 cells. These were then analyzed by polyclonal anti- (38). Paxillin and pp125FAK are known to colocalize at the phosphotyrosine (aPY) antibodies, polyclonal anti-ppl25FAK anti- our in vitro bodies, and a monoclonal anti-paxillin antibody. Bands that appeared focal adhesion plaques (20, 24, 28, 30). Moreover, at around 65 kDa in the anti-phosphotyrosine blot are due to an experiment showed that Csk can bind to paxillin phosphor- artifact, as in Fig. 1 (described in the text). ylated by c-Src (Fig. 2e). Thus, it seems likely that paxillin phosphorylated by c-Src anchors Csk in the vicinity of 34). Little change was observed in the specific activity of the activated c-Src in vivo. The binding ofCsk to pp125FAK seems v-Src kinase, which lacks Tyr-527, under similar conditions to be independent ofpaxillin, because a significant amount of (data not shown). pp125FAK was detected with the GST-Csk SH3/2 protein in the absence of paxillin (Fig. 3). DISCUSSION The focal adhesion contacts become much weaker during From mutational analysis, we have shown that both the SH2 mitosis, and fibroblasts become round to accomplish mitosis. The kinase activity of c-Src is activated during mitosis, pre- and SH3 domains of Csk are necessary for the suppression of sumably by the reduction of Tyr-527 phosphorylation (34). c-Src kinase activity in v-Crk/c-Src cells, in addition to Csk's Tyrosine phosphorylation of both paxillin and pp125FAK is kinase activity. In normal cells, tyrosine-phosphorylated decreased during mitosis. Therefore, our model suggests that paxillin and pp125FAK are the major proteins which bind to mitotic activation ofc-Src is mainly due to the activation ofthe the Csk SH2 domain. c-Src fraction which is normally suppressed by Csk through its interaction with tyrosine-phosphorylated paxillin and a b c 12 3 1 2 3 1 2 3 pp125FAK at the focal adhesion plaques. That only 10-20%o of c-Src kinase is activated during mitosis (34) suggests that this c-Src A Fak _enolase 4P_ dX fraction ofc-Src might localize or interact with the architecture a pY blot ax pY blot of focal adhesions. Consistent with our hypothesis, a nonmyristoylated form of c-Src which does not localize to mem- ...... brane and/or cytoskeletal fractions is not activated during ai Fak; blot oL Pax blot mitosis (21, 39). However, many other factors might be atSrc blot involved in the mitotic activation of c-Src, in addition to FIG. 4. Change in the kinase activity of c-Src in 3Y1(c-src) during Tyr-527 dephosphorylation (40). cell adhesions. The levels of the kinase activity of c-Src (a) and the The requirement for binding of the Csk SH2 domain to levels of tyrosine phosphorylation of pp125FAK (b) and paxillin (c) paxillin and pp125FAK to suppress the morphological trans- were analyzed with cells grown to semiconfluence on culture dishes formation of 3Y1(v-crk/c-src) cells indicates that the c-Src in the presence of serum (lane 1), trypsinized and kept in suspension activity colocalized with these proteins becomes suppressed for 1 hr (lane 2), or spread onto fibronectin for 1 hr after being kept when Csk is overexpressed (4). Indeed, comparable to the in suspension, in the absence of serum (lane 3). c-Src was immuno- seems to be Ab327 after to in vitro degree ofthe mitotic activation, 10-20% of c-Src precipitated by and, being washed, subjected activated in 3Y1(v-crk/c-src) cells (4). Further, v-Crk can kinase assay in the presence of 10 1ACi of [y.32P]ATP (3000 mCi/ and mmol; 1 Ci = 37 GBq) with acid-denatured enolase as substrate (a prevent Csk from interacting with paxillin (H.S. H.H., Upper). Each pp125FAK and paxillin sample was immunoprecipitated unpublished work). Thus, c-Src, which is normally sup- by anti-paxillin antibody or anti-ppl25FAK antibody and subjected to pressed by Csk through its interaction by paxillin, might be sequential immunoblotting analysis using anti-phosphotyrosine an- activated in the presence of v-Crk. tibody (b and c Upper) and anti-pp125FAK antibody (b Lower) or We have also shown that the kinase activity of c-Src is anti-paxillin antibody (c Lower). The bands that appeared below regulated by cell adhesion to the ECM; the kinase activity of paxillin in the anti-phosphotyrosine blot in c are an artifact, as in c-Src increases when cells are kept in suspension (Fig. 4). Figs. 1 and 3. The relative intensity of each band quantitated with a Under these conditions, little tyrosine phosphorylation of densitometer (LKB UltroScan XL) in a Upper is 1.0 (lane 1), 2.8 is detectable There- (lane 2), and 0.97 (lane 3) for autophosphorylation and 1.0 (lane 1), paxillin and pp125FAK (ref. 20; Fig. 4). 2.2 (lane 2), and 0.95 (lane 3) for enolase. The relative amounts of fore, cell adhesion to the ECM and integrity of the focal c-Src protein in a Lower are 1.0 (lane 1), 1.3 (lane 2), and 0.98 (lane adhesion contacts contribute to the regulation of a certain 3). fraction of the Src family kinases, possibly by affecting the Downloaded by guest on October 1, 2021 3988 Cell Biology: Sabe et al. Proc. Natl. Acad. Sci. USA 91 (1994) accessibility of Csk kinase activity to the Src kinases. Such 10. Cantley, L. C., Auger, K. R., Carpenter, C., Duckworth, B., a mechanism, again, might be related to the mitotic activation Graziani, A., Kapeller, R. & Soltoff, S. (1991) Cell 64, 281-302. of the kinase activity of c-Src. 11. Koch, C. A., Anderson, D., Moran, M. F., Ellis, C. & Pawson, T. The protein (1991) Science 252, 668-674. level ofpaxillin seems to be reduced when cells 12. Waksman, G., Kominos, D., Robertson, S. C., Pant, N., Baltimore, are arrested during mitosis by nocodazole treatment (Fig. 3). D., Birge, R. B., Cowburn, D., Hanafusa, H., Mayer, B. J., Over- The degree of such a reduction of the level of paxillin varied duin, M., Reth, M. D., Rios, C. B., Silverman, L. & Kurian, J. in repeated experiments, although tyrosine phosphorylation (1992) Nature (London) 358, 646-653. was always almost completely absent (data not shown). Such 13. Pawson, T. & Gish, G. D. (1992) CeU 71, 359-362. a decrease in paxillin during mitosis could be at least partly 14. Songyang, Z., Shoelson, S. E., Chaudhuri, M., Gish, G., Pawson, due to the prolonged incubation of cells with nocodazole. T., Haser, W. G., King, F., Roberts, T., Ratnofsky, S., Lechleider, By screening a random peptide R. L., Neel, B. G., Birge, R. B., Fajardo, J. E., Chou, M. M., library, Tyr(P)-(Thr/Ala)- Hanafusa, H., Schaffhausen, B. & Cantley, L. C. (1993) Cell 72, Xaa-Xaa was identified as a high-affinity binding sequence of 767-778. the Csk SH2 domain (45). This sequence is found within the 15. Bar-Sagi, D., Rotin, D., Batzer, A., Mandiyan, V. & Schlessinger, kinase domain of pp125FAK (Tyr-Ala-Glu-Ile, aa 397-400; J. (1993) Cell 74, 83-91. Tyr-Ala-Tyr-Gln, aa 526-529; refs. 24 and 44) and the auto- 16. Gout, I., Dhand, R., Hiles, I. D., Fry, M. J., Panayotou, G., Das, phosphorylation site of the Src family kinases (Tyr-Thr-Ala- P., Truong, O., Totty, N. F., Hsuan, J., Booker, G. W., Campbell, Arg, aa 416-419 in c-Src; ref. 41). The primary structure of I. D. & Waterfield, M. D. (1993) Cell 75, 25-36. 17. Shenoy, S., Choi, J.-K., Bargodia, S., Copeland, T. D., Maller, paxillin is unknown. 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