(CANCER RESEARCH54, 2563-2567, May 15, 1994] Advances in Brief

Cellular Interactions of CRKL, an SH2-SH3 Adaptor Protein'

Johanna ten Hoeve, Vesa Kaartinen, Thoas Fioretos, Leena Haati@ja,Jan-Willem Voncken, Nora Heisterkamp, and John Groffen2

SectionofMolecular Diagnosis, Department of Pathology, ChildrensHospital ofLos Angeles,Los Angeles,California 90027

Abstract Ref. 9). Thus recruited to the plasma membrane, mSOSl can activate p2l'@. Recently, it has been demonstrated that GRB2 also binds Chronic myelogenous leukemia is characterized by a specific chromo BCR/ABL via Y'77, a tyrosine residue encoded by BCR exon 1 somal translocation, t(9;22), in which the ABL protooncogene and the BCR gene become juxtaposed. The chlmenc BCR/ABL gene produces a phosphorylated only in BCR/ABL expressing cells (10, 11). P210 fusion protein with deregulated tyrosine kinase activity. We have Several other “adaptorproteins―have been identified to date (12). recently isolated a complementary DNA, CRKL, which could code for an CRK was initially discovered as the oncogene v-crk (13). CRK adaptor protein consisting of one SH2 and two SH3 domains and lacking consists of an amino terminal SH2 domain and two tandem SH3 any catalytic domain. In the current study, we show that CRKL is highly domains. Deletion of the carboxy terminal SH3 domain leads to phosphorylated in the chronic myelogenous leukemia cell line 1362 and transformation, which is accompanied by an increase in cellular that it is a substrate for the p2lO BCR/ABL and p145 ABL kinases. phosphotyrosine (14, 15). We have recently isolated a gene, CRKL, BCR/ABL and ABL are coimmunoprecipitated with CRKL in vivo, dem encoding a protein with a 60% overall homology to CRK (16). In the onstrating that relatively stable complexes are formed. In addition, the current study, we have identified the CRKL protein product as a nucleotide exchange factor mSOS1 was found to be coimmunoprecipitated 38-kDa protein expressed in a variety of cell types. We present data with CRKL These findings establish a putative signal transduction path way through which BCR/ABL mediates its oncogenic activity. which show that P38 is phosphorylated on tyrosine by ABL and BCR/ABL and demonstrate that it forms complexes in vivo with both ABL and BCR/ABL. In addition, CRKL is capable of binding to Introduction mSOS1. These data provide evidence that CRKL is part of a signal transduction pathway involving ABL and show one mechanism CML3 and Ph-positive acute lymphoblastic leukemia are charac through which BCR/ABL might exert its oncogenic effect. terized by a specific chromosomal translocation, t(9;22). The ABL protooncogene from chromosome 9 is juxtaposed to the BCR gene on Materials and Methods chromosome 22 by this translocation. The chimeric BCR/ABL gene produces P210 or P190 fusion proteins (reviewed in Refs. 1—3).ABL Antisera. Antisera were prepared against GST fusion proteins containing encodes a P145 nonreceptor protein tyrosine kinase and contains two overlapping regions of the CRKL protein. For CH15 antiserum, an several other functional domains, including an SH3, an SH2, and an RsaI-RsaI CRKL cDNA fragment (encoding amino acid residues 128—247) F-actin binding domain (reviewed in Ref. 4). Likewise, the BCR was inserted into pGEX-2T (Pharmacia); for CH16 antiserum, a smaller Sau3A-RsaI CRKL cDNA fragment (encoding amino acid residues 184—247) protein is multifunctional. The domain encoded by BCR exon-1 has was used. The resulting GST fusion proteins were purified on glutathione senne/threonine kinase activity, can dimerize, and binds the ABL SH2 Sepharose columns (Pharmacia) and used to immunize rabbits. Anti-p-Tyr domain in a nonphosphotyrosine-dependent manner (1, 5); the central (Ab-2), cx-BCR (BCR Ab-2), and a-ABL (ABL Ab-3) monoclonal antibodies part of the molecule has homology to GTP exchange factors (6), and were from Oncogene Science, Inc.; the a-SOS polyclonal rabbit antiserum was its carboxy terminus has GTPase-activating protein activity towards from Upstate Biotechnology, Inc. The RC-20 a-p-Tyr antibodies were from small p21@s@likemolecules, including p21rac (7). Both BCR and ABL Transduction Laboratories. The rabbit polyclonal antiserum directed against are thus implicated in signal transduction. The BCR/ABL p210 con the tyrosine kinase FER (CH6) has been described previously (17). tains the BCR exon 1 encoded domain, the exchange factor homology Expression in COS-1 Cells. FER, ABL, BCR, BCR/ABL P210, and domain, and the majority of the ABL protein; therefore, the oncogenic BCR/ABLP190constructsfortransientexpressionin COS-l cells consistedof effect of BCR/ABL must be related to the perturbation of the normal complete cDNAs inserted into a modified SV4O-basedmammalian expression vector, pCDX (18). The entire CRKL coding region was inserted into the BCR and/or ABL signaling pathways. eukaryotic expression vector pSG5 (Stratagene). Constructs were introduced Aspects of a signaling cascade involving the epidermal growth into COS-1 cells using the DEAE-dextran method essentially as described factor receptor, a receptor protein tyrosine kinase, have recently been (19). Cells were harvested 60—72h after transfection. elucidated. Upon ligand binding, the epidermal growth factor receptor Preparation of Extracts and Immunoprecipitations. Cells were sus is autophosphorylated on tyrosine, thereby providing a binding site for pended in 0.5 ml ice-cold lysis buffer [25 mMNa-phosphate (jH 7.2), 150 mM GRB2. GRB2, which consists of a single SH2 domain flanked by two NaG, 1% Triton X-100, 5 mMEDTA, 50 mMNaF, 2 mM PMSF, 2 m@i SH3 domains, associates with tyrosine phosphorylated proteins Na3VO4, 20 p@g/mlleupeptin,and 20 @Wmlaprotinin]and left on ice for through its 5H2 domain and with the RAS guanidine-nucleotide 15—45mm. Cells were further disrupted by passage through 18 and 25 gauge exchange factor mSOS1 through its SH3 domains (Ref. 8; reviewed in needles, and nuclei and debris were pelleted (30 min at 12,000 X g, 4°C). COS-1cell extractsor K562 cell extractscontaining250 or 500 @.tgofprotein, respectively, were incubated for 90 min at 4°Cwith 5 @lofantiserum; immune Received 3/30/94; accepted 4/6/94. complexes were collected after an additional 30-mm incubation with 5 @.d The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with protein A agarose beads (BRL), washed four times with lysis buffer, and boiled 18 U.S.C. Section 1734 solely to indicate this fact. in sodium dodecyl sulfate sample buffer [60 mM Tris (pH 6.8), 2% SDS, 10% 1 This study was supported by USPHS Grant CA47456 (to J. G.). glycerol, 100 mM DTF, 0.01% bromophenol bluej prior to gel electrophoresis. 2 To whom requests for reprints should be addressed, at Department of Pathology, Western Blotting and Immunodetection. Proteins were transferred to Childrens Hospital of Los Angeles, 4650 Sunset Boulevard, Los Angeles, CA 90027. 3 The abbreviations used are: CML, chronic myelogenous leukemia; cDNA, comple Hybond-ECL (Amersham) using a mini trans-blot apparatus (Bio-Rad). Mem mental)' DNA; kDa, kilodalton(s). branes were blocked overnight at 4°CinTBST [20 m@tTris-HCI (pH 8.0), 150 2563

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A B contains antibodies against GST epitopes) and horseradish peroxidase-labeled 12345 12345 secondary antibodies in binding buffer and developed as described above. Northern Blot Analysis. RNAs were isolated using guanidine-isothiocya nate (19). Total RNA (15 @g)wasrun on a formaldehyde-agarose gel and blotted to nitrocellulose (20). Posthybridization washes were performed in O.3X standard saline citrate at 65°C.

Results

Expression of CRKL. Northern blot analysis on RNAs isolated j from different mouse tissues showed that murine crkl, similar to c-crk in chicken (15), was ubiquitously expressed as a 6-kilobase mRNA in all tissues tested (not shown). Among human cell lines, CRKL is relatively abundantly expressed in the CML cell line K562 (Fig. IA, I Lane 2) and at a lower level in the hepatoma cell line HepG2 (Fig. IA, Lane 5). In contrast, CRK mRNA expression was higher in HepG2 Fig. 1. Northern blot analysis of CRKL expression. Human RNAs are from the cell lines A498 (kidney carcinoma), Lane 1; K562 (myelogenous leukemia), Lane 2; CHAGO than in K562 (not shown). (bronchogenic carcinoma), Lane 3; A172 (glioblastoma), Lane 4; and HepG2 (hepatoma), Two overlapping regions within the central domain of CRKL were Lane 5. A, blot hybridized to a human CRKL cDNA containing the entire coding region. expressed as GST fusion proteins in bacteria and used to raise anti B, ethidium-stained gel serves as a loading control. sera. CH15 is directed against the NH2-terminal 5H3 domain and the region between the SIB domains, whereas CH16 is raised against the latterjunction region only (Fig. 2A). The junction region polypeptide mM NaG, and 0.1% Tween] plus 5% nonfat milk (Bio-Rad) and incubated for 2 h at room temperature with the antisera; bound antibodies were detected with of64aminoacidresiduesisdivergedconsiderablybetweenCRKand horseradish peroxidase-labeled secondary antibodies (Bio-Rad). Membranes to CRKL (16). be used with a-p-Tyr antibodies were blocked in TBST plus 2% bovine serum Both antisera specifically detected proteins of approximately 38 albumin plus 1% ovalbumin. RC-20 was used according to the manufacturer's kDa (P38) in COS-1 cells transiently transfected with a construct recommendations. Blots were developed using the ECL reagents (Amersham) expressing full-length CRKL (Fig. 28, Lanes +) and in K562 cells and exposed to Hyperfilm-ECL (Amersham). (Fig. 2B, Lanes K). COS-1 cells also contained a substantial amount Blots were strippedfor 30 min at 55°Cin2%sodiumdodecyl sulfate, 100 of endogenous P38 protein (Fig. 28, Lanes —).When the same blot mM @3-mercaptoethanol, and 62.5 mM Tris-HC1 (pH 6.8). was stripped and reacted with anti-phosphotyrosine (a-p-Tyr) In the Western blot binding assay, purified GST and GST-CRKL fusion monoclonal antibodies, bands of an apparently similar molecular proteins were used. The GST-CRKL fusion protein contains the entire CRKL mass were detected in K562 and the CRKL-transfected COS-1 cell, coding region lacking only the first six NH2-terminal amino acids. Western and were substantially weaker in A498 and untransfected COS-1 blots were blocked for 1 h in PBS plus 0.1% Tween plus 5% milk and cells (not shown). incubated for 2 h at room temperature with the GST or GST-CRKL proteins (at 1 or 2 @&Wml,respectively)in bindingbuffer[25 mMNa-phosphate(jH 7.2), CRKL Is Phosphorylated on Tyrosine by ABL and BCR/ABL 150 mM NaG, 0.1% Tween 70, 2.5 mM EDTA, 20 mM NaP, 1% nonfat milk, The protein of 38-kDa detected by the a-p-Tyr antibody was one of 1 mMdithiotreitol,10 @tg/mileupeptin,and 10 @glmlaprotinin).BoundGST the most prominent p-Tyr containing proteins detected in that cell line. proteins were detected by subsequentincubationswith CH15 (which also To confirm its identity, cellular extracts of K562 were incubated with

A CH15

CH16

IC Fig. 2. Identification of CRKL protein. A, the NL ______@ segments of CRKL against which the antisera were SH2 @SH3 raised. The relative locations of the SH2 and SH3 domains are as indicated. B, Western blot analysis of CRKL expression. Cellular extracts include those of the humanmyelogenousleukemiacell line K562 (K); the human kidney carcinoma cell line A498 (A); COS-1 cells (-); and COS-1 cells tranSfCCted B with CRKL (+). Left, location of 43- and 29-kDa K standards. Arrow, position of the group of P38 pro — + K A — + K A + teins. Antisera used are indicated beneath each panel. @ 43— %[email protected] @-@-

p-I@. ,—4@@P38 29— prel5 CH15 CH16 2564

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CELLULARINTERACflON5OFCRKL

A to

C)co 0@OziO 00‘0Z

Fig. 3. CRKL is phosphorylatedon tyrosine in cells expressing ABL and BCR/ABLA, analysis of IØM@.•.ø @@ the CML cell line K562. Antisera used for immu .— . @P38 noprecipitation are shown above the lanes, and those for Western blot analysis are shown below g___@ .. . each panel. Lane 1 contains total cellular extracts. NRS, nonrelated rabbit serum. The prominent band 1 2 3 4 5 1 2 3 4 5 around50kDais immunoglobulin.B,Westernblot CHI6 a—P--TYR analysis of cotransfections with CRKL, BCR/ABL, andABLin COS-1cells.Extractsincludethoseof B @j@J @ K562 (Lane 1) and COS-1 cells (Lane 2); and of ,- 0 (.@ _J -J< < @J@j @@@@ COS-1 cells transfected with CRKL, BCR/ABL (0J1)o (@) @i I @< -J @ P210+CRKL, BCR/ABL P210, CRKL+ABL, and 0 0 00 0 0 ABL (Lanes 3-7, respectively). Left antisera used. 00 @ Righz@location of BCR/ABL P210, ABL P145, and r-@ ‘I@ -P210 CRKL P38. C, P38 consists of closely migrating @ forms. Lanes 1 and 2, K562 extracts probed with a-A9t. 1 2 3 4 5 6 CH15 and CH16, respectively. Lanes 3 and 4, cx tracts ofCOS-1 cells transfeCtedwith CRKL+ABL andwithCRKLaloneandprobedwithCH16.Lanes 5 and 6, the K562 and CRKL+ABL extracts (Lanes @ II ; @@@ 2 and 3) reprobedwith a-p-Tyr (051) antibodies. CHI6 — — -P38 @ •: !k@4

@ a-p-Typ,@ : . •- • ---4 @-P38

2 3 4 5 6 7

CRKL antisera, and immunoprecipitates were run in duplicate. The ABL, P145 ABL, and P38 CRKL was confirmed using anti-ABL Western blot reacted with CH16 antiserum showed specific immuno monoclonal antibodies and CH16 (Fig. 3B). Tyrosine-phosphorylated precipitation of P38; the duplicate filter reacted with the a-p-Tyr P38 was clearly visible in K562 (Fig. 3B, Lane 1), in cells cotrans monoclonaldemonstratedthatCRKL wasindeedtheprominentp-Tyr fected with CRKL+BCR/ABL P210 (Fig. 3B, Lane 4), with containing P38 in K562 (Fig. 3A). CRKL+BCR/ABL P190 (not shown), and with CRKL+ABL (Fig. To investigate whether CRKL can act as a substrate for BCR/ABL, 3B, Lane 6; also see Fig. 4). cotransfections of CRKL, BCR/ABL P210, BCR/ABL P190, and The phosphorylation of CRKL resulted in mobility shifts. On ABL into COS-1 cells were performed. The presence of P210 BCR/ gels with higher resolution, the P38 band in K562 resolved into

@ CRKL @R-.Aa.

@ @) U) 5) (0 @ II ii― @@@ A C.) 00 CRKL@FER CRKL@FER @ D I') to Fig. 4. Western blot analysis ofCHl5 and CH16 I I immunoprecipitates from transfected COS-1 cells. 0 @ A-C, coimmunoprecipitation of ABL and BCR/ 0 ABL with CRKL antisera. Top, transfected COS-1 cell extracts used.Lanes 1, 3, 5, and 7, total cellular ciii: 4@4 4:@ -P38 extracts;Lanes2, 4, 6, and8, CH15immunopre cipitates; Lane 9, a CH16 immunoprecipitate; Lane 10, an immunoprecipitate with CH15 pre immune serum (A andB only). Lanes containing ‘Pu,— @@I4 -P210 -P94 @ immunoprecipitatesare characterizedby the pro @..@. . .@ -P146 minent immunoglobulin band at around 50 kDa. Left, the antisera subsequently used on the same a-ABL Western blot. Right, locations of P145, P210, and P38. D, the tyrosine kinase PER does not coiinmunoprecipitate with CRKL. Extracts of COS-1 cells cotransfected with CRKL+FER were loaded directly (left lanes) or after im @ munoprecipitation with CH15 (right lanes). CR . @:1 -P210 Antisera used for Western blotting are indicated @ beneath each panel. The locations of P94@ and -P146 P38°@'areas indicated. P38- .@ a-P—TVR CH15 T@t@:! ••@@@ø-P38 a-FER

1 2 3 4 6 6 7 8 9 10 2565

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1994 American Association for Cancer Research. CELLULAR INTERACTIONS OF CRKL two closely migrating forms I and II detected by CH16 (Fig. 3C, A Lane 2). CH15 detected forms I and II and a third protein product, III (Fig. 3C, Lane 1). Form II was present in COS-1 cells 1234 transfected with CRKL, and form I appeared in cells cotransfected with CRKL+ABL (Fig. 3C, Lanes 4 and 3, respectively). Form I 200- contained phosphotyrosine (Fig. 3C, Lanes 5 and 6). This suggests that I and II are differentially phosphorylated forms of one @@-PI7O@ protein, CRKL. P38 Forms a Specific Complex with ABL and BCR/ABL The phosphorylation of CRKL on tyrosine in BCR/ABL and ABL over expressing cells suggested that they may interact directly. To confirm this, we determined whether ABL and/or BCR/ABL were present in immune complexes of P38 with the CRKL antisera. CH15 and CH16 antisera did not react directly with either ABL or BCR/ABL (compare 97— Fig. 4, A and B, Lanes 3, 5, and 7). CH15 immunoprecipitation followed by Western blot analysis with ABL antibodies showed ABL P145 to be present in immunoprecipitates of COS-1 cells expressing CRKL and CRKL+ABL (Fig. 4B, Lanes 2 and 8). CH16 antiserum also coimmunoprecipitated ABL P145 in cells expressing CRKL+ABL (Fig. 4B, Lane 9). Similarly, BCR/ABL P210 was recovered in CH15 and CH16 immunoprecipitates from BCR/ABL P210+CRKL and BCR/ABL P210 expressing cells (Fig. 4B, Lanes 4 B and 6; and results not shown). Anti-p-Tyr antibodies showed that K562 P210, P145, and P38 present in the CH15 and CH16 immunoprecipi tates are phosphorylated on tyrosine (Fig. 4C, Lanes 4, 6, 8, and 9). to to The identity of these proteins was confirmed by coelectrophoresis of the same extracts without prior immunoprecipitation (Fig. 4B, Lanes 1, 3, 5, and 7). Control preimmune serum did not precipitate CRKL or ABL (Fig. 4, A andB, Lanes 10). The interaction of CRKL with BCR/ABL and ABL appears to be specific. The nonreceptor tyrosine kinase FER is related to ABL and, as ABL, is located both in the cytoplasm and in the nucleus (17, 21, —P170 22). CH15 immunoprecipitates of COS-1 cells transfected with CRKL+FER did not contain detectable amounts of P94@ (Fig. 4D). Identification of CRKL-binding Proteins. Total cellular extracts of ABL-expressing COS-1 cells were Western blotted and incubated with GST or GST-CRKL fusion proteins to examine the binding capability of CRKL to other proteins. GST alone bound very little protein (Fig. 5A, Lane 1), whereas GST-CRKL bound to four proteins larger than 100 kDa (Fig. 5A, Lane 2). The most prominent of these — @:4 —P38 corresponded in migration to the overexpressed Pl45@@,1(Fig. 5A, Lane 3). In a similar experiment, GST-CRKL also bound to BCR/ ABL P210 (not shown). A second protein showing a relatively strong 123 reaction with GST-CRKL had a mobility similar to that of P170°°° Fig. 5. Binding of CRKL to cellular proteins. A, Western blot-binding assay using detected on the same membrane by a-SOS antibodies (compare Fig. GST-CRKL Western blots containing extracts ofCOS-1 cells transfected with ABL were 5A, Lanes 2 and 4). The SIB binding domain of nucleotide exchange incubated with GST, with the GST-CRKL fusion protein, or with a-ABL or a-SOS-i antibodies as indicated below each lane. B, coimmunoprecipitation of CRKL and P170'°° factor mSOS1 has recently been shown to bind not only GRB2, but from K562 extracts. Lane 1, total cellular extract. Lanes 2 and 3, immunoprecipitates. The also the CRK SH3 domain in vitro (23). To verify a potential inter top partof the membranewasincubatedwith a-SOS antibodiesandthe bottompartwith action of CRKL with mSOSl in whole cells, cellular extracts of K562 the CH16 antiserum. were incubated with CH15 or control preimmune antiserum, Western blotted, and probed with CH16 or a-SOS antibodies. A P170 protein immunoreactive with the a-SOS antibodies was present in the CH15 of ABL or BCR/ABL, also contains phosphotyrosine, we suggest that immunoprecipitate (Fig. 5B). tyrosine phosphorylation of CRKL by ABL could be a normal cellular process. That raises the question to what extent such a normal cellular Discussion pathway involving CRKL might be perturbed in Ph-positive leuke mia. Since BCR/ABL P210 retains the capability of ABL to Our results demonstrate that CRKL binds to ABL and BCR/ABL in complex with CRKL, it is plausible that the deregulated kinase vivo. Interestingly, interactions between CRK and ABL have also activity of BCR/ABL causes excessive phosphorylation of CRKL been demonstrated.4 One of the consequences of binding of BCR/ on tyrosine. Significantly, in the BCR/ABL p210-expressingCML ABL or ABL and CRKL is the phosphorylation of CRKL on tyrosine. cell line K562, the majority of p38 CRKL was found to be tyrosine Since a small fraction of endogenous CRKL, without overexpression phosphorylated. The presence of a prominent tyrosine-phosphory lated p36-p4l protein in K562 has also been shown by others using 4 H. Hanafusa, personal communication. immunoblotting and anti-phosphotyrosine antibodies (24, 25). This 2566

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1994 American Association for Cancer Research. CELLULAR INTERACflON5 OF CRKL protein is phosphorylated on tyrosine specifically in CML cell S. R.,Aaronson,S. A., andEva,A. A regionofproto-dblessentialforits transforming lines but not in matched cell line controls (24). Our data strongly activity shows sequence similarity to a yeast cell cycle gene, CDC24, and the human breakpoint cluster gene, her. N. Biol., 3: 372—379,1991. suggest that the p36-p4l identified by these investigators is CRKL. 7. Diekmann, D., Brill, S., Garrett, M. D., Totty, N., Hsuan, J., Monfries, C., Hall, C., A second possible mechanism of CRKL and CRK involvement in LAm, L, and Hall, A. Bcr encodes a GTPase-activating protein for p21'°°.Nature leukemia is through gene dosage. A duplication of the Ph chro (Lond.), 351: 400—402,1991. 8. Lowenstein, E. J., Daly, R. J., Batzer, A. G., Li, W., Margolis, B., Lammers, R., mosome is frequently seen in blast phase CML, and an i(17q), Ullrich, A., Skoinick, E. Y., Bar-Sagi, D., and Schlessinger, J. The SH2 and SH3 which involves a deletion of l7p and duplication of 17q, is detected in domain-containing protein GRB2 links receptor tyrosine kinases to ras signalling. around 20% of patients in acute phase of CML Both abnormalities Cell, 70: 431—442,1992. 9. McCormick,F. How receptorsturnRas on. Nature(Land.)363: 15—16,1993. modify the gene copy number of either CRKL or CRK (26, 27). In this 10. Pendergast,A. M., Quilhiam,L A., Cripe, L D., Bassing, C. H., Dai, Z., Li, N., context@K562 is an example ofa CML cell line having an increased copy &tzer, A., Rabun, K. M., Der, C. J., Schlessinger, J., and Gishizky, M. L BCR number and mRNA expression of CRKL over CRK. ABL-induced oncogenesis is mediated by direct interaction with the SH2 domain of the GRB-2 adaptorprotein.Cell, 75: 175—185,1993. Both GRB2 and CRKL interact with BCR/ABL, albeit apparently 11. Pull, L, Liu, J., Gish, G., Mbamalu,G., Bowtell, D., Pelicci, P. G., Arlinghaus,A., through a different mechanism; GRB2 binds BCR/ABL through the Pawson, T. Bcr-Abl oncoproteinsbind directly to activatorsof the Ras signalling phosphorylated tyrosine residue Y@ in the BCR part of the protein pathway. EMBO J., 13: 764—773,1994. 12. Koch, C. A., Anderson, D., Moran, M. F., Ellis, C., Pawson, T. SH2 and SH3 (10, 11), while CRKL is likely to bind the ABL part of BCR/ABL. domains:elementsthatcontrolinteractionsofcytoplasmicsignalingproteins.Science Both GRB2 (11, 12) and CRKL (this study) are able to complex with (Washington DC), 252: 668—674,1991. mSOS1. Upon activation of receptor tyrosine kinases, GRB2/SOS 13. Mayer, B. J., Hamaguchi, M., and Hanafusa, H. A novel viral oncogene with structural similarity to phospholipase C. Nature (Land.), 332: 272—275,1988. complexes are recruited to the plasma membrane where RAS is 14. Mayer, B. J., and Hanafusa, H. Mutagenic analysis of the v-crk oncogene: require. located, thereby activating the RAS pathway. The deregulated BCR/ mentfor SH2 andSH3 domainsandcorrelationbetweenincreasedcellularphospho ABL kinase also seems to activate the RAS pathway through GRB2/ tyrosineandtransformation.J.Virol.,64: 3581—3589,1990. 15. Reichman, C. T., Mayer, B. J., Keshav, S., and Hanafusa, H. The product of the SOS (10, 11). The present finding of association of CRKL with cellularcrkgene consists primarilyof SH2 andSH3 regions.Cell Growth& Differ., mSOS1 suggests that CRKL might also regulate the RAS pathway. 3: 451—460,1992. Independent of this, it is clear that CRKL can bind to BCR/ABL and 16. ten Hoeve, J., Morris, C., Heisterkamp, N., and Groffen, J. Isolation and chromosomal localizationof CRKL,a humanCRK-likegene. Oncogene,8: 2469—2474,1993. becomes phosphorylated on tyrosine as a result of this interaction. It 17. Han, Q-L, Heisterkamp, N., and Groffen, J. Isolation and sequence analysis of a will be of interestto identify the functionalsignificance of the phos novel human tyrosine kinase gene. MoL Cell. Biol., 9: 1587—1593,1989. phorylation of CRKL and to further unravel the roles of both adaptor 18. Okayama,H., andBerg,P. A cDNA cloning vectorthatpermitsexpressionof cDNA inserts in mammalian cells. Mol. Cell. Biol., 3: 280-289, 1983. proteins in the leukemic processes initiated by the BCR/ABL fusion 19. Chomczynski, P., and Sacchi. N. Single-step method of RNA isolation by acid protein. guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem., 162: 156— 159,1987. Acknowledgments 20. Sambrook, J., Fritsch, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual,Ed. 2. Cold SpringHarbor,NY: Cold SpringHarborLaboratory,1989. 21. Hao, Q-L, Ferris, D. K., White, G., Heisterkamp, N., and Groffen, J. Nuclear and We thank S. Feller and H. Hanafusafor sharing unpublisheddata and cytoplasmic location of the FER tyrosine kinase. Mol. Cell. Biol., 11: 1180—1183, materials;R. Arlinghausfor COS-1 cells and pSG5; and K. C. Chen for 1991. photographic art work. 22. Van Ellen, R., Jackson, P., and Baltimore, D. The mouse type IV c-ablgene product is a nuclear protein, and activation of transforming ability is associated with cyto References plasmic localization. Cell, 58: 669—678, 1989. 23. Gout, I., Dhand, R., Hiles, I. D., Fry, M. J., Panayotou, G., Das, P., Truong, 0., Tony, 1. Sawyers,C. L, Denny,C. T., andWitte,0. N. Leukemiaandthedisruptionofnormal N. F., Hsuan, J., Booker, G. W., Campbell, I. D., and Waterfield, M. D. The GTPase hematopoiesis.Cell64: 337—350,1991. dynamin bindsto and is activated by a subset ofSH3 domains. Cell, 75: 25—36,1993. 2. Kantarjian, H. M., Deisseroth, A., Kurzrock, R., Estrov, Z., and Talpaz, M. Chronic 24. Freed, E., and Hunter,T. A 41-kilodalton protein is a potential substratefor the myelogenous leukemia: a concise update. Blood, 82: 691—703,1993. p2l0t@@@@Nprotein-tyrosinekinase in chronic myelogenous leukemia cells. Mol. Cell. 3. Heisterkamp, N., Voncken, J-W., van Schaick, H., and Groffen, J. Ph-positive Biol., 12: 1312—1323,1992. leukemia. In: I. R Kirsch (ed.), The Causes and Consequences of Chromosomal 25. Naldini, L, Stacchini, A., Cirillo, D. M., Aglietta, M., Gavosto, F., and Comoglio, Aberrations, pp. 359-376. Boca Raton, FL: CRC Press, 1993. P. M. Phosphotyrosineantibodiesidentifythe p210°@tyrosinekinase and proteins 4. wang, J. Y. J. Abl tyrosinekinase in signal transductionandcell-cycle regulation. phosphorylated on tyrosine in human chronic myelogenous leukemia cells. Mol. Cell. Qirr. Opin. Genet. Dcv., 3: 35—43,1993. Biol.,6:1803—1811,1986. 5. McWhirter, J., Galasso, D., Wang, J. Y. J. 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Johanna ten Hoeve, Vesa Kaartinen, Thoas Fioretos, et al.

Cancer Res 1994;54:2563-2567.

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