p130CAS Forms a Signaling Complex with the Adapter Protein CRKL in Hematopoietic Cells Transformed by the BCR/ABL Oncogene

Abstract

The Philadelphia chromosome (Ph) translocation generates a chimeric tyrosine kinase oncogene, BCR/ABL, which causes chronic myelogenous leukemia (CML) and a type of acute lymphoblastic leukemia (ALL). In primary samples from virtually all patients with CML or Ph+ALL, the CRKL adapter protein is tyrosine phosphorylated and physically associated with p210BCR/ABL. CRKL has one SH2 domain and two SH3 domains and is structurally related to c-CRK-II (CRK) and the v-Crk oncoprotein. We have previously shown that CRKL, but not the related adapter protein c-CRK, is tyrosine phosphorylated in cell lines transformed by BCR/ABL, and that CRKL binds to BCR/ABL through the CRKL-SH3 domains. Furthermore, the CRKL-SH2 domain has been shown to bind one or more cellular proteins, one of which is p120CBL. Here we demonstrate that another cellular protein linked to BCR/ABL through the CRKL-SH2 domain is p130CAS. p130CAS was found to be tyrosine phosphorylated and associated with CRKL in BCR/ABL expressing cell lines and in samples obtained from CML and ALL patients, but not in samples from controls. In both normal and BCR/ABL transformed cells, p130CAS was detected in focal adhesion-like structures, as was BCR/ABL. In normal cells, the focal adhesion proteins tensin, p125FAK, and paxillin constitutively associated with p130CAS. However, in BCR/ABL transformed cells, the interaction between p130CAS and tensin was disrupted, while the associations between p130CAS, p125FAK, and paxillin were unaffected. These results suggest that the BCR/ABL oncogene could alter the function of p130CAS in at least three ways: tyrosine phosphorylation, inducing constitutive binding of CRKL to a domain in p130CAS containing Tyr-X-X-Pro motifs (substrate domain), and disrupting the normal interaction of p130CAS with the focal adhesion protein tensin. These alterations in the structure of signaling proteins in focal adhesion like structures could contribute to the known adhesion abnormalities in CML cells. The Philadelphia chromosome (Ph) translocation generates a chimeric tyrosine kinase oncogene, BCR/ABL, which causes chronic myelogenous leukemia (CML) and a type of acute lymphoblastic leukemia (ALL). In primary samples from virtually all patients with CML or Ph+ALL, the CRKL adapter protein is tyrosine phosphorylated and physically associated with p210BCR/ABL. CRKL has one SH2 domain and two SH3 domains and is structurally related to c-CRK-II (CRK) and the v-Crk oncoprotein. We have previously shown that CRKL, but not the related adapter protein c-CRK, is tyrosine phosphorylated in cell lines transformed by BCR/ABL, and that CRKL binds to BCR/ABL through the CRKL-SH3 domains. Furthermore, the CRKL-SH2 domain has been shown to bind one or more cellular proteins, one of which is p120CBL. Here we demonstrate that another cellular protein linked to BCR/ABL through the CRKL-SH2 domain is p130CAS. p130CAS was found to be tyrosine phosphorylated and associated with CRKL in BCR/ABL expressing cell lines and in samples obtained from CML and ALL patients, but not in samples from controls. In both normal and BCR/ABL transformed cells, p130CAS was detected in focal adhesion-like structures, as was BCR/ABL. In normal cells, the focal adhesion proteins tensin, p125FAK, and paxillin constitutively associated with p130CAS. However, in BCR/ABL transformed cells, the interaction between p130CAS and tensin was disrupted, while the associations between p130CAS, p125FAK, and paxillin were unaffected. These results suggest that the BCR/ABL oncogene could alter the function of p130CAS in at least three ways: tyrosine phosphorylation, inducing constitutive binding of CRKL to a domain in p130CAS containing Tyr-X-X-Pro motifs (substrate domain), and disrupting the normal interaction of p130CAS with the focal adhesion protein tensin. These alterations in the structure of signaling proteins in focal adhesion like structures could contribute to the known adhesion abnormalities in CML cells. INTRODUCTIONChronic myelogenous leukemia (CML) 1The abbreviations used are: CMLchronic myelongenous leukemiaALLacute lymphoblastic leukemiaCASCrk associated substrateGSTglutathione S-transferasePhPhiladelphia chromosomeSH2Src homology region 2SH3Src homology region 3PAGEpolyacrylamide gel electrophoresis. and some acute lymphoblastic leukemias (ALL, Ph+) are caused by the activated tyrosine kinase fusion protein BCR/ABL. There are multiple proteins which are tyrosine phosphorylated in response to BCR/ABL transformation (1Andoniou C.E. Thien C.B.F. Langdon W.Y. EMBO J. 1994; 13: 4515-4523Crossref PubMed Scopus (217) Google Scholar, 2de Jong R. ten Hoeve J. Heisterkamp N. Groffen J. J. Biol. Chem. 1995; 270: 21468-21471Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 3Matulonis U. Salgia R. Okuda K. Druker B. Griffin J. Exp. Hematol. 1993; 21: 1460-1466PubMed Google Scholar, 4Salgia R. Brunkhorst B. Pisick E. Li J.L. Lo S.H. Chen L.B. Griffin J.D. Oncogene. 1995; 11: 1149-1155PubMed Google Scholar). We and others have shown that one of the most prominent and consistently tyrosine-phosphorylated proteins is an adapter protein termed CRKL (CRK-like) (5ten Hoeve J. Arlinghaus R.B. Guo J.Q. Heisterkamp N. Groffen J. Blood. 1994; 84: 1731-1736Crossref PubMed Google Scholar, 6Nichols G.L. Raines M.A. Vera J.C. Lacomis L. Tempst P. Golde D.W. Blood. 1994; 84: 2912-2918Crossref PubMed Google Scholar, 7Oda T. Heaney C. Hagopian J.R. Okuda K. Griffin J.D. Druker B.J. J. Biol. Chem. 1994; 269: 22925-22928Abstract Full Text PDF PubMed Google Scholar). CRKL has a 60% homology to c-CRK-II (CRK), the human homologue of v-Crk (8Mayer B.J. Hanafusa H. J. Virol. 1990; 64: 3581-3589Crossref PubMed Google Scholar, 9ten Hoeve J. Morris C. Heisterkamp N. Groffen J. Oncogene. 1993; 8: 2469-2474PubMed Google Scholar). The v-crk oncogene was originally cloned from the CT10 virus and can transform fibroblasts and other cells. The v-Crk oncoprotein contains both one SH2 and one SH3 domain, while c-CRK-II and CRKL contain one SH2 and two SH3 domains (10Matsuda M. Tanaka S. Nagata S. Kojima A. Kurata T. Shibuya M. Mol. Cell. Biol. 1992; 12: 3482-3489Crossref PubMed Scopus (245) Google Scholar, 11Matsuda M. Mayer B.J. Hanafusa H. Mol. Cell. Biol. 1991; 11: 1607-1613Crossref PubMed Scopus (94) Google Scholar). A second product of the c-CRK gene, c-CRK-I, has one SH2 and one SH3 domain, but to date, only one form of CRKL has been observed and no oncogenic forms have been described. v-crk transformation in 3Y1 fibroblasts leads to tyrosine phosphorylation of multiple proteins (12Matsuda M. Reichman C.T. Hanafusa H. J. Virol. 1992; 66: 115-121Crossref PubMed Google Scholar). In the v-crk transformed fibroblasts, c-Abl has been shown to bind to v-Crk and is believed to be activated by this interaction (13Ren R. Ye Z.S. Baltimore D. Genes Dev. 1994; 8: 783-795Crossref PubMed Scopus (290) Google Scholar).Another prominent binding protein for the v-Crk oncoprotein has been shown to be p130CAS (Crk associated substrate) (14Sakai R. Iwamatsu A. Hirano N. Ogawa S. Tanaka T. Mano H. Yazaki Y. Hirai H. EMBO J. 1994; 13: 3748-3756Crossref PubMed Scopus (592) Google Scholar). p130CAS was originally cloned from the v-crk transformed 3Y1 cell line and shown to be heavily tyrosine phosphorylated in these cells (14Sakai R. Iwamatsu A. Hirano N. Ogawa S. Tanaka T. Mano H. Yazaki Y. Hirai H. EMBO J. 1994; 13: 3748-3756Crossref PubMed Scopus (592) Google Scholar, 15Sakai R. Iwamatsu A. Hirano N. Ogawa S. Tanaka T. Nishida J. Yazaki Y. Hirai H. J. Biol. Chem. 1994; 269: 32740-32746Abstract Full Text PDF PubMed Google Scholar). Analysis of cDNA for p130CAS reveals multiple domains. The N terminus contains an SH3 domain, followed by a “substrate domain” consisting of 14 Tyr-X-X-Pro (Y-X-X-P) motifs, each of which are potential CRK/CRKL-SH2 binding sites (15Sakai R. Iwamatsu A. Hirano N. Ogawa S. Tanaka T. Nishida J. Yazaki Y. Hirai H. J. Biol. Chem. 1994; 269: 32740-32746Abstract Full Text PDF PubMed Google Scholar). There is also a pp60c-Src binding site in the C terminus of the molecule (16Nakamoto T. Sakai R. Ozawa K. Yazaki Y. Hirai H. J. Biol. Chem. 1996; 271: 8959-8965Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). p130CAS also has been shown to be tyrosine phosphorylated in response to v-Src (14Sakai R. Iwamatsu A. Hirano N. Ogawa S. Tanaka T. Mano H. Yazaki Y. Hirai H. EMBO J. 1994; 13: 3748-3756Crossref PubMed Scopus (592) Google Scholar, 17Kanner S.B. Reynolds A.B. Wang H.C. Vines R.R. Parsons J.T. EMBO J. 1991; 10: 1689-1698Crossref PubMed Scopus (157) Google Scholar, 18Petch L.A. Bockholt S.M. Bouton A. Parsons J.T. Burridge K. J. Cell Sci. 1995; 108: 1371-1379Crossref PubMed Google Scholar). In v-src transformed cells, p130CAS is localized to focal adhesions (18Petch L.A. Bockholt S.M. Bouton A. Parsons J.T. Burridge K. J. Cell Sci. 1995; 108: 1371-1379Crossref PubMed Google Scholar, 19Lo S.H. Weisberg E. Chen L.B. Bioessays. 1994; 16: 817-823Crossref PubMed Scopus (117) Google Scholar), and recently, several laboratories have shown that p130CAS is tyrosine phosphorylated in response to β1 integrin cross-linking (17Kanner S.B. Reynolds A.B. Wang H.C. Vines R.R. Parsons J.T. EMBO J. 1991; 10: 1689-1698Crossref PubMed Scopus (157) Google Scholar, 18Petch L.A. Bockholt S.M. Bouton A. Parsons J.T. Burridge K. J. Cell Sci. 1995; 108: 1371-1379Crossref PubMed Google Scholar, 20Nojima Y. Morino N. Mimura T. Hamasaki K. Furuya H. Sakai R. Sato T. Tachibana K. Morimoto C. Yazaki Y. Hirai H. J. Biol. Chem. 1995; 270: 15398-15402Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, 21Vuori K. Ruoslahti E. J. Biol. Chem. 1995; 270: 22259-22262Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). Although the function of p130CAS in either normal or transformed cells is unknown, these observations suggest that p130CAS may be involved in integrin signaling.Since p130CAS apparently plays a prominent role in transformation by the v-crk oncogene, we examined the interaction of p130CAS with c-CRK and CRKL in hematopoietic cells transformed by BCR/ABL and in normal hematopoietic cells. We find that p130CAS is tyrosine phosphorylated in most cell lines transformed by BCR/ABL and associated with the SH2 domain of CRKL, but not with c-CRK. This potentially links p130CAS to BCR/ABL since CRKL is constitutively associated with BCR/ABL through its SH3 domain. This interaction was unique to BCR/ABL transformed cell lines and was not observed in normal cells. Furthermore, in primary cells from patients with CML and Ph+ acute lymphocytic leukemia (ALL), p130CAS co-immunoprecipitates with CRKL. In both normal and BCR/ABL transfected cells, we found that p130CAS localized in focal adhesion-like structures, as did BCR/ABL. Our results are of interest as they extend the known interactions between BCR/ABL and cellular signal transduction molecules mediated by CRKL.DISCUSSIONChronic myelogenous leukemia was the first disorder identified with a specific abnormal chromosomal translocation, the Philadelphia chromosome, creating a fusion of two genes, BCR and ABL (exons b2a2 or b3a2), and thus generating the oncoprotein p210BCR/ABL (30Chissoe S.L. Bodenteich A. Wang Y.F. Wang Y.P. Burian D. Clifton S.W. Crabtree J. Freeman A. Iyer K. Li J.A. Ma Y.C. Mclaury H.J. Pan H.Q. Sarhan O.H. Toth S. Wang Z.L. Zhang G.Z. Heisterkamp N. Groffen J. Roe B.A. Genomics. 1995; 27: 67-82Crossref PubMed Scopus (171) Google Scholar). In an alternative fusion of BCR and ABL (exons b1a2), there is production of the oncoprotein p190BCR/ABL which can be seen in some acute lymphoblastic leukemias (30Chissoe S.L. Bodenteich A. Wang Y.F. Wang Y.P. Burian D. Clifton S.W. Crabtree J. Freeman A. Iyer K. Li J.A. Ma Y.C. Mclaury H.J. Pan H.Q. Sarhan O.H. Toth S. Wang Z.L. Zhang G.Z. Heisterkamp N. Groffen J. Roe B.A. Genomics. 1995; 27: 67-82Crossref PubMed Scopus (171) Google Scholar). BCR/ABL is a potent tyrosine kinase which translocates to the cytoskeleton and disrupts the actin cytoskeleton (31McWhirter J. Wang J. Mol. Cell. Biol. 1991; 11: 1553-1565Crossref PubMed Google Scholar, 32McWhirter J. Wang J. EMBO J. 1993; 12: 1533-1546Crossref PubMed Scopus (286) Google Scholar, 33Van Etten R. Jackson P. Baltimore D. Sanders M. Matsudaira P. Janmey P. J. Cell Biol. 1994; 124: 325-340Crossref PubMed Scopus (237) Google Scholar). There is growing evidence that CML cells have adhesion defects and that these defects contribute significantly to the clinical phenotype (34Gordon M.Y. Dowding C.R. Riley G.P. Goldman J.M. Greaves M.F. Nature. 1987; 328: 342-344Crossref PubMed Scopus (396) Google Scholar, 35Verfaillie C.M. McCarthy J.B. McGlave P.B. J. Clin. Invest. 1992; 90: 1232-1241Crossref PubMed Scopus (243) Google Scholar). There are multiple proteins which are phosphorylated by BCR/ABL in primary cells from subjects with CML or ALL and in cell lines generated by transfection of BCR/ABL (33Van Etten R. Jackson P. Baltimore D. Sanders M. Matsudaira P. Janmey P. J. Cell Biol. 1994; 124: 325-340Crossref PubMed Scopus (237) Google Scholar). We and others have previously shown that a potentially key signaling intermediate is CRKL (5ten Hoeve J. Arlinghaus R.B. Guo J.Q. Heisterkamp N. Groffen J. Blood. 1994; 84: 1731-1736Crossref PubMed Google Scholar, 6Nichols G.L. Raines M.A. Vera J.C. Lacomis L. Tempst P. Golde D.W. Blood. 1994; 84: 2912-2918Crossref PubMed Google Scholar, 7Oda T. Heaney C. Hagopian J.R. Okuda K. Griffin J.D. Druker B.J. J. Biol. Chem. 1994; 269: 22925-22928Abstract Full Text PDF PubMed Google Scholar).CRKL is a 39-kDa protein with one SH2 and two SH3 domains (9ten Hoeve J. Morris C. Heisterkamp N. Groffen J. Oncogene. 1993; 8: 2469-2474PubMed Google Scholar). has a homology to and to the of adapter proteins S.M. R.B. Hanafusa H. Baltimore D. Sci. 1994; Full Text PDF PubMed Scopus Google Scholar). The of c-CRK-I, and CRKL. CRKL and c-CRK-II contain an SH2 domain and two SH3 domains at the and v-Crk both have a of the most SH3 S.M. B. Hanafusa H. Oncogene. 1995; 10: Google Scholar). CRKL has been shown to have in binding as c-CRK S.M. B. Hanafusa H. Oncogene. 1995; 10: Google Scholar). However, in BCR/ABL containing cells, is CRKL and not c-CRK which is tyrosine phosphorylated (5ten Hoeve J. Arlinghaus R.B. Guo J.Q. Heisterkamp N. Groffen J. Blood. 1994; 84: 1731-1736Crossref PubMed Google Scholar, 6Nichols G.L. Raines M.A. Vera J.C. Lacomis L. Tempst P. Golde D.W. Blood. 1994; 84: 2912-2918Crossref PubMed Google Scholar, 7Oda T. Heaney C. Hagopian J.R. Okuda K. Griffin J.D. Druker B.J. J. Biol. Chem. 1994; 269: 22925-22928Abstract Full Text PDF PubMed Google Scholar). In to the between c-CRK and CRKL in hematopoietic cells, we have to they have a with the cloned p130CAS (14Sakai R. Iwamatsu A. Hirano N. Ogawa S. Tanaka T. Mano H. Yazaki Y. Hirai H. EMBO J. 1994; 13: 3748-3756Crossref PubMed Scopus (592) Google Scholar, 15Sakai R. Iwamatsu A. Hirano N. Ogawa S. Tanaka T. Nishida J. Yazaki Y. Hirai H. J. Biol. Chem. 1994; 269: 32740-32746Abstract Full Text PDF PubMed Google Scholar). Our results that p130CAS is tyrosine and with CRKL, but not c-CRK in BCR/ABL transformed cell lines and samples from patients with CML and Ph+ The of between p130CAS and BCR/ABL to CRKL has not been shown in this and be is of interest that p130CAS is not significantly tyrosine phosphorylated in cells and the are Jong R. ten Hoeve J. Heisterkamp N. Groffen J. J. Biol. Chem. 1995; 270: 21468-21471Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, S. R. Mol. Cell. Biol. 1996; 16: PubMed Scopus Google Scholar). There is a that cells have other abnormalities which p130CAS from there is of tyrosine phosphorylation of proteins in CML primary cells. This could also the tyrosine phosphorylation of p130CAS by BCR/ABL. has also been shown that cells and this is related to p130CAS phosphorylation R. T. 1990; PubMed Scopus Google Scholar). to be to the and of was identified as a prominent tyrosine-phosphorylated of the v-Src and v-Crk (17Kanner S.B. Reynolds A.B. Wang H.C. Vines R.R. Parsons J.T. EMBO J. 1991; 10: 1689-1698Crossref PubMed Scopus (157) Google Scholar, R.B. Mayer B.J. Hanafusa H. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar). has also been shown that p130CAS is tyrosine phosphorylated in response to adhesion and cross-linking of β1 (17Kanner S.B. Reynolds A.B. Wang H.C. Vines R.R. Parsons J.T. EMBO J. 1991; 10: 1689-1698Crossref PubMed Scopus (157) Google Scholar, 18Petch L.A. Bockholt S.M. Bouton A. Parsons J.T. Burridge K. J. Cell Sci. 1995; 108: 1371-1379Crossref PubMed Google Scholar, 20Nojima Y. Morino N. Mimura T. Hamasaki K. Furuya H. Sakai R. Sato T. Tachibana K. Morimoto C. Yazaki Y. Hirai H. J. Biol. Chem. 1995; 270: 15398-15402Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, 21Vuori K. Ruoslahti E. J. Biol. Chem. 1995; 270: 22259-22262Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). p130CAS has been shown to with the focal adhesion proteins and tensin in and in S.H. Weisberg E. Chen L.B. Bioessays. 1994; 16: 817-823Crossref PubMed Scopus (117) Google Scholar, Sci. U. S. A. 1995; PubMed Scopus Google Scholar). p130CAS may be in transformation by M. A. Hirai H. E. Mol. Cell. Biol. 1995; PubMed Scopus Google Scholar). has been shown that c-Abl may p130CAS in In normal cells, p130CAS is localized to both the and the in v-src and v-crk transformed fibroblasts, is localized to the focal adhesion (14Sakai R. Iwamatsu A. Hirano N. Ogawa S. Tanaka T. Mano H. Yazaki Y. Hirai H. EMBO J. 1994; 13: 3748-3756Crossref PubMed Scopus (592) Google Scholar, 17Kanner S.B. Reynolds A.B. Wang H.C. Vines R.R. Parsons J.T. EMBO J. 1991; 10: 1689-1698Crossref PubMed Scopus (157) Google Scholar, 19Lo S.H. Weisberg E. Chen L.B. Bioessays. 1994; 16: 817-823Crossref PubMed Scopus (117) Google was originally cloned from 3Y1 fibroblasts transformed with v-crk (14Sakai R. Iwamatsu A. Hirano N. Ogawa S. Tanaka T. Mano H. Yazaki Y. Hirai H. EMBO J. 1994; 13: 3748-3756Crossref PubMed Scopus (592) Google Scholar, 15Sakai R. Iwamatsu A. Hirano N. Ogawa S. Tanaka T. Nishida J. Yazaki Y. Hirai H. J. Biol. Chem. 1994; 269: 32740-32746Abstract Full Text PDF PubMed Google Scholar). There are two tyrosine phosphorylated proteins in v-crk transformed 3Y1 paxillin and p130CAS R.B. Reichman C. Hanafusa H. Mol. Cell. Biol. 1993; 13: PubMed Scopus Google Scholar). protein and the cDNA p130CAS has been p130CAS contains an SH3 domain which has been shown to bind to the in the terminus of In the of the molecule is the “substrate domain” with motifs for binding the domain (14Sakai R. Iwamatsu A. Hirano N. Ogawa S. Tanaka T. Mano H. Yazaki Y. Hirai H. EMBO J. 1994; 13: 3748-3756Crossref PubMed Scopus (592) Google Scholar). Mayer B.J. Hirai H. Sakai R. Biol. 1995; Full Text Full Text PDF PubMed Scopus Google have shown that the domain binds to the domain of p130CAS in Lo S.H. Weisberg E. Chen L.B. Bioessays. 1994; 16: 817-823Crossref PubMed Scopus (117) Google have shown that the SH2 domain of tensin is also of binding p130CAS in v-src transformed cells. We that the CRKL-SH2 domain binds to the domain of examined the of p130CAS with focal adhesion proteins in normal and BCR/ABL transformed hematopoietic cells. There was a constitutive of and paxillin with p130CAS. the focal adhesion protein tensin associated with p130CAS in normal hematopoietic cells, but not in BCR/ABL transformed cells. is an SH2 containing protein which has three and is believed to be involved in to the actin cytoskeleton S.H. Janmey Chen L.B. J. Cell. Biol. 1994; PubMed Scopus Google Scholar). also is tyrosine phosphorylated in response to S.H. S. Y. Janmey Chen L.B. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). In this we have shown that tensin and p130CAS form a in normal cells and that binding is in BCR/ABL transformed cells. The for this could be that tensin p130CAS could be in either of these molecules could be to another protein not with each or the tyrosine phosphorylation of either of these molecules could potentially The in binding between p130CAS and tensin could be an BCR/ABL and this is we have shown that p130CAS is in hematopoietic cells, is tyrosine phosphorylated in response to BCR/ABL, and with the unique adapter protein CRKL as shown by p130CAS also constitutively with focal adhesion proteins as paxillin and However, p130CAS with tensin in normal but not BCR/ABL expressing cells. is known that CML cells have abnormalities of adhesion through β1 and potentially p130CAS could be involved in these INTRODUCTIONChronic myelogenous leukemia (CML) 1The abbreviations used are: CMLchronic myelongenous leukemiaALLacute lymphoblastic leukemiaCASCrk associated substrateGSTglutathione S-transferasePhPhiladelphia chromosomeSH2Src homology region 2SH3Src homology region 3PAGEpolyacrylamide gel electrophoresis. and some acute lymphoblastic leukemias (ALL, Ph+) are caused by the activated tyrosine kinase fusion protein BCR/ABL. There are multiple proteins which are tyrosine phosphorylated in response to BCR/ABL transformation (1Andoniou C.E. Thien C.B.F. Langdon W.Y. EMBO J. 1994; 13: 4515-4523Crossref PubMed Scopus (217) Google Scholar, 2de Jong R. ten Hoeve J. Heisterkamp N. Groffen J. J. Biol. Chem. 1995; 270: 21468-21471Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 3Matulonis U. Salgia R. Okuda K. Druker B. Griffin J. Exp. Hematol. 1993; 21: 1460-1466PubMed Google Scholar, 4Salgia R. Brunkhorst B. Pisick E. Li J.L. Lo S.H. Chen L.B. Griffin J.D. Oncogene. 1995; 11: 1149-1155PubMed Google Scholar). We and others have shown that one of the most prominent and consistently tyrosine-phosphorylated proteins is an adapter protein termed CRKL (CRK-like) (5ten Hoeve J. Arlinghaus R.B. Guo J.Q. Heisterkamp N. Groffen J. Blood. 1994; 84: 1731-1736Crossref PubMed Google Scholar, 6Nichols G.L. Raines M.A. Vera J.C. Lacomis L. Tempst P. Golde D.W. Blood. 1994; 84: 2912-2918Crossref PubMed Google Scholar, 7Oda T. Heaney C. Hagopian J.R. Okuda K. Griffin J.D. Druker B.J. J. Biol. Chem. 1994; 269: 22925-22928Abstract Full Text PDF PubMed Google Scholar). CRKL has a 60% homology to c-CRK-II (CRK), the human homologue of v-Crk (8Mayer B.J. Hanafusa H. J. Virol. 1990; 64: 3581-3589Crossref PubMed Google Scholar, 9ten Hoeve J. Morris C. Heisterkamp N. Groffen J. Oncogene. 1993; 8: 2469-2474PubMed Google Scholar). The v-crk oncogene was originally cloned from the CT10 virus and can transform fibroblasts and other cells. The v-Crk oncoprotein contains both one SH2 and one SH3 domain, while c-CRK-II and CRKL contain one SH2 and two SH3 domains (10Matsuda M. Tanaka S. Nagata S. Kojima A. Kurata T. Shibuya M. Mol. Cell. Biol. 1992; 12: 3482-3489Crossref PubMed Scopus (245) Google Scholar, 11Matsuda M. Mayer B.J. Hanafusa H. Mol. Cell. Biol. 1991; 11: 1607-1613Crossref PubMed Scopus (94) Google Scholar). A second product of the c-CRK gene, c-CRK-I, has one SH2 and one SH3 domain, but to date, only one form of CRKL has been observed and no oncogenic forms have been described. v-crk transformation in 3Y1 fibroblasts leads to tyrosine phosphorylation of multiple proteins (12Matsuda M. Reichman C.T. Hanafusa H. J. Virol. 1992; 66: 115-121Crossref PubMed Google Scholar). In the v-crk transformed fibroblasts, c-Abl has been shown to bind to v-Crk and is believed to be activated by this interaction (13Ren R. Ye Z.S. Baltimore D. Genes Dev. 1994; 8: 783-795Crossref PubMed Scopus (290) Google Scholar).Another prominent binding protein for the v-Crk oncoprotein has been shown to be p130CAS (Crk associated substrate) (14Sakai R. Iwamatsu A. Hirano N. Ogawa S. Tanaka T. Mano H. Yazaki Y. Hirai H. EMBO J. 1994; 13: 3748-3756Crossref PubMed Scopus (592) Google Scholar). p130CAS was originally cloned from the v-crk transformed 3Y1 cell line and shown to be heavily tyrosine phosphorylated in these cells (14Sakai R. Iwamatsu A. Hirano N. Ogawa S. Tanaka T. Mano H. Yazaki Y. Hirai H. EMBO J. 1994; 13: 3748-3756Crossref PubMed Scopus (592) Google Scholar, 15Sakai R. Iwamatsu A. Hirano N. Ogawa S. Tanaka T. Nishida J. Yazaki Y. Hirai H. J. Biol. Chem. 1994; 269: 32740-32746Abstract Full Text PDF PubMed Google Scholar). Analysis of cDNA for p130CAS reveals multiple domains. The N terminus contains an SH3 domain, followed by a “substrate domain” consisting of 14 Tyr-X-X-Pro (Y-X-X-P) motifs, each of which are potential CRK/CRKL-SH2 binding sites (15Sakai R. Iwamatsu A. Hirano N. Ogawa S. Tanaka T. Nishida J. Yazaki Y. Hirai H. J. Biol. Chem. 1994; 269: 32740-32746Abstract Full Text PDF PubMed Google Scholar). There is also a pp60c-Src binding site in the C terminus of the molecule (16Nakamoto T. Sakai R. Ozawa K. Yazaki Y. Hirai H. J. Biol. Chem. 1996; 271: 8959-8965Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). p130CAS also has been shown to be tyrosine phosphorylated in response to v-Src (14Sakai R. Iwamatsu A. Hirano N. Ogawa S. Tanaka T. Mano H. Yazaki Y. Hirai H. EMBO J. 1994; 13: 3748-3756Crossref PubMed Scopus (592) Google Scholar, 17Kanner S.B. Reynolds A.B. Wang H.C. Vines R.R. Parsons J.T. EMBO J. 1991; 10: 1689-1698Crossref PubMed Scopus (157) Google Scholar, 18Petch L.A. Bockholt S.M. Bouton A. Parsons J.T. Burridge K. J. Cell Sci. 1995; 108: 1371-1379Crossref PubMed Google Scholar). In v-src transformed cells, p130CAS is localized to focal adhesions (18Petch L.A. Bockholt S.M. Bouton A. Parsons J.T. Burridge K. J. Cell Sci. 1995; 108: 1371-1379Crossref PubMed Google Scholar, 19Lo S.H. Weisberg E. Chen L.B. Bioessays. 1994; 16: 817-823Crossref PubMed Scopus (117) Google Scholar), and recently, several laboratories have shown that p130CAS is tyrosine phosphorylated in response to β1 integrin cross-linking (17Kanner S.B. Reynolds A.B. Wang H.C. Vines R.R. Parsons J.T. EMBO J. 1991; 10: 1689-1698Crossref PubMed Scopus (157) Google Scholar, 18Petch L.A. Bockholt S.M. Bouton A. Parsons J.T. Burridge K. J. Cell Sci. 1995; 108: 1371-1379Crossref PubMed Google Scholar, 20Nojima Y. Morino N. Mimura T. Hamasaki K. Furuya H. Sakai R. Sato T. Tachibana K. Morimoto C. Yazaki Y. Hirai H. J. Biol. Chem. 1995; 270: 15398-15402Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, 21Vuori K. Ruoslahti E. J. Biol. Chem. 1995; 270: 22259-22262Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). Although the function of p130CAS in either normal or transformed cells is unknown, these observations suggest that p130CAS may be involved in integrin signaling.Since p130CAS apparently plays a prominent role in transformation by the v-crk oncogene, we examined the interaction of p130CAS with c-CRK and CRKL in hematopoietic cells transformed by BCR/ABL and in normal hematopoietic cells. We find that p130CAS is tyrosine phosphorylated in most cell lines transformed by BCR/ABL and associated with the SH2 domain of CRKL, but not with c-CRK. This potentially links p130CAS to BCR/ABL since CRKL is constitutively associated with BCR/ABL through its SH3 domain. This interaction was unique to BCR/ABL transformed cell lines and was not observed in normal cells. Furthermore, in primary cells from patients with CML and Ph+ acute lymphocytic leukemia (ALL), p130CAS co-immunoprecipitates with CRKL. In both normal and BCR/ABL transfected cells, we found that p130CAS localized in focal adhesion-like structures, as did BCR/ABL. Our results are of interest as they extend the known interactions between BCR/ABL and cellular signal transduction molecules mediated by CRKL.