BCR/ABL-induced Leukemogenesis Causes Phosphorylation of Hef1 and Its Association with Crkl
1997; Elsevier BV; Volume: 272; Issue: 51 Linguagem: Inglês
10.1074/jbc.272.51.32649
ISSN1083-351X
AutoresRon de Jong, Arnoud van Wijk, Leena Haataja, Nora Heisterkamp, John Groffen,
Tópico(s)Protein Degradation and Inhibitors
ResumoBCR/ABL is considered responsible for the development of Philadelphia chromosome-positive leukemia. Experimental animal models, such as transgenic mice, have demonstrated unambiguously that Bcr/Abl is capable of inducing leukemogenesis. The adaptor molecule Crkl is a major in vivo substrate of the deregulated Bcr/Abl tyrosine kinase and functions as a molecular link with other signaling proteins. While associated in vivowith Bcr/Abl through its SH3 domain, Crkl can interact simultaneously via its SH2 domain with other tyrosine-phosphorylated proteins.Here we report the identification of prominently tyrosine-phosphorylated proteins with a molecular mass of approximately 110 kDa, which bind specifically to the Crkl SH2 domain in leukemic tissues of P190BCR/ABL transgenic mice. We demonstrate that these proteins are identical to Hef1/Cas-L, which is related to p130Cas. The proto-oncoprotein p120Cbl and Hef1, but not p130Cas, were detectably phosphorylated on tyrosine in P190Bcr/Abl-expressing leukemic cells and were found in complex with Crkl, showing the existence of protein complexes in P190Bcr/Abl leukemic cells, consisting of P190Bcr/Abl, Crkl, and Hef1 or p120Cbl. This supports a model in which Crkl acts as mediator between Bcr/Abl and downstream effectors. Since Hef1 is involved in the β1-integrin signaling pathway, our study demonstrates that Bcr/Abl could specifically interfere with normal β1-integrin signaling. BCR/ABL is considered responsible for the development of Philadelphia chromosome-positive leukemia. Experimental animal models, such as transgenic mice, have demonstrated unambiguously that Bcr/Abl is capable of inducing leukemogenesis. The adaptor molecule Crkl is a major in vivo substrate of the deregulated Bcr/Abl tyrosine kinase and functions as a molecular link with other signaling proteins. While associated in vivowith Bcr/Abl through its SH3 domain, Crkl can interact simultaneously via its SH2 domain with other tyrosine-phosphorylated proteins. Here we report the identification of prominently tyrosine-phosphorylated proteins with a molecular mass of approximately 110 kDa, which bind specifically to the Crkl SH2 domain in leukemic tissues of P190BCR/ABL transgenic mice. We demonstrate that these proteins are identical to Hef1/Cas-L, which is related to p130Cas. The proto-oncoprotein p120Cbl and Hef1, but not p130Cas, were detectably phosphorylated on tyrosine in P190Bcr/Abl-expressing leukemic cells and were found in complex with Crkl, showing the existence of protein complexes in P190Bcr/Abl leukemic cells, consisting of P190Bcr/Abl, Crkl, and Hef1 or p120Cbl. This supports a model in which Crkl acts as mediator between Bcr/Abl and downstream effectors. Since Hef1 is involved in the β1-integrin signaling pathway, our study demonstrates that Bcr/Abl could specifically interfere with normal β1-integrin signaling. Several experimental mouse models have been developed to investigate the oncogenic action of Bcr/Abl, which is causative of the development of chronic myeloid leukemia (CML) 1The abbreviations used are: CML, chronic myeloid leukemia; ALL, acute lymphoblastic leukemia; GST, glutathione S-transferase; mAb, monoclonal antibody; SH2, Src homology region-2; SH3, Src homology region-3. 1The abbreviations used are: CML, chronic myeloid leukemia; ALL, acute lymphoblastic leukemia; GST, glutathione S-transferase; mAb, monoclonal antibody; SH2, Src homology region-2; SH3, Src homology region-3. and Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL), in vivo. Transgenic P190BCR/ABL mice reproducibly develop lymphoblastic leukemia/lymphoma involving cells of pre-B-cell origin or their progenitors (1Heisterkamp N. Jenster G. ten Hoeve J. Zovich D. Pattengale P.K. Groffen J. Nature. 1990; 344: 251-254Crossref PubMed Scopus (590) Google Scholar, 2Voncken J.W. Griffiths S. Greaves M.F. Pattengale P.K. Heisterkamp N. Groffen J. Cancer Res. 1992; 52: 4534-4539PubMed Google Scholar). Established transgenic mice strains expressing P190Bcr/Abl provide an unlimited source and unique opportunity for studying the signal transduction pathways affected by the Bcr/Abl oncoprotein in vivo.The tyrosine kinase activity located in the Abl segment of Bcr/Abl is dramatically increased (3Konopka J.B. Watanabe S.M. Witte O.N. Cell. 1984; 37: 1035-1042Abstract Full Text PDF PubMed Scopus (674) Google Scholar), and this is critical for its transforming capacity. Although it remains unclear which intracellular signaling pathways are crucial to the in vivo oncogenic activity of Bcr/Abl, numerous signaling proteins have been implicated by studies involving patient-derived cell lines or stably transfected cells transformed by Bcr/Abl. However, only few signaling proteins are tyrosine-phosphorylated by Bcr/Abl or in complex with Bcr/Abl in leukemic cells isolated directly from Ph+ patients, indicating the importance of studying the molecular mechanisms behind Bcr/Abl-induced leukemogenesis in an in vivo context. Signaling proteins shown to be affected by Bcr/Abl include p130Cas (4Salgia R. Pisick E. Sattler M. Li J.-L. Uemura N. Wong W.-K. Burky S.A. Hirai H. Chen L.B. Griffin J.D. J. Biol. Chem. 1996; 271: 25198-25203Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar), p120CBL (5de 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), p68paxillin (6Salgia R. Uemura N. Okuda K. Li J.-L. Pisick E. Sattler M. de Jong R. Druker B. Heisterkamp N. Chen L.B. Groffen J. Griffin J.D. J. Biol. Chem. 1995; 270: 29145-29150Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar), Grb-2 (7Pendergast A.M. Quilliam L.A. Cripe L.D. Bassing C.H. Dai Z. Li N. Batzer A. Rabun K.M. Der C.J. Schlessinger J. Gishizky M. Cell. 1993; 75: 175-185Abstract Full Text PDF PubMed Scopus (591) Google Scholar), p62Dok (8Carpino N. Wisniewski D. Strife A. Marshak D. Kobayashi R. Stillman B. Clarkson B. Cell. 1997; 88: 197-204Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar) and p39Crkl (9ten Hoeve J. Arlinghaus R.B. Guo J.Q. Heisterkamp N. Groffen J. Blood. 1994; 84: 1731-1736Crossref PubMed Google Scholar, 10Oda 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, 11Nichols G.L. Raines M.A. Vera J.C. Lacomis L. Tempst P. Golde D.W. Blood. 1994; 84: 2912-2918Crossref PubMed Google Scholar).The human Crkl protein has a high degree of homology with the adaptor protein Crk (12ten Hoeve J. Morris C. Heisterkamp N. Groffen J. Oncogene. 1993; 8: 2469-2474PubMed Google Scholar, 13Matsuda M. Tanaka S. Nagata S. Kojima A. Kurata T. Shibuya M. Mol. Cell. Biol. 1992; 12: 3482-3489Crossref PubMed Scopus (245) Google Scholar). Crkl consists of an SH2 and two SH3 domains in the absence of other functional motifs. It is ubiquitously expressed and tyrosine-phosphorylated abundantly during early embryogenesis, but tyrosine phosphorylation is very restricted in adult tissues (14de Jong R. Haataja L. Voncken J.W. Heisterkamp N. Groffen J. Oncogene. 1995; 11: 1469-1474PubMed Google Scholar). We and others have demonstrated that Crkl is constitutively tyrosine-phosphorylated in CML and ALL patient material containing an active Bcr/Abl protein (e.g. chronic and blast phase samples) but not in remission-stage samples or in several types of leukemia lacking Bcr/Abl (9ten Hoeve J. Arlinghaus R.B. Guo J.Q. Heisterkamp N. Groffen J. Blood. 1994; 84: 1731-1736Crossref PubMed Google Scholar, 10Oda 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, 11Nichols G.L. Raines M.A. Vera J.C. Lacomis L. Tempst P. Golde D.W. Blood. 1994; 84: 2912-2918Crossref PubMed Google Scholar). Moreover, it is also abnormally tyrosine-phosphorylated in leukemic samples of P190- and P210BCR/ABL transgenic mice (14de Jong R. Haataja L. Voncken J.W. Heisterkamp N. Groffen J. Oncogene. 1995; 11: 1469-1474PubMed Google Scholar). This indicates that Crkl is a signaling protein likely to be of significance in vivoto the development of Ph+ leukemia.The N-terminal SH3 domain of Crkl interacts specifically with proline-rich sites present in the proto-oncoprotein Abl and in Bcr/Abl (15ten Hoeve J. Kaartinen V. Fioretos T. Haataja L. Voncken J.-W. Heisterkamp N. Groffen J. Cancer Res. 1994; 54: 2563-2567PubMed Google Scholar, 16Senechal K. Halpern J. Sawyers C.L. J. Biol. Chem. 1996; 271: 23255-23261Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar), while the Crkl SH2 domain recognizes specific phosphotyrosine binding sites in its target proteins. We and others have identified p120Cbl as a 120-kDa tyrosine-phosphorylated protein, that specifically interacts with the Crkl SH2 domain in cells expressing the Bcr/Abl protein, including Ph+ patient material (5de 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, 17Sattler M. Salgia R. Okuda K. Uemura N. Durstin M.A. Pisick E. Xu G. Li J.-L. Prasad K.V. Griffin J.D. Oncogene. 1996; 12: 839-846PubMed Google Scholar). Furthermore, Crkl is also associated through its SH2 domain with phosphorylated cytoskeletal proteins paxillin (6Salgia R. Uemura N. Okuda K. Li J.-L. Pisick E. Sattler M. de Jong R. Druker B. Heisterkamp N. Chen L.B. Groffen J. Griffin J.D. J. Biol. Chem. 1995; 270: 29145-29150Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar) and Crk-associated substrate p130Cas (4Salgia R. Pisick E. Sattler M. Li J.-L. Uemura N. Wong W.-K. Burky S.A. Hirai H. Chen L.B. Griffin J.D. J. Biol. Chem. 1996; 271: 25198-25203Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar) in Bcr/Abl-transformed cell lines and in Ph+ patient samples. Previously, analogous interactions have been described for family members Crk and v-Crk, which can form stable complexes with phosphorylated p130Cas and paxillin through their SH2 domains (18Matsuda M. Mayer B.J. Hanafusa H. Mol. Cell. Biol. 1991; 11: 1607-1613Crossref PubMed Scopus (94) Google Scholar, 19Birge R.B. Fajardo J.E. Mayer B.J. Hanafusa H. J. Biol. Chem. 1992; 267: 10588-10595Abstract Full Text PDF PubMed Google Scholar, 20Birge R.B. Fajardo J.E. Reichman C. Shoelson S.E. Songyang Z. Cantley L.C. Hanafusa H. Mol. Cell. Biol. 1993; 13: 4648-4656Crossref PubMed Scopus (247) Google Scholar, 21Sakai 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, 22Vuori K. Hirai H. Aizawa S. Ruoslahti E. Mol. Cell. Biol. 1996; 16: 2606-2613Crossref PubMed Google Scholar, 23Petruzelli L. Takami M. Herrera R. J. Biol. Chem. 1996; 271: 7796-7801Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Both paxillin and p130Cas are normally tyrosine-phosphorylated after integrin-mediated cell adhesion, are localized to focal adhesions, and are constitutively phosphorylated in v-src- and v-crk-transformed cells (18Matsuda M. Mayer B.J. Hanafusa H. Mol. Cell. Biol. 1991; 11: 1607-1613Crossref PubMed Scopus (94) Google Scholar, 19Birge R.B. Fajardo J.E. Mayer B.J. Hanafusa H. J. Biol. Chem. 1992; 267: 10588-10595Abstract Full Text PDF PubMed Google Scholar, 20Birge R.B. Fajardo J.E. Reichman C. Shoelson S.E. Songyang Z. Cantley L.C. Hanafusa H. Mol. Cell. Biol. 1993; 13: 4648-4656Crossref PubMed Scopus (247) Google Scholar, 21Sakai 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, 22Vuori K. Hirai H. Aizawa S. Ruoslahti E. Mol. Cell. Biol. 1996; 16: 2606-2613Crossref PubMed Google Scholar, 23Petruzelli L. Takami M. Herrera R. J. Biol. Chem. 1996; 271: 7796-7801Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 24Turner C.E. Bioassays. 1994; 16: 47-52Crossref PubMed Scopus (148) Google Scholar, 25Harte M.T. Hildebrand J.D. Burnham M.R. Bouton A.H. Parsons J.T. J. Biol. Chem. 1996; 271: 13649-13655Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar). Thus, Crkl might link different signaling proteins to Bcr/Abl through its SH2 and SH3 domains. In addition, these data implicate a role for cytoskeleton reorganization and integrin-mediated signaling pathways in the development of Ph+ leukemia, which correlates with the aberrant cell adhesion properties of Ph+ cells (26Bazzoni G. Carlesso N. Griffin J. Hemler M. J. Clin. Invest. 1996; 98: 521-528Crossref PubMed Scopus (109) Google Scholar) (reviewed in Verfaille et al. (27Verfaille C.M. Hurley R. Lundell B.I. Zhao C. Bhatia R. Acta Haematol. 1997; 97: 40-52Crossref PubMed Scopus (71) Google Scholar)).In studying leukemic tissues isolated from transgenic mice expressing the transforming P190Bcr/Abl protein, we noted the presence of a group of 110-kDa tyrosine-phosphorylated proteins, collectively designated as pp110, that associated with the Crkl SH2 domain in vitro. We demonstrate here that pp110 is Hef1/Cas-L, a p130Cas-related protein which is inducibly phosphorylated on tyrosine after β1-integrin ligation in lymphoid cells. We show that Hef1 is hyperphosphorylated in leukemia/lymphomas induced by P190Bcr/Abl in transgenic mice, and, similar to p120Cbl, is in complex with Bcr/Abl-bound Crkl. These findings lend support to an involvement of integrin-mediated signal transduction pathways during Bcr/Abl-induced leukemogenesis.DISCUSSIONWe previously reported that Crkl binds the tyrosine-phosphorylated proto-oncoprotein CBL in a CML cell line, and this corresponds to a 120-kDa protein also seen in Ph+ patient material (5de 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). In the current study, we have demonstrated the presence of hyperphosphorylated proteins of approximately 110 kDa bound to the Crkl SH2 domain in lymphoma/leukemia tissue derived from P190BCR/ABL transgenic mice, in addition to the p120Cbl. The pp110 was identified as Hef1/Cas-L, a p130Cas-related protein. Interestingly, although some pp110 was also detectable in P210Bcr/Abl-expressing patient samples, preliminary data suggest that expression of Hef1 and its tyrosine phosphorylation might be more characteristic for P190Bcr/Abl-induced leukemia/lymphoma since pp110 was also prominently present in Ph+ blood samples of ALL patients (data not shown). This likely reflects the hematopoietic cell lineage transformed by the P190Bcr/Abl protein, since expression of Hef1/Cas-L proteins is restricted to isolated peripheral lymphocytes and lymphocyte cell lines (32Minegishi M. Tachibana K. Sato T. Iwata S. Nojima Y. Morimoto C. J. Exp. Med. 1996; 184: 1365-1375Crossref PubMed Scopus (147) Google Scholar). In concordance with this, the CML cell line K562 did not contain detectable (phosphorylated) Hef1. Taken together, the Crkl-binding protein Hef1 might represent a signaling mediator of Bcr/Abl in Ph-positive cells of the lymphoid lineage.It has been reported that p130Cas is phosphorylated in Bcr/Abl-expressing cells and is associated with Crkl in CML patient samples (4Salgia R. Pisick E. Sattler M. Li J.-L. Uemura N. Wong W.-K. Burky S.A. Hirai H. Chen L.B. Griffin J.D. J. Biol. Chem. 1996; 271: 25198-25203Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). In addition, the product of the Crkl-related gene Crk has been shown to interact with p130Cas: in v-Crk-transformed cells, v-Crk is associated with constitutively phosphorylated p130Cas (18Matsuda M. Mayer B.J. Hanafusa H. Mol. Cell. Biol. 1991; 11: 1607-1613Crossref PubMed Scopus (94) Google Scholar, 19Birge R.B. Fajardo J.E. Mayer B.J. Hanafusa H. J. Biol. Chem. 1992; 267: 10588-10595Abstract Full Text PDF PubMed Google Scholar, 21Sakai 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), and integrin stimulation induces the phosphorylation of p130Cas and its association with c-Crk (22Vuori K. Hirai H. Aizawa S. Ruoslahti E. Mol. Cell. Biol. 1996; 16: 2606-2613Crossref PubMed Google Scholar, 23Petruzelli L. Takami M. Herrera R. J. Biol. Chem. 1996; 271: 7796-7801Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Therefore, we investigated whether p130Cas was also hyperphosphorylated in the leukemic tissues of P190BCR/ABL transgenic mice. In contrast to Hef1, p130Cas was not detectably phosphorylated on tyrosine or complexed with Crkl in these samples. It has been suggested that these structurally similar proteins have a different function which depends on the differentiation state of the cell, since Cas appears to be phosphorylated only in more terminally differentiated B cells after B cell receptor or β1-integrin stimulation (35Manie S.N. Beck A.R.P. Astier A. Law S.F. Canty T. Hirai H. Druker B.J. Avraham H. Haghayeghi N. Sattler M. Salgia R. Griffin J.D. Golemis E.A. Freedman A.S. J. Biol. Chem. 1997; 272: 4230-4236Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). This concept would correlate with the observed lack of Cas tyrosine phosphorylation and the fact that preB cells or their progenitors are the target cell type transformed by P190Bcr/Abl in transgenic mice (2Voncken J.W. Griffiths S. Greaves M.F. Pattengale P.K. Heisterkamp N. Groffen J. Cancer Res. 1992; 52: 4534-4539PubMed Google Scholar).Hef1 was originally discovered as a p105 protein that becomes tyrosine-phosphorylated upon β1-integrin ligation (36Nojima Y. Rothstein D.M. Sugati K. Schlossman S.F. Morimoto C. J. Exp. Med. 1992; 175: 1045-1053Crossref PubMed Scopus (142) Google Scholar) and was subsequently cloned from a cDNA library expressed in yeast (31Law S.F. Estojak J. Wang B. Mysliwiec T. Kruh G. Golemis E.A. Mol. Cell. Biol. 1996; 16: 3327-3337Crossref PubMed Scopus (221) Google Scholar) and, independently, using the cross-reacting p130Cas mAb from Transduction Laboratory (32Minegishi M. Tachibana K. Sato T. Iwata S. Nojima Y. Morimoto C. J. Exp. Med. 1996; 184: 1365-1375Crossref PubMed Scopus (147) Google Scholar). Hef1 and p130Cas share a high overall sequence similarity and contain several similar protein structures. The SH3 domains of Hef1 and Cas recognize proline-rich sequences in the focal adhesion kinase FAK and the related RAFTK (31Law S.F. Estojak J. Wang B. Mysliwiec T. Kruh G. Golemis E.A. Mol. Cell. Biol. 1996; 16: 3327-3337Crossref PubMed Scopus (221) Google Scholar, 32Minegishi M. Tachibana K. Sato T. Iwata S. Nojima Y. Morimoto C. J. Exp. Med. 1996; 184: 1365-1375Crossref PubMed Scopus (147) Google Scholar, 35Manie S.N. Beck A.R.P. Astier A. Law S.F. Canty T. Hirai H. Druker B.J. Avraham H. Haghayeghi N. Sattler M. Salgia R. Griffin J.D. Golemis E.A. Freedman A.S. J. Biol. Chem. 1997; 272: 4230-4236Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 37Polte T.R. Hanks S.K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10678-10682Crossref PubMed Scopus (386) Google Scholar). Similar as in p130Cas, repeated YXXP motifs in Hef1 conform to the consensus SH2 binding site of the Crk family (38Songyang Z. Shoelson S.E. Chaudhuri M. Gish G. Pawson T. Haser W.G. King F. Roberts T. Ratnofsky R. Lechleider R.J. Neel B.G. Birge R.B. Fajardo J.E. Chou M.M. Hanafusa H. Schaffhausen B. Cantley L.C. Cell. 1993; 72: 767-778Abstract Full Text PDF PubMed Scopus (2373) Google Scholar) are located in the central domain of Hef1. Analogous to the above mentioned Crk-p130Cas interaction, an increased association between tyrosine-phosphorylated Hef1 and Crkl was recently demonstrated after B cell receptor or β1-integrin stimulation in several different types of B cells (35Manie S.N. Beck A.R.P. Astier A. Law S.F. Canty T. Hirai H. Druker B.J. Avraham H. Haghayeghi N. Sattler M. Salgia R. Griffin J.D. Golemis E.A. Freedman A.S. J. Biol. Chem. 1997; 272: 4230-4236Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Our current results show that the association between Crkl and Hef1 is mediated by the Crkl SH2 domain and phosphotyrosine residues in Hef1, most likely involving the YXXP motifs.Besides the prominently phosphorylated Hef1, we also observed the phosphorylation of the proto-oncoprotein p120Cbl and its in vivo association with Crkl and P190Bcr/Abl in P190Bcr/Abl-induced leukemia/lymphoma. The association between Crkl and Hef1 or Cbl in leukemic tissue from P190BCR/ABL transgenic mice supports a model in which one of the important functions of Crkl in Ph+ leukemia is to act as a molecular link between Bcr/Abl and downstream signaling proteins. In this model, stable complexes exist in Bcr/Abl-induced leukemia, involving Bcr/Abl, Crkl, and either one of the Crkl SH2 binding partners Hef1, p130Cas, p120Cbl, or paxillin, depending on the Bcr/Abl protein and the transformed cell type. It is expected that individual Crkl proteins can only complex with one of the SH2-binding partners on an either/or basis. Crkl is constitutively bound to Bcr/Abl through its SH3 domain and tethers its tyrosine-phosphorylated SH2-domain targets to Bcr/Abl, which leads to a significant increase in their degree of tyrosine-phosphorylation because of the deregulated kinase activity of Bcr/Abl. Although this model favors trimolecular complexes, different Crkl SH2 targets might assemble in multimer complexes since Bcr/Abl contains an oligomerization domain (39McWhirter J.R. Galasso D.L. Wang J.Y.J. Mol. Cell. Biol. 1993; 13: 7587-7595Crossref PubMed Scopus (371) Google Scholar). The initial association of Hef1 and Cbl with the Crkl SH2 domain in P190Bcr/Abl-induced leukemia requires a low level transient tyrosine-phosphorylation, which is likely provided by normal signaling events such as stimulation by growth factors or integrin signaling, after which tyrosine-phosphorylation would be constitutively switched on, leading to a continuous activation of the signaling pathways in which Hef1 and Cbl partake.An analogous model for successive phosphorylation has been proposed for the c-Abl-associated Crk, which through its SH2 domain binds to phosphorylated Abl kinase substrates and enhances their processive phosphorylation in vitro and in cultured cells (40Mayer B.J. Baltimore D. Mol. Cell. Biol. 1994; 14: 2883-2894Crossref PubMed Scopus (158) Google Scholar, 41Mayer B.J. Hiray H. Sakai R. Curr. Biol. 1995; 5: 296-305Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). In addition to the indirect association mediated by Crkl, a direct interaction between Bcr/Abl and Hef1 might also occur, as was previously suggested for Cbl (17Sattler M. Salgia R. Okuda K. Uemura N. Durstin M.A. Pisick E. Xu G. Li J.-L. Prasad K.V. Griffin J.D. Oncogene. 1996; 12: 839-846PubMed Google Scholar) and which might be the basis for the observed association between Hef1 and v-Abl (31Law S.F. Estojak J. Wang B. Mysliwiec T. Kruh G. Golemis E.A. Mol. Cell. Biol. 1996; 16: 3327-3337Crossref PubMed Scopus (221) Google Scholar). The Abl SH2 domain can bind directly in vitro to Hef1 in a Far Western assay, which is consistent with the resemblance between the Crk family SH2 and ABL SH2 domain binding preferences (38Songyang Z. Shoelson S.E. Chaudhuri M. Gish G. Pawson T. Haser W.G. King F. Roberts T. Ratnofsky R. Lechleider R.J. Neel B.G. Birge R.B. Fajardo J.E. Chou M.M. Hanafusa H. Schaffhausen B. Cantley L.C. Cell. 1993; 72: 767-778Abstract Full Text PDF PubMed Scopus (2373) Google Scholar). This direct interaction could enhance the stability of the aforementioned complexes.Our identification of another protein involved in integrin-mediated signaling that is connected with Bcr/Abl in vivo, substantiates the suggestion that Bcr/Abl transformation utilizes the intracellular components of the integrin signal transduction pathway. Previously, we reported that paxillin, a cytoskeletal protein involved in integrin signaling and cell adhesion, is tyrosine-phosphorylated and associated with the Crkl-SH2 domain in Bcr/Abl-transformed cells and in a CML cell line (6Salgia R. Uemura N. Okuda K. Li J.-L. Pisick E. Sattler M. de Jong R. Druker B. Heisterkamp N. Chen L.B. Groffen J. Griffin J.D. J. Biol. Chem. 1995; 270: 29145-29150Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Furthermore, in Bcr/Abl-expressing cells, Crkl is associated through its SH2 domain with phosphorylated p130Cas (4Salgia R. Pisick E. Sattler M. Li J.-L. Uemura N. Wong W.-K. Burky S.A. Hirai H. Chen L.B. Griffin J.D. J. Biol. Chem. 1996; 271: 25198-25203Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar), which is also implicated in integrin signaling. These data are consistent with the localization of Bcr/Abl to the actin cytoskeleton (42McWhirter J.R. Wang J.Y.J. Mol. Cell. Biol. 1991; 11: 1553-1565Crossref PubMed Google Scholar), and together with the observed increase in tyrosine phosphorylation of other focal adhesion proteins in P210Bcr/Abl-expressing myeloid cell lines (43Salgia R. Brunkhorst B. Pisick E. Li J.-L. Lo S.H. Chen L.B. Griffin J.D. Oncogene. 1995; 11: 1149-1155PubMed Google Scholar), this suggests that actin cytoskeleton rearrangement might contribute to the aberrant adhesion properties of Ph+ leukemic cells. CML progenitor cells from patients are clearly abnormal in that they have defects in their adherence to bone marrow stroma (44Gordon M.Y. Dowding C.R. Riley G.P. Goldman J.M. Greaves M.F. Nature. 1987; 328: 342-344Crossref PubMed Scopus (396) Google Scholar). The present finding that P190Bcr/Abl phosphorylates Hef1, a protein normally involved in β1- integrin signaling, provides a mechanism for how Bcr/Abl can modulate CML progenitor cell adherence to the stroma. Several experimental mouse models have been developed to investigate the oncogenic action of Bcr/Abl, which is causative of the development of chronic myeloid leukemia (CML) 1The abbreviations used are: CML, chronic myeloid leukemia; ALL, acute lymphoblastic leukemia; GST, glutathione S-transferase; mAb, monoclonal antibody; SH2, Src homology region-2; SH3, Src homology region-3. 1The abbreviations used are: CML, chronic myeloid leukemia; ALL, acute lymphoblastic leukemia; GST, glutathione S-transferase; mAb, monoclonal antibody; SH2, Src homology region-2; SH3, Src homology region-3. and Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL), in vivo. Transgenic P190BCR/ABL mice reproducibly develop lymphoblastic leukemia/lymphoma involving cells of pre-B-cell origin or their progenitors (1Heisterkamp N. Jenster G. ten Hoeve J. Zovich D. Pattengale P.K. Groffen J. Nature. 1990; 344: 251-254Crossref PubMed Scopus (590) Google Scholar, 2Voncken J.W. Griffiths S. Greaves M.F. Pattengale P.K. Heisterkamp N. Groffen J. Cancer Res. 1992; 52: 4534-4539PubMed Google Scholar). Established transgenic mice strains expressing P190Bcr/Abl provide an unlimited source and unique opportunity for studying the signal transduction pathways affected by the Bcr/Abl oncoprotein in vivo. The tyrosine kinase activity located in the Abl segment of Bcr/Abl is dramatically increased (3Konopka J.B. Watanabe S.M. Witte O.N. Cell. 1984; 37: 1035-1042Abstract Full Text PDF PubMed Scopus (674) Google Scholar), and this is critical for its transforming capacity. Although it remains unclear which intracellular signaling pathways are crucial to the in vivo oncogenic activity of Bcr/Abl, numerous signaling proteins have been implicated by studies involving patient-derived cell lines or stably transfected cells transformed by Bcr/Abl. However, only few signaling proteins are tyrosine-phosphorylated by Bcr/Abl or in complex with Bcr/Abl in leukemic cells isolated directly from Ph+ patients, indicating the importance of studying the molecular mechanisms behind Bcr/Abl-induced leukemogenesis in an in vivo context. Signaling proteins shown to be affected by Bcr/Abl include p130Cas (4Salgia R. Pisick E. Sattler M. Li J.-L. Uemura N. Wong W.-K. Burky S.A. Hirai H. Chen L.B. Griffin J.D. J. Biol. Chem. 1996; 271: 25198-25203Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar), p120CBL (5de 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), p68paxillin (6Salgia R. Uemura N. Okuda K. Li J.-L. Pisick E. Sattler M. de Jong R. Druker B. Heisterkamp N. Chen L.B. Groffen J. Griffin J.D. J. Biol. Chem. 1995; 270: 29145-29150Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar), Grb-2 (7Pendergast A.M. Quilliam L.A. Cripe L.D. Bassing C.H. Dai Z. Li N. Batzer A. Rabun K.M. Der C.J. Schlessinger J. Gishizky M. Cell. 1993; 75: 175-185Abstract Full Text PDF PubMed Scopus (591) Google Scholar), p62Dok (8Carpino N. Wisniewski D. Strife A. Marshak D. Kobayashi R. Stillman B. Clarkson B. Cell. 1997; 88: 197-204Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar) and p39Crkl (9ten Hoeve J. Arlinghaus R.B. Guo J.Q. Heisterkamp N. Groffen J. Blood. 1994; 84: 1731-1736Crossref PubMed Google Scholar, 10Oda 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, 11Nichols G.L. Raines M.A. Vera J.C. Lacomis L. Tempst P. Golde D.W. Blood. 1994; 84: 2912-2918Crossref PubMed Google Scholar). The human Crkl protein has a high degree of homology with the adaptor protein Crk (12ten Hoeve J. Morris C. Heisterkamp N. Groffen J. Oncogene. 1993; 8: 2469-2474PubMed Google Scholar, 13Matsuda M. Tanaka S. Nagata S. Kojima A. Kurata T. Shibuya M. Mol. Cell. Biol. 1992; 12: 3482-3489Crossref PubMed Scopus (245) Google Scholar). Crkl consists of an SH2 and two SH3 domains in the absence of other functional motifs. It is ubiquitously expressed and tyrosine-p
Referência(s)