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 p210(BCR/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 p120(CBL). Here we demonstrate that another cellular protein linked to BCR/ABL through the CRKL-SH2 domain is p130(CAS). p130(CAS) 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, p130(CAS) was detected in focal adhesion-like structures, as was BCR/ABL. In normal cells, the focal adhesion proteins tensin, p125(FAK), and paxillin constitutively associated with p130(CAS). However, in BCR/ABL transformed cells, the interaction between p130(CAS) and tensin was disrupted, while the associations between p130(CAS), p125(FAK), and paxillin were unaffected. These results suggest that the BCR/ABL oncogene could alter the function of p130(CAS) in at least three ways: tyrosine phosphorylation, inducing constitutive binding of CRKL to a domain in p130(CAS) containing Tyr-X-X-Pro motifs (substrate domain), and disrupting the normal interaction of p130(CAS) 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.
Highlights
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)
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
Chronic 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, and generating the oncoprotein p210BCR/ABL [30]
Summary
Cell Lines, and Cell Culture—The murine myeloid cell line 32Dcl, murine pre-B cell line BaF3, human megakaryocytic cell line Mo7e, human T-cell line H9, human B-cell line Nalm, and CML cell lines BV-173, and K562 were maintained in culture as described previously [22]. The BCR/ABL expressing hematopoietic cell lines were generated by transfection with p210BCR/ABL or p190BCR/ABL cDNA and maintained as described previously [22]. Protein samples were analyzed by SDS-polyacrylamide gel electrophoresis and electrophoretically transferred and immunoblotted as described using a chemiluminescense technique [25]. Immunochemical detection of tyrosine-phosphorylated proteins in Western blotting utilized monoclonal antibody 4G10 (a generous gift from Dr Brian Druker, Oregon Health Science University, Portland, OR). GST fusion proteins of SH3 domain and substrate domain of p130CAS have been described [16]. Far Western Blotting—Immunoprecipitations were performed using anti-p130CAS mouse monoclonal antibody as described previously and separated on 7.5% SDS-PAGE gels [24]. The blots were washed and placed into anti-GST mouse monoclonal antibody (Santa Cruz Biotechnology) for 1 h. The membranes were processed per established methods using enhanced chemiluminescense technique (Amersham) [22]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
