Abstract
The Philadelphia chromosome translocation generates a chimeric oncogene, BCR/ABL, which causes chronic myelogenous leukemia (CML). In primary neutrophils from patients with CML, the major novel tyrosine-phosphorylated protein is CRKL, an SH2-SH3-SH3 linker protein which has an overall homology of 60% to CRK, the human homologue of the v-crk oncogene product. Anti-CRKL immunoprecipitates from CML cells, but not normal cells, were found to contain p210BCR/ABL and c-ABL. Several other phosphoproteins were also detected in anti-CRKL immunoprecipitates, one of which has been identified as paxillin, a 68-kDa focal adhesion protein which we have previously shown to be phosphorylated by p210BCR/ABL. Using GST-CRKL fusion proteins, the SH3 domains of CRKL were found to bind c-ABL and p210BCR/ABL, while the SH2 domain of CRKL bound to paxillin, suggesting that CRKL could physically link p210BCR/ABL to paxillin. Paxillin contains three tyrosines in Tyr-X-X-Pro (Y-X-X-P) motifs consistent with amino acid sequences predicted to be optimal for binding to the CRKL-SH2 domain (at positions Tyr-31, Tyr-118, and Tyr-181). Each of these tyrosine residues was mutated to a phenylalanine residue, and in vitro binding assays indicated that paxillin tyrosines at positions 31 and 118, but not 181, are likely to be involved in CRKL-SH2 binding. These results suggest that the p210BCR/ABL oncogene may be physically linked to the focal adhesion-associated protein paxillin in hematopoietic cells by CRKL. This interaction could contribute to the known adhesive defects of CML cells.
Highlights
Chronic myelogenous leukemia (CML)1 is characterized by the production of an active tyrosine kinase fusion protein, p210BCR/ABL
In cell lines and primary cells, p210BCR/ABL translocates to the cytoskeleton [1] and causes the tyrosine phosphorylation of several cellular proteins, including the SH2
In cell lines either derived from patients with advanced phase CML or generated by transfecting the BCR/ABL oncogene, there are a number of cellular proteins which are constitutively tyrosine-phosphorylated, such as p120rasGAP [4], p120c-CBL [5], p52SHC [6], p93FES [7], p95VAV [8], p68paxillin [9], and p72SHPTP2 [10]
Summary
Cell Lines and Cell Culture—The 32Dcl cell line was obtained from Dr Joel Greenberger (University of Pittsburgh) and cultured in RPMI 1640 containing 10% fetal calf serum and 15% WEHI-condition media (as a source of IL-3). 32Dcl expressing p210BCR/ABL clones were generated using previously described methods [18]. Precipitations were performed with 1–10 g of fusion protein on glutathione beads using the lysate from 5–10 ϫ 106 cells as described [9]. The full-length wild type human paxillin (amino acids 1–527) and tyrosine mutant paxillin were expressed in the vector pGEX-2TK (Pharmacia Biotech Inc.), and paxillin fusion proteins were produced. For the CRKL-SH2 fusion protein binding experiment, 1 g of wild type or mutant GST-paxillin fusion protein on glutathione beads was phosphorylated for 30 min at 25 °C in vitro with 2 units of v-Abl kinase (Oncogene Science) in 100 l of kinase reaction buffer (containing 1 mM MgCl2, 1 mM MnCl2, 0.1 mM ATP in 20 mM HEPES solution, pH 7.2). The precipitated samples were subjected to SDS-PAGE, electrotransferred to Immobilon-polyvinylidene difluoride membranes, and immunoblotted with anti-paxillin antibody
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