CRKL Links p210BCR/ABL with Paxillin in Chronic Myelogenous Leukemia Cells

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.

Identification of Tyr-762 in the platelet-derived growth factor α-receptor as the binding site for Crk proteins

“…Subsequent studies have shown that overexpression of v-Crk or CrkI, but not CrkII, leads to transformation of cells (Matsuda et al, 1992). CrkL has been implicated in development of chronic myelogenous leukemia (Nichols et al, 1994;Oda et al, 1994;Salgia et al, 1995;ten Hoeve et al, 1994). The presence of both SH2 and SH3 domains in Crk proteins is of crucial importance for their function as adaptor molecules (Tanaka et al, 1993(Tanaka et al, , 1995.…”

Expression of the focal adhesion protein paxillin in lung cancer and its relation to cell motility

“…Expression of paxillin was found in the NSCLC cell lines; and in the SCLC cell lines, the expression of paxillin was lower as compared to NSCLC cell lines. As controls, CRKL (Salgia et al, 1995c) and b-actin were expressed in all cell lines at a consistent level. Western blot analysis was also used to determine the expression of paxillin and several other focal adhesion proteins in NSCLC and SCLC cell lines ( Figure 5).…”

Role of the adapter protein CRKL in signal transduction of normal hematopoietic and BCR/ABL-transformed cells

“…55,56 The CRK SH2 domain binds to pYXXP (phosphoTyr-X-XPro) motifs. 57 Similarly, the CRKL SH2 domain has recently been shown to bind pYXXP motifs in the focal adhesion protein paxillin 33 and the proto-oncoprotein CBL. 58 Also, the SH2 domain of CRKL has been found to bind to HEF1, or CAS.…”

“…81 BCR/ABL-transformed cells have an altered F-actin structure and have tyrosine phosphorylation of focal adhesion proteins such as tensin, talin, vinculin, RAFTK, p125 FAK , and paxillin. 28,33,86 CRKL is the key signaling molecule which links BCR/ABL to the focal adhesion protein paxillin. 33 Paxillin is a 68 kDa multi-functional protein with three YXXP motifs at the N-terminus and four LIM domains at the C-terminus.…”