FIGURE 2.

Bcr-Abl-induced tyrosine phosphorylation of the WAVE2 complex is inhibited in mitotic cells. A, Abi1-mediated tyrosine phosphorylation of WAVE2 by Bcr-Abl. Upper panel, WAVE2 was immunoprecipitated (IP) from Ba/F3 cells and the Ba/F3 cells transformed by either wild type p185^Bcr-Abl (p185^wt) or mutant p185^ΔSH3ΔC (ΔSH3ΔC) defective in binding to Abi1. The immunoprecipitates were analyzed by Western blotting (WB) for WAVE2 and phosphotyrosine (pTyr) as indicated. Lower panel, the lysates of Ba/F3 and p185^wt cells expressing wild type GFP-Abi1 (BaF3 and p185wt), as well as p185^wt cells expressing mutant GFP-Abi1PPLL (PPLL) defective in binding to Bcr-Abl, were immunoprecipitated by GFP antibody, and the immunoprecipitates were analyzed by Western blotting for phosphotyrosine. The same membrane was then stripped and reprobed with anti-WAVE2 antibody. B, Abi1 forms a complex with other components of WAVE2 complex in both interphase and mitotic cells. The p185^wt cells were untreated (asynchronous cells (asy)) or synchronized at M-phase (M) as described under “Experimental Procedures.” The cell lysates were immunoprecipitated with control IgG (Ctrl) or anti-Abi1 antibody (Abi1). The immunoprecipitates were subjected to Western blot analysis using the indicated antibodies. C, Bcr-Abl-induced tyrosine phosphorylation of Abi1 and WAVE2 is inhibited during mitosis. The lysates from asynchronous or M-phase-synchronized cells were immunoprecipitated by control IgG, anti-Abi1, or anti-WAVE2 antibodies as indicated. The immunoprecipitates were subjected to Western blot analysis using the antibodies for Abi1, WAVE2, or tyrosine-phosphorylated proteins. D, profile of protein tyrosine phosphorylation in p185^wt cells treated with or without nocodazole. The p185^wt cells were treated with and without 200 ng/ml nocodazole in the presence or absence of 1 μm imatinib (Ima) for 12 h. The lysates containing 80 μg of total proteins were separated on 8% SDS-PAGE and analyzed by Western blotting using anti-phosphotyrosine antibody. E, the tyrosine kinase activity of Bcr-Abl is not affected during mitosis. The p185^wt cells were treated with or without 200 ng/ml nocodazole for 12 h, and cell lysates were immunoprecipitated by control IgG, anti-Abl antibody (left panel), or anti-Crkl antibody (right panel). The immunoprecipitates were separated on SDS-PAGE and subjected to Western blot analysis using the indicated antibodies.