FIGURE 5.

Phosphorylation of Ser-216 in Abi1 inhibits Bcr-Abl-induced tyrosine phosphorylation and membrane translocation of WAVE2 complex and attenuates F-actin assembly. A, Ser-216 phosphorylation of Abi1 inhibits its tyrosine phosphorylation stimulated by Bcr-Abl. Cell lysates from asynchronous (Asy) and mitotic p185^wt cells (M) were immunoprecipitated (IP) by anti-Abi1 and anti-pSer-216 antibodies. The immunoprecipitates were analyzed by Western blotting (WB) using the indicated antibodies. B, phosphomimetic mutation of Ser-216 in Abi1 does not affect its complex formation with Hem1, Sra, or WAVE2. The lysates from the p185^wt cells expressing wild type GFP-Abi1 (wt) or GFP-Abi1S216D (SD) were immunoprecipitated with a control IgG or the anti-GFP antibody. The immunoprecipitates were subjected to Western blot analysis using the indicated antibodies. C, phosphomimetic mutation of the Ser-216 is sufficient to attenuate Bcr-Abl-induced tyrosine phosphorylation of Abi1. The p185^wt cells were transfected by vectors expressing wild type GFP-Abi1 (wt) or the mutant forms of GFP-Abi1 in which the Ser-216 was replaced by alanine (SA) or aspartic acid (SD). The cell lysates were immunoprecipitated by a control IgG (Ctrl) or an antibody against GFP. The immunoprecipitates were subjected to Western blot analysis using anti-phosphotyrosine (pTyr) or anti-GFP antibodies as indicated. D, expression of the phosphomimetic mutant Abi1 reduced Bcr-Abl-induced tyrosine phosphorylation of WAVE2. p185^wt cells expressing wild type GFP-Abi1 or GFP-Abi1 with the mutation of Ser-216 to aspartic acid were immunoprecipitated by a control IgG or anti-WAVE2 antibody (WAVE). The immunoprecipitates were subjected to Western blot analysis using the antibodies against WAVE2 and phosphotyrosine as indicated. E, phosphomimetic mutation of Ser-216 alters the subcellular distribution of Abi1 in p185^wt cells. The p185^wt cells expressing GFP, GFP-Abi1, and GFP-Abi1S216D (GFP-SD) were stained with DAPI. The GFP and GFP fusion proteins (green), as well as the nuclei (blue), were visualized, and images were captured by fluorescence microscopy. F, expression of Abi1S216D attenuates Bcr-Abl-stimulated actin polymerization. The p185^wt (p185 ctrl) and p185^wt cells expressing Abi1S216D (p185 S216D) were stained with Alexa-conjugated phalloidin and DAPI to visualize F-actin (red) and nuclei (blue), respectively. The stained cells were analyzed by fluorescence microscopy (upper panel). The cells showing F-actin-enriched spots were counted, and the percentage of these cells compared with total cells in the area was calculated and expressed as the average percentage of three randomly selected areas (lower panel). *, p < 0.01. The data are representative of three independent experiments.