Myosin VI SI isoform is phosphorylated by c-Src kinase. (A) Alignment of the tail domain sequences of the myosin VI SI and myosin VI NI isoforms. The DYD motif comprises part of the SI (9 aa) and an adjacent aspartate (D1113) and tyrosine (Y1114) residue (red, aa 1,113–1,123). (B, top) Prediction of tyrosine phosphorylation sites in the region 1,100–1,150 aa in the myosin VI SI (left) and myosin VI NI (right) isoforms. Highlighted in red is the Y1114 residue that displays a higher phosphorylation score in myosin VI SI than in myosin VI NI (0.912 vs. 0.726). (bottom) Prediction of kinase-specific phosphorylation sites in myosin VI SI (left) and myosin VI NI (right) isoforms. Highlighted in red is the Y1114 residue predicted to be phosphorylated by c-Src protein kinase in myosin VI SI. (C) Schematic comparison between GFP-MyoVI-SIfull and GFP-MyoVI-SIfullΔDYD (see Fig. 3 for colored references) with the deleted DYD motif (aa 1,113–1,115) in red. (D) Immunoprecipitation of GFP-MyoVI from cells coexpressing GFP-MyoVI-SIfull, GFP-MyoVI-NIfull, or GFP-MyoVI-SIfullΔDYD with c-SrcY527F-GFP. (top) Western blotting using an anti–myosin VI antibody. Arrows indicate GFP-MyoVI. (middle) Phosphorylation of immunoprecipitated GFP-MyoVI detected by Western blotting using antiphosphotyrosine (pY) antibody. (bottom) c-SrcY527F-GFP expression detected by Western blotting using a specific antiphospho-Src antibody (pY416-Src, 84 kD). (E) Quantification of phosphorylated GFP-MyoVI expressed as a percentage of phosphorylated GFP-MyoVI-SIfull (n = 3). IP, immunoprecipitation; WB, Western blot. Error bars are means ± SEM. **, P < 0.01; ***, P < 0.001.