Tyrosine phosphorylation of ROCKII. (A) D2 cells were plated on hydrogel (HG) or FN-coated dishes in serum-containing medium for 2 h. Cells were treated with 50 μM pervanadate for 20 min and harvested for immunoprecipitation with anti-ROCKII (C-20) antibody or normal IgG. Western blots of the immunoprecipitates with anti-phosphotyrosine (4G10) antibody followed by reprobing with anti-ROCKII (H-85) antibody are shown. (B and C) HEK293T cells were transiently transfected with control vector or the expression plasmids of wild-type and various mutants of myc-ROCKII as indicated. Cells were treated with 100 μM pervanadate for 30 min before harvest for immunoprecipitation with anti-myc antibody or normal IgG. The immunoprecipitates were analyzed by Western blot as described in A. (D) Alignment of the vertebrate ROCKII sequence corresponding to the residues 712–732 of human ROCKII. Residues corresponding to Y722 of human ROCKII are indicated by asterisk and bold letters.