Y722 Phosphorylation reduces the level of ROCKII binding to GTP-RhoA. (A) HEK293T cells expressing myc-ROCKII were treated with or without 100 μM pervanadate (PV) for 30 min. Equal amounts of cell lysates were incubated with the indicated amount of GTPγS-loaded GST-RhoA protein followed by glutathione-Sepharose beads pulldown. A representative Western blot probed by myc antibody of the pulldown samples is shown at the top. Quantification of amounts of myc-ROCKII in the pulldown materials relative to the total input from three independent experiments is shown at the bottom. The inset indicates the phosphorylation status of myc-ROCKII by 4G10 antibody. (B) Myc-ROCKII immunoprecipitates from pervanadate-treated cells were incubated with or without λ protein phosphatase at 30°C for 20 min before incubation with 2 μg of GTPγS-loaded GST-RhoA protein for 30 min. After extensive wash, GST-RhoA protein pulled down by myc-ROCKII protein beads was detected by Western blotting with anti-RhoA antibody (top). *, heavy chain of IgG. Relative amount of RhoA pulled down by myc-ROCKII beads from three independent experiments is also shown (bottom). (C) Wild-type or Y722F mutant of myc-ROCKII from pervanadate-treated cells were used for the GTPγS-GST-RhoA pulldown assay. (D) Y722F and Y722D myc-ROCKII from cells without pervanadate treatment were used for GTPγS-GST-RhoA pulldown assay. All data are represented as mean ± SD (*, P < 0.1; **, P < 0.01; n ≥ 3).