Disrupting Y722 phosphorylation of ROCKII augments RhoA-dependent ROCK activation in cells. (A and B) NIH3T3 cells were cotransfected with pSRE-Luc, pCMV-HRC, and wild type, Y722F myc-ROCKII, or pcDNA3. After transfection for 24 h, cells were serum starved and treated with or without 10 μM of nocodazole for 4 h. (A) Endogenous RhoA activities in the cells were measured by GST-RBD pulldown assay. A representative Western blot for the assay is shown on the left. Relative endogenous RhoA activity from three independent experiments is shown on the right. (B) Transfected cells were pretreated with or without 20 μM of Y27632 before nocodazole stimulation. The reporter activity was determined and expressed as induction folds relative to the nontreated cells. Values are mean ± SD of three independent experiments. (*, P < 0.1). (C) NIH3T3 cells stably expressing wild-type or Y722F myc-ROCKII were selected and these cells were serum starved and treated with or without 10 μM of nocodazole for 1 h, fixed, and stained with rhodamine-phalloidin and HOECHST 33342. Bar, 20 μm. (D) A parallel set of cells were harvested for Western blot analysis. The relative ratio of phospho-MLC to total MLC is shown below. (E) Cells were trypsinized and replated to culture dishes and monitored for 5 h by time-lapse microscopy. Areas of spreading were measured using MetaMorph software and expressed as mean ± SD. (F) A parallel set of cells were treated with Y27632 for the spreading assay. Videos 1 and 2 correspond to E and F, respectively.