Y722 phosphorylation of ROCKII is associated with cell adhesion. (A) D2 cells in suspension and plated onto FN-coated coverslips for 0.5 and 2 h were fixed and stained with anti-pY722ROCKII antibody, which had been preincubated with or without 1 μg/ml of Y722 peptide or pY722 peptide. Insets show enlargements of the boxed ares. Bar, 20 μm. (B) D2 cells were transfected with the pEGFP and wild-type or Y722F myc-ROCKII (1:5 ratio). Transfected cells were plated on FN-coated dishes and monitored by time-lapse microscopy for 60 min after plating. Arrows indicate the GFP-positive transfected cells. Video 3 corresponds to this figure. Bars, 20 μm. (C) Transfected cells, as described in B, were preincubated with or without 2 mM MnCl2 or 20 μM Y27632 for 30 min and then assayed for their adhesion to FN-coated plates within 30 min as described in Materials and methods. Data are represented as mean ± SD (**, P < 0.05). (D) NIH3T3 cells were transfected with the expression vector of GFP-paxillin. After serum starvation, cells were treated with 10 μM nocodazole for 1 h and fixed for immunofluorescence staining of pY722-ROCKII. Bar, 20 μm.
