ROCKII Ser ¹³⁶⁶ phosphorylation reflects the activation status

ROCK (Rho-associated protein kinase), a downstream effector of RhoA, plays an important role in many cellular processes. Accumulating evidence has shown the involvement of ROCK activation in the pathogenesis of many diseases. However, a reagent capable of detecting ROCK activation directly is lacking. In the present study, we show autophosphorylation of ROCKII in an in vitro kinase reaction. The phosphorylation sites were identified by MS, and the major phosphorylation site was found to be at the highly conserved residue Ser ¹³⁶⁶. A phospho-specific antibody was generated that can specifically recognize ROCKII Ser ¹³⁶⁶ phosphorylation. We found that the extent of Ser ¹³⁶⁶phosphorylation of endogenous ROCKII is correlated with that of myosin light chain phosphorylation in cells in response to RhoA stimulation, showing that Ser ¹³⁶⁶ phosphorylation reflects its kinase activity. In addition, ROCKII Ser ¹³⁶⁶ phosphorylation could be detected in human breast tumours by immunohistochemical staining. The present study provides a new approach for revealing the ROCKII activation status by probing ROCKII Ser ¹³⁶⁶phosphorylation directly in cells or tissues.