title: ROCKII Ser1366 phosphorylation reflects the activation status.
abstract: 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 Ser1366. A phospho-specific antibody was generated that can specifically recognize ROCKII Ser1366 phosphorylation. We found that the extent of Ser1366 phosphorylation of endogenous ROCKII is correlated with that of myosin light chain phosphorylation in cells in response to RhoA stimulation, showing that Ser1366 phosphorylation reflects its kinase activity. In addition, ROCKII Ser1366 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 Ser1366 phosphorylation directly in cells or tissues.
authors.affiliation: Institute of Biochemistry and Molecular Biology, National Yang-Ming University, No. 155, Sec. 2, Linong Street, Taipei, Taiwan, ROC.
authors.forename: Hsiang-Hao
authors.initials: HH
authors.lastname: Chuang
authors.forename: Chih-Hsuan
authors.initials: CH
authors.lastname: Yang
authors.forename: Yeou-Guang
authors.initials: YG
authors.lastname: Tsay
authors.forename: Chih-Yi
authors.initials: CY
authors.lastname: Hsu
authors.forename: Ling-Ming
authors.initials: LM
authors.lastname: Tseng
authors.forename: Zee-Fen
authors.initials: ZF
authors.lastname: Chang
authors.forename: Hsiao-Hui
authors.initials: HH
authors.lastname: Lee
mesh_terms: D000577:Amides / Q000494:pharmacology; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D000906:Antibodies / Q000302:isolation & purification; D015153:Blotting, Western; D004789:Enzyme Activation; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011485:Protein Binding; D011725:Pyridines / Q000494:pharmacology; D011817:Rabbits; D012694:Serine / Q000378:metabolism*; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
chemical_list: D000577:Amides; D000906:Antibodies; D011725:Pyridines; C066737:RHOA protein, human; C108830:Y 27632; D012694:Serine; C515852:ROCK2 protein, human; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein