ROCKII Ser 1366 phosphorylation reflects the activation status
Journal
Biochemical Journal
Journal Volume
443
Journal Issue
1
Pages
145-151
Date Issued
2012
Author(s)
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 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. ? The Authors Journal compilation ? 2012 Biochemical Society.
Subjects
Autophosphorylation; Marker; Rho-associated protein kinase (ROCK); RhoA
SDGs
Other Subjects
myosin light chain; phosphospecific antibody; protein kinase; Rho associated protein kinase II; Rho kinase; RhoA guanine nucleotide binding protein; serine; unclassified drug; 4 (1 aminoethyl) n (4 pyridyl)cyclohexanecarboxamide; amide; antibody; pyridine derivative; Rho kinase; RhoA guanine nucleotide binding protein; RHOA protein, human; ROCK2 protein, human; serine; animal cell; article; autophosphorylation; breast tumor; controlled study; enzyme activation; enzyme active site; enzyme activity; human; human cell; immunohistochemistry; in vitro study; nonhuman; priority journal; protein expression; protein localization; protein phosphorylation; amino acid sequence; amino acid substitution; animal; cell strain HEK293; drug antagonism; genetics; immunology; immunoprecipitation; isolation and purification; metabolism; molecular genetics; mouse; phosphorylation; protein binding; rabbit; site directed mutagenesis; Western blotting; Amides; Amino Acid Sequence; Amino Acid Substitution; Animals; Antibodies; Blotting, Western; Enzyme Activation; HEK293 Cells; Humans; Immunoprecipitation; Mice; Molecular Sequence Data; Mutagenesis, Site-Directed; Phosphorylation; Protein Binding; Pyridines; Rabbits; rho-Associated Kinases; rhoA GTP-Binding Protein; Serine
Type
journal article