The COOH terminus of Rho-kinase negatively regulates rho-kinase activity.
Rho-kinase is implicated in the phosphorylation of myosin light chain downstream of Rho, which is thought to induce smooth muscle contraction and stress fiber formation in non-muscle cells. Here, we examined the mode of action of inhibitors of Rho-kinase. The chemical compounds such as HA1077 and Y-32885 inhibited not only the Rho-kinase activity but also the activity of protein kinase N, one of the targets of Rho, but had less of an effect on the activity of myotonic dystrophy kinase-related Cdc42-binding kinase beta (MRCKbeta). The COOH-terminal portion of Rho-kinase containing Rho-binding (RB) and pleckstrin homology (PH) domains (RB/PH (TT)), in which point mutations were introduced to abolish the Rho binding activity, interacted with Rho-kinase and thereby inhibited the Rho-kinase activity, whereas RB/PH (TT) had no effect on the activity of protein kinase N or MRCKbeta, suggesting that the COOH-terminal region of Rho-kinase is a possible negative regulatory region of Rho-kinase. The expression of RB/PH (TT) specifically blocked the stress fiber and focal adhesion formation induced by the active form of Rho or Rho-kinase in NIH 3T3 cells, but not that induced by the active form of MRCKbeta or myosin light chain. Thus, RB/PH (TT) appears to specifically inhibit Rho-kinase in vivo.
Amano
M
M
Division of Signal Transduction, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma 630-0101, Japan.
Chihara
K
K
Nakamura
N
N
Kaneko
T
T
Matsuura
Y
Y
Kaibuchi
K
K
D016475:3T3 Cells; D000818:Animals; D002846:Chromatography, Affinity; D003599:Cytoskeleton / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D018994:Myosin Light Chains / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D054460:rho-Associated Kinases
D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D018994:Myosin Light Chains; D010446:Peptide Fragments; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases