Rho-associated kinase of chicken gizzard smooth muscle

Jianhua Feng, Masaaki Ito, Yasuko Kureishi, Kazuhito Ichikawa, Mutsuki Amano, Naoki Isaka, Katsuya Okawa, Akihiro Iwamatsu, Kozo Kaibuchi, David J. Hartshorne, Takeshi Nakano

Research output: Contribution to journalArticlepeer-review

217 Citations (Scopus)

Abstract

Rho-associated kinase (Rho-kinase) from chicken gizzard smooth muscle was purified to apparent homogeneity (160 kDa on SDS-polyacrylamide gel electrophoresis) and identified as the ROKα isoform. Several substrates were phosphorylated. Rates with myosin phosphatase target subunit 1 (MYPT1), myosin, and the 20-kDa myosin light chain were higher than other substrates. Thiophosphorylation of MYPT1 inhibited myosin phosphatase activity. Phosphorylation of myosin at serine 19 increased actin-activated Mg±-ATPase activity, i.e. similar to myosin light chain kinase. Myosin phosphorylation was increased at higher ionic strengths, possibly by formation of 6 S myosin. Phosphorylation of the isolated light chain and myosin phosphatase was decreased by increasing ionic strength. Rhokinase was stimulated 1.5-2-fold by guanosine 5'-O-3(thio)triphosphate-RhoA, whereas limited tryptic hydrolysis caused a 5-6-fold activation, independent of RhoA. Several kinase inhibitors were screened and most effective were Y-27632, staurosporine, and H-89. Several lipids caused slight activation of Rho-kinase, but arachidonic acid (30-50 μM) induced a 5-6-fold activation, independent of RhoA. These results suggest that Rho-kinase of smooth muscle may be involved in the contractile process via phosphorylation of MYPT1 and myosin. Activation by arachidonic acid presents a possible regulatory mechanism for Rho-kinase.

Original languageEnglish
Pages (from-to)3744-3752
Number of pages9
JournalJournal of Biological Chemistry
Volume274
Issue number6
DOIs
Publication statusPublished - 05-02-1999
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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