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Rho-associated kinase of chicken gizzard smooth muscle
1First Department of Internal Medicine, Mie University School of Medicine, Tsu, 514-8507, Japan.
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 ROKalpha 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. Rho-kinase 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 microM) 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.
Insights
Chicken gizzard Rho-associated kinase (Rho-kinase) phosphorylates myosin phosphatase target subunit 1 and myosin, impacting smooth muscle contraction. Arachidonic acid activates Rho-kinase, suggesting a regulatory role.
Area of Science:
- Biochemistry
- Molecular Biology
- Cellular Physiology
Background:
- Rho-associated kinase (Rho-kinase) is a key regulator of smooth muscle contraction.
- Understanding Rho-kinase's substrates and activators is crucial for elucidating contractile mechanisms.
Purpose of the Study:
- To purify and characterize chicken gizzard Rho-kinase (ROKalpha).
- To identify Rho-kinase substrates and investigate their phosphorylation.
- To explore activators of Rho-kinase, including RhoA and lipids.
Main Methods:
- Purification of Rho-kinase from chicken gizzard smooth muscle.
- SDS-polyacrylamide gel electrophoresis for protein homogeneity assessment.
- Phosphorylation assays using various substrates (MYPT1, myosin, myosin light chain).
- Enzyme kinetics and inhibitor screening.
- Lipid activation assays.
Main Results:
- Rho-kinase (ROKalpha) was purified and identified.
- Myosin phosphatase target subunit 1 (MYPT1) and myosin were identified as major substrates.
- Phosphorylation of MYPT1 inhibited myosin phosphatase activity.
- Myosin phosphorylation by Rho-kinase increased actin-activated Mg+-ATPase activity.
- Arachidonic acid significantly activated Rho-kinase independently of RhoA.
Conclusions:
- Chicken gizzard Rho-kinase plays a role in smooth muscle contraction through MYPT1 and myosin phosphorylation.
- Arachidonic acid activation suggests a novel regulatory pathway for Rho-kinase in smooth muscle.