Abstract
Calponin, an F-actin-associated protein implicated in the regulation of smooth muscle contraction, is known to be phosphorylated in vitro by protein kinase C (PKC) and Ca(2+)/calmodulin dependent protein kinase II (CaM kinase II). Unphosphorylated calponin binds to F-actin and inhibits the actin-activated myosin ATPase activity; these properties are lost on phosphorylation. In the present study, we found that Rho-kinase phosphorylated basic calponin stoichiometrically in vitro. We identified the sites of phosphorylation of calponin by Rho-kinase as Thr-170, Ser-175, Thr-180, Thr-184, and Thr-259, and prepared antibodies that specifically recognized calponin phosphorylated at Thr-170 and Thr-184. We showed that the phosphorylation of calponin by Rho-kinase inhibited the binding of calponin to F-actin. Taken together, these results suggest that calponin is a substrate of Rho-kinase and that Rho-kinase regulates the interaction of calponin with F-actin.
Copyright 2000 Academic Press.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Actins / metabolism
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Amino Acid Sequence
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Animals
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Antibodies / immunology
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Blotting, Western
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Calcium-Binding Proteins / immunology
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Calcium-Binding Proteins / metabolism*
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Calponins
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Cattle
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Chromatography, High Pressure Liquid
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Guanosine 5'-O-(3-Thiotriphosphate) / metabolism
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Intracellular Signaling Peptides and Proteins
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Microfilament Proteins
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Molecular Sequence Data
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Peptide Mapping
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Phosphorylation
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Phosphoserine / immunology
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Phosphoserine / metabolism
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Phosphothreonine / immunology
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Phosphothreonine / metabolism
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Protein Binding
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Protein Isoforms / metabolism
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Protein Serine-Threonine Kinases / metabolism*
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Rabbits
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Recombinant Fusion Proteins / metabolism
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rho-Associated Kinases
Substances
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Actins
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Antibodies
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Calcium-Binding Proteins
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Intracellular Signaling Peptides and Proteins
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Microfilament Proteins
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Protein Isoforms
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Recombinant Fusion Proteins
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Phosphothreonine
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Phosphoserine
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Guanosine 5'-O-(3-Thiotriphosphate)
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Protein Serine-Threonine Kinases
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rho-Associated Kinases