Abstract
Calponin, a thin filament-associated protein, inhibits actin-activated myosin ATPase activity, and this inhibition is reversed by phosphorylation. Calponin phosphorylation by protein kinase C and Ca2+/calmodulin-dependent protein kinase II has been shown in purified protein systems but has been difficult to demonstrate in more physiological preparations. We have previously shown that calponin is phosphorylated in a cell-free homogenate of swine carotid artery. The goal of this study was to determine whether protein kinase C and/or Ca2+/calmodulin-dependent protein kinase II catalyzes calponin phosphorylation. Ca2+-dependent calponin phosphorylation was not inhibited by calmodulin antagonists. In contrast, both Ca2+- and phorbol dibutyrate/1-oleoyl-2-acetyl-sn-glycerol dependent calponin phosphorylation were inhibited by the pseudosubstrate inhibitor of protein kinase C and staurosporine. Our results also demonstrate that stimulation with either Ca2+, phorbol dibutyrate, or 1-oleoyl-2-acetyl-sn-glycerol activates endogenous protein kinase C. We interpret our results as clearly demonstrating that the physiological kinase for calponin phosphorylation is protein kinase C and not Ca2+/calmodulin-dependent protein kinase II. We also present data showing that the direct measurement of 32P incorporation into calponin and the indirect measurement of calponin phosphorylation using nonequilibrium pH gradient gel electrophoresis provide similar quantitative values of calponin phosphorylation.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Animals
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Antiemetics / pharmacology
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Calcium / pharmacology
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Calcium-Binding Proteins / metabolism*
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Calcium-Calmodulin-Dependent Protein Kinase Type 2
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Calcium-Calmodulin-Dependent Protein Kinases / metabolism
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Calmodulin / antagonists & inhibitors
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Calmodulin-Binding Proteins / metabolism*
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Calponins
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Carcinogens / pharmacology
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Carotid Arteries / chemistry
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Carotid Arteries / enzymology*
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Chelating Agents / pharmacology
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Diglycerides / pharmacology
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Egtazic Acid / pharmacology
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Electrophoresis / methods
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Enzyme Inhibitors / pharmacology
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Imidazoles / pharmacology
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Microfilament Proteins
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Okadaic Acid / pharmacology
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Organ Culture Techniques
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Peptide Fragments / pharmacology
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Phorbol 12,13-Dibutyrate / pharmacology
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Phosphorus Radioisotopes
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Phosphorylation
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Protein Kinase C / antagonists & inhibitors
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Protein Kinase C / metabolism*
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Protein Kinase C / pharmacology
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Staurosporine / pharmacology
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Sulfonamides / pharmacology
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Swine
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Trifluoperazine / pharmacology
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Vasodilator Agents / pharmacology
Substances
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Antiemetics
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Calcium-Binding Proteins
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Calmodulin
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Calmodulin-Binding Proteins
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Carcinogens
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Chelating Agents
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Diglycerides
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Enzyme Inhibitors
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Imidazoles
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Microfilament Proteins
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Peptide Fragments
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Phosphorus Radioisotopes
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Sulfonamides
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Vasodilator Agents
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protein kinase C (19-31)
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Okadaic Acid
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Trifluoperazine
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Phorbol 12,13-Dibutyrate
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calmidazolium
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Egtazic Acid
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W 7
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1-oleoyl-2-acetylglycerol
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Protein Kinase C
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Calcium-Calmodulin-Dependent Protein Kinase Type 2
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Calcium-Calmodulin-Dependent Protein Kinases
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Staurosporine
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Calcium