Premium
Protein kinase C–catalyzed calponin phosphorylation in swine carotid arterial homogenate
Author(s) -
Rokolya Aniko,
Walsh Michael P.,
Singer Harold A.,
Moreland Robert S.
Publication year - 1998
Publication title -
journal of cellular physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.529
H-Index - 174
eISSN - 1097-4652
pISSN - 0021-9541
DOI - 10.1002/(sici)1097-4652(199809)176:3<545::aid-jcp11>3.0.co;2-z
Subject(s) - calponin , protein kinase c , phosphorylation , calmodulin , protein kinase a , kinase , biochemistry , microbiology and biotechnology , protein phosphorylation , biology , chemistry , actin , enzyme
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 Ca 2+ /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 Ca 2+ /calmodulin‐dependent protein kinase II catalyzes calponin phosphorylation. Ca 2+ ‐dependent calponin phosphorylation was not inhibited by calmodulin antagonists. In contrast, both Ca 2+ ‐ 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 Ca 2+ , 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 Ca 2+ /calmodulin‐dependent protein kinase II. We also present data showing that the direct measurement of 32 P incorporation into calponin and the indirect measurement of calponin phosphorylation using nonequilibrium pH gradient gel electrophoresis provide similar quantitative values of calponin phosphorylation. J. Cell. Physiol. 176:545–552, 1998. © 1998 Wiley‐Liss, Inc.