PHOSPHORYLATION OF CALPONIN IN AIRWAY SMOOTH-MUSCLE

Calponin is an actin-binding protein known to be a substrate in vitro for several protein kinases and phosphoprotein phosphatases. We tested the hypothesis that calponin is phosphorylated in vivo using canine t racheal smooth muscle strips metabolically labeled with P-32(i). Calpo nin was gel purified from muscles stimulated with 1 mu M carbachol. Ph osphorylation increased to 2.0 times the basal level of 178 +/- 26 cou nts per minute (cpm)/mu g calponin within 30 s to 350 +/- 64 cpm/mu g. Two-dimensional nonequilibrium pH gradient gel electrophoresis resolv ed four charge isoforms of calponin in unstimulated muscle. Stimulatio n with carbachol induced an additional more acidic isoform. Phosphoryl ation of calponin in vitro with protein kinase C (PKC) also induced fo rmation of additional acidic isoforms. The functional effect of phosph orylation was demonstrated using an in vitro motility assay in which u nphosphorylated calponin (2 mu M) caused a profound inhibition of acti n sliding. Calponin phosphorylated by PKC did not inhibit actin slidin g. The results show that phosphorylation of calponin occurs in intact tracheal smooth muscle and that phosphorylation of calponin in vitro a lleviates the inhibitory effect of calponin on actomyosin function.