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 tracheal smooth muscle strips metabolically labeled with 32Pi. Calponin was gel purified from muscles stimulated with 1 microM carbachol. Phosphorylation increased to 2.0 times the basal level of 178 +/- 26 counts per minute (cpm)/microgram calponin within 30 s to 350 +/- 64 cpm/micrograms. Two-dimensional nonequilibrium pH gradient gel electrophoresis resolved four charge isoforms of calponin in unstimulated muscle. Stimulation with carbachol induced an additional more acidic isoform. Phosphorylation of calponin in vitro with protein kinase C (PKC) also induced formation of additional acidic isoforms. The functional effect of phosphorylation was demonstrated using an in vitro motility assay in which unphosphorylated calponin (2 microM) caused a profound inhibition of actin sliding. Calponin phosphorylated by PKC did not inhibit actin sliding. The results show that phosphorylation of calponin occurs in intact tracheal smooth muscle and that phosphorylation of calponin in vitro alleviates the inhibitory effect of calponin on actomyosin function.
Pohl
J
J
Department of Pharmacology, University of Nevada School of Medicine, Reno 89557, USA.
Winder
S J
SJ
Allen
B G
BG
Walsh
M P
MP
Sellers
J R
JR
Gerthoffer
W T
WT
D000199:Actins / Q000502:physiology; D000818:Animals; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D055598:Chemical Phenomena; D002621:Chemistry; D004285:Dogs; D004586:Electrophoresis; D015180:Electrophoresis, Gel, Two-Dimensional; D005260:Female; D007536:Isomerism; D008297:Male; D008433:Mathematics; D008840:Microfilament Proteins; D009130:Muscle, Smooth / Q000378:metabolism*; D010766:Phosphorylation; D014132:Trachea / Q000378:metabolism*; D000096985:Calponins
D000199:Actins; D002135:Calcium-Binding Proteins; D008840:Microfilament Proteins; D000096985:Calponins