Calponin phosphorylation in vitro and in intact muscle.
Calponin, a thin-filament-associated protein implicated in the regulation of smooth-muscle contraction, is phosphorylated in vitro by protein kinase C and Ca2+/calmodulin-dependent protein kinase II [Winder and Walsh (1990) J. Biol. Chem. 265, 10148-10155] and dephosphorylated by a type 2A protein phosphatase [Winder, Pato and Walsh (1992) Biochem. J. 286, 197-203]. Unphosphorylated calponin binds to actin and inhibits the actin-activated myosin MgATPase; these properties are lost on phosphorylation. Although both serine and threonine residues in calponin are phosphorylated, the major site of phosphorylation by either kinase is Ser-175. Calponin also undergoes phosphorylation when bound to actin in synthetic thin filaments, in a reconstituted actomyosin system, in washed myofibrils and in tissue extracts; this results in dissociation of calponin from actin. Tryptic phosphopeptide mapping indicates that the same sites are phosphorylated in the bound as in the isolated protein. Toad stomach calponin exists in at least three isoforms which differ in charge but exhibit the same molecular mass on SDS/PAGE. In a toad stomach extract, all three isoforms are phosphorylated by protein kinase C or Ca2+/calmodulin-dependent protein kinase II as shown by two-dimensional gel electrophoresis (non-equilibrium pH-gradient gel electrophoresis and SDS/PAGE). Calponin phosphorylation also occurs in intact toad stomach smooth-muscle strips metabolically labelled with 32Pi and stimulated to contract with carbachol. These results support the hypothesis that calponin may be regulated in vivo by phosphorylation-dephosphorylation.
Winder
S J
SJ
MRC Group in Signal Transduction, University of Calgary, Alberta, Canada.
Allen
B G
BG
Fraser
E D
ED
Kang
H M
HM
Kargacin
G J
GJ
Walsh
M P
MP
D000595:Amino Acid Sequence; D000818:Animals; D001001:Anura; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D066298:In Vitro Techniques; D008840:Microfilament Proteins; D008969:Molecular Sequence Data; D009119:Muscle Contraction; D009124:Muscle Proteins / Q000378:metabolism*; D009130:Muscle, Smooth / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D000096985:Calponins
D002135:Calcium-Binding Proteins; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D008840:Microfilament Proteins; D009124:Muscle Proteins; D010446:Peptide Fragments; D000096985:Calponins