Calponin phosphorylation in vitro and in intact muscle

Abstract

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.

Cytoskeletal targeting of calponin in differentiated, contractile smooth muscle cells of the ferret

“…The signal for the postulated agonist-induced decrease in actin affinity is still unknown, but the CaP redistribution has been shown to be dependent on protein kinase C (PKC) activity (Parker et al 1994), suggesting a phosphorylation event. CaP can be phosphorylated by PKC in vitro (Winder et al 1993). Also, several groups have reported that CaP can be phosphorylated in vivo (Winder et al 1993;Mino, Yuasa, Naka & Tanaka, 1995;Pohl et al 1997).…”

“…Also, several groups have reported that CaP can be phosphorylated in vivo (Winder et al 1993;Mino, Yuasa, Naka & Tanaka, 1995;Pohl et al 1997). Winder et al (1993) also showed that upon phosphorylation in vitro, CaP's binding affinity to actin is diminished significantly. Phosphorylation may trigger a loss of CaP binding affinity to actin.…”

“…dependent kinase II (Nakamura, Mino, Yamamoto, Naka & Tanaka, 1993;Winder, Allen, Fraser, Kang & Kargacin & Walsh, 1993). Phosphorylated CaP is neither capable of binding to actin nor capable of inhibiting actomyosin ATPase.…”

The Effects of Smooth Muscle Calponin on the Strong and Weak Myosin Binding Sites of F-actin

“…Calponin is a 34-kDa smooth muscle protein that binds actin (3), myosin (12), Ca 2ϩ -binding proteins (13,14), and tropomyosin (15). In vitro calponin inhibits the actomyosin ATPase activity upon binding to actin, and this inhibition is reversed by its phosphorylation (16) or by its interaction with Ca 2ϩ -binding proteins such as calmodulin or caltropin (13,14). Consequently, it has been suggested that calponin may have a role in controlling smooth muscle contraction.…”

A role for serine-175 in modulating the molecular conformation of calponin

“…Therefore it has been proposed that additional regulatory mechanisms may exist at the level of the thin filament [18][19][20], and calponin may play a role in this signal transduction pathway. For example, calponin inhibits the actin-activated Mg-ATPase activity of phosphorylated smooth muscle myosin, and this inhibition is regulated by phosphorylation-dephosphorylation of calponin [5,6,[21][22][23]. In itro motility assays demonstrated that calponin inhibits the relative movement of actin and myosin filaments [24,25].…”