Direct association of calponin with specific domains of PKC-

Somara S, Bitar KN. Direct association of calponin with specific domains of PKC. Am J Physiol Gastrointest Liver Physiol 295: G1246 –G1254, 2008. First published October 23, 2008; doi:10.1152/ajpgi.90461.2008.—Calponin contributes to the regulation of smooth muscle contraction through its interaction with F-actin and inhibition of the actin-activated Mg-ATPase activity of phosphorylated myosin. Previous studies have shown that the contractile agonist acetylcholine induced a direct association of translocated calponin and PKCin the membrane. In the present study, we have determined the domain of PKCinvolved in direct association with calponin. In vitro binding assay was carried out by incubating glutathione S-transferase-calponin aa 92-229 with His-tagged proteins of individual domains and different combinations of domains of PKC. Calponin was found to bind directly to the full-length PKC. Calponin bound to C2 and C4 domains but not to C1 and C3 domains of PKC. When incubated with proteins of different combination of domains, calponin bound to C2-C3, C3-C4, and C2-C3-C4 but not to C1-C2 or C1-C2-C3. To determine whether these in vitro bindings mimic the in vivo associations, and in vivo binding assay was performed by transfecting colonic smooth muscle cells with Histagged proteins of individual domains and different combinations of domains of PKC. Coimmunoprecipitation of calponin with Histagged truncated forms of PKCshowed that C1-C2, C1-C2-C3, C2-C3, and C3-C4 did not associate with calponin. Calponin associated only with full-length PKCand with C2-C3-C4 in cells in the resting state, and this association increased upon stimulation with acetylcholine. These data suggest that calponin bound to fragments that may mimic the active form of PKCand that the functional association of PKCwith calponin requires both C2 and C4 domains during contraction of colonic smooth muscle cells.