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NEUROREGULATION AND MOTILITY

Direct association and translocation of PKC- with calponin

Department of Pediatrics, University of Michigan, Ann Arbor, Michigan 48109

Submitted 1 November 2003 ; accepted in final form 9 January 2004

Calponin has been implicated in the regulation of smooth muscle contraction through its interaction with F-actin and inhibition of the actin-activated MgATPase activity of phosphorylated myosin. Calponin has also been shown to interact with PKC. We have studied the interaction of calponin with PKC- and with the low molecular weight heat-shock protein (HSP)27 in contraction of colonic smooth muscle cells. Particulate fractions from isolated smooth muscle cells were immunoprecipitated with antibodies to calponin and Western blot analyzed with antibodies to HSP27 and to PKC-. Acetylcholine induced a sustained increase in the immunocomplexing of calponin with HSP27 and of calponin with PKC- in the particulate fraction, indicating an association of the translocated proteins in the membrane. To examine whether the observed interaction in vivo is due to a direct interaction of calponin with PKC-, a cDNA of 1.3 kb of human calponin gene was PCR amplified. PCR product encoding 622 nt of calponin cDNA (nt 351–972 corresponding to amino acids 92–229) was expressed as fusion glutathione S-transferase (GST) protein in the vector pGEX-KT. We have studied the direct association of GST-calponin fusion protein with recombinant PKC- in vitro. Western blot analysis of the fractions collected after elution with reduced glutathione buffer (pH 8.0) show a coelution of GST-calponin with PKC-, indicating a direct association of GST-calponin with PKC-. These data suggest that there is a direct association of translocated calponin and PKC- in the membrane and a role for the complex calponin-PKC--HSP27, in contraction of colonic smooth muscle cells.

heat-shock protein 27; smooth muscle; contraction; cytoskeleton

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