Direct association and translocation of PKC-alpha with calponin.
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-alpha 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-alpha. Acetylcholine induced a sustained increase in the immunocomplexing of calponin with HSP27 and of calponin with PKC-alpha 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-alpha, 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-alpha 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-alpha, indicating a direct association of GST-calponin with PKC-alpha. These data suggest that there is a direct association of translocated calponin and PKC-alpha in the membrane and a role for the complex calponin-PKC-alpha-HSP27, in contraction of colonic smooth muscle cells.
Patil
Suresh B
SB
Department of Pediatrics, University of Michigan, Ann Arbor, MI 48109, USA.
Pawar
Mercy D
MD
Bitar
Khalil N
KN
D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D001692:Biological Transport; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000276:immunology / Q000502:physiology*; D003106:Colon / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D005769:Gastrointestinal Motility / Q000187:drug effects / Q000502:physiology; D005982:Glutathione Transferase / Q000235:genetics; D006360:Heat-Shock Proteins / Q000276:immunology / Q000378:metabolism; D006801:Humans; D008840:Microfilament Proteins; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D011493:Protein Kinase C / Q000276:immunology / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D014335:Tropomyosin / Q000378:metabolism; D000096985:Calponins
D000109:Acetylcholine; D000906:Antibodies; D002135:Calcium-Binding Proteins; D005982:Glutathione Transferase; D006360:Heat-Shock Proteins; D008840:Microfilament Proteins; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D014335:Tropomyosin; D000096985:Calponins; C496628:PRKCA protein, human