Regulation of protein kinase C by the cytoskeletal protein calponin.
Previous studies from this laboratory have shown that, upon agonist activation, calponin co-immunoprecipitates and co-localizes with protein kinase Cepsilon (PKCepsilon) in vascular smooth muscle cells. In the present study we demonstrate that calponin binds directly to the regulatory domain of PKC both in overlay assays and, under native conditions, by sedimentation with lipid vesicles. Calponin was found to bind to the C2 region of both PKCepsilon and PKCalpha with possible involvement of C1B. The C2 region of PKCepsilon binds to the calponin repeats with a requirement for the region between amino acids 160 and 182. We have also found that calponin can directly activate PKC autophosphorylation. By using anti-phosphoantibodies to residue Ser-660 of PKCbetaII, we found that calponin, in a lipid-independent manner, increased auto-phosphorylation of PKCalpha, -epsilon, and -betaII severalfold compared with control conditions. Similarly, calponin was found to increase the amount of (32)P-labeled phosphate incorporated into PKC from [gamma-(32)P]ATP. We also observed that calponin addition strongly increased the incorporation of radiolabeled phosphate into an exogenous PKC peptide substrate, suggesting an activation of enzyme activity. Thus, these results raise the possibility that calponin may function in smooth muscle to regulate PKC activity by facilitating the phosphorylation of PKC.
Leinweber
B
B
Boston Biomedical Research Institute, Watertown, Massachusetts 02472, the Northeastern Ontario Regional Cancer Centre, 41 Ramsey Lake Road, Sudbury, Ontario P3E 5J1, Canada.
Parissenti
A M
AM
Gallant
C
C
Gangopadhyay
S S
SS
Kirwan-Rhude
A
A
Leavis
P C
PC
Morgan
K G
KG
D002135:Calcium-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D003598:Cytoskeletal Proteins / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D008840:Microfilament Proteins; D010766:Phosphorylation; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D000096985:Calponins
D002135:Calcium-Binding Proteins; D003598:Cytoskeletal Proteins; D008840:Microfilament Proteins; D011493:Protein Kinase C; D011994:Recombinant Proteins; D000096985:Calponins