title: Inhibitory properties of the regulatory domains of human protein kinase Calpha and mouse protein kinase Cepsilon.
abstract: Two fusion proteins in which the regulatory domains of human protein kinase Calpha (Ralpha; amino acids 1-270) or mouse protein kinase Cepsilon (Repsilon; amino acids 1-385) were linked in frame with glutathione S-transferase (GST) were examined for their abilities to inhibit the catalytic activities of protein kinase Calpha (PKCalpha) and other protein kinases in vitro. Both GST-Ralpha and GST-Repsilon but not GST itself potently inhibited the activities of lipid-activated rat brain PKCalpha. In contrast, the fusion proteins had little or no inhibitory effect on the activities of the Ser/Thr protein kinases cAMP-dependent protein kinase, cGMP-dependent protein kinase, casein kinase II, myosin light chain kinase, and mitogen activated protein kinase or on the src Tyr kinase. GST-Ralpha and GST-Repsilon, on a molar basis, were 100-200-fold more potent inhibitors of PKCalpha activity than was the pseudosubstrate peptide PKC19-36. In addition, a GST-Ralpha fusion protein in which the first 32 amino acids of Ralpha were deleted (including the pseudosubstrate sequence from amino acids 19-31) was an effective competitive inhibitor of PKCalpha activity. The three GST-R fusion proteins also inhibited protamine-activated PKCalpha and proteolytically activated PKCalpha (PKM), two lipid-independent forms of PKCalpha; however, the IC50 values for inhibition were 1 order of magnitude greater than the IC50 values obtained in the presence of lipid. These results suggest that part of the inhibitory effect of the GST-R fusion proteins on lipid-activated PKCalpha may have resulted from sequestration of lipid activators. Nonetheless, as evidenced by their abilities to inhibit the lipid-independent forms of the enzyme, the GST-R fusion proteins also inhibited PKCalpha catalytic activity through direct interactions. These data indicate that the R domains of PKCalpha and PKCepsilon are specific inhibitors of protein kinase Calpha activity and suggest that regions of the R domain outside the pseudosubstrate sequence contribute to autoinhibition of the enzyme.
authors.affiliation: Department of Research, Northeastern Ontario Regional Cancer Center, Sudbury, Ontario P3E 5J1, Canada.
authors.forename: A M
authors.initials: AM
authors.lastname: Parissenti
authors.forename: A F
authors.initials: AF
authors.lastname: Kirwan
authors.forename: S A
authors.initials: SA
authors.lastname: Kim
authors.forename: C M
authors.initials: CM
authors.lastname: Colantonio
authors.forename: B P
authors.initials: BP
authors.lastname: Schimmer
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D047390:Casein Kinase II; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism; D005982:Glutathione Transferase / Q000096:biosynthesis; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry; D011493:Protein Kinase C / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D051744:Protein Kinase C-epsilon; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism
chemical_list: D007527:Isoenzymes; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D011494:Protein Kinases; C497256:Prkce protein, mouse; C497257:Prkce protein, rat; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D017869:Cyclic GMP-Dependent Protein Kinases; C496628:PRKCA protein, human; C497255:PRKCE protein, human; C496630:Prkca protein, mouse; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D051744:Protein Kinase C-epsilon; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D009219:Myosin-Light-Chain Kinase