title: Inhibition of protein kinase C catalytic activity by additional regions within the human protein kinase Calpha-regulatory domain lying outside of the pseudosubstrate sequence.
abstract: The N-terminal pseudosubstrate site within the protein kinase Calpha (PKCalpha)-regulatory domain has long been regarded as the major determinant for autoinhibition of catalytic domain activity. Previously, we observed that the PKC-inhibitory capacity of the human PKCalpha-regulatory domain was only reduced partially on removal of the pseudosubstrate sequence [Parissenti, Kirwan, Kim, Colantonio and Schimmer (1998) J. Biol. Chem. 273, 8940-8945]. This finding suggested that one or more additional region(s) contributes to the inhibition of catalytic domain activity. To assess this hypothesis, we first examined the PKC-inhibitory capacity of a smaller fragment of the PKCalpha-regulatory domain consisting of the C1a, C1b and V2 regions [GST-Ralpha(39-177): this protein contained the full regulatory domain of human PKCalpha fused to glutathione S-transferase (GST), but lacked amino acids 1-38 (including the pseudosubstrate sequence) and amino acids 178-270 (including the C2 region)]. GST-Ralpha(39-177) significantly inhibited PKC in a phorbol-independent manner and could not bind the peptide substrate used in our assays. These results suggested that a region within C1/V2 directly inhibits catalytic domain activity. Providing further in vivo support for this hypothesis, we found that expression of N-terminally truncated pseudosubstrate-less bovine PKCalpha holoenzymes in yeast was capable of inhibiting cell growth in a phorbol-dependent manner. This suggested that additional autoinhibitory force(s) remained within the truncated holoenzymes that could be relieved by phorbol ester. Using tandem PCR-mediated mutagenesis, we observed that mutation of amino acids 33-86 within GST-Ralpha(39-177) dramatically reduced its PKC-inhibitory capacity when protamine was used as substrate. Mutagenesis of a broad range of sequences within C2 (amino acids 159-242) also significantly reduced PKC-inhibitory capacity. Taken together, these observations support strongly the existence of multiple regions within the PKCalpha-regulatory domain that play a direct role in the inhibition of catalytic domain activity.
authors.affiliation: Tumor Biology Research Program, Northeastern Ontario Regional Cancer Centre, 41 Ramsey Lake Road, Sudbury, ON, Canada P3E 5J1.
authors.forename: Angie F
authors.initials: AF
authors.lastname: Kirwan
authors.forename: Ashley C
authors.initials: AC
authors.lastname: Bibby
authors.forename: Thierry
authors.initials: T
authors.lastname: Mvilongo
authors.forename: Heimo
authors.initials: H
authors.lastname: Riedel
authors.forename: Thomas
authors.initials: T
authors.lastname: Burke
authors.forename: Sherri Z
authors.initials: SZ
authors.lastname: Millis
authors.forename: Amadeo M
authors.initials: AM
authors.lastname: Parissenti
mesh_terms: D000818:Animals; D001921:Brain / Q000201:enzymology*; D002118:Calcium / Q000378:metabolism; D020134:Catalytic Domain; D002417:Cattle; D002455:Cell Division / Q000187:drug effects; D017931:DNA Primers / Q000737:chemistry; D004789:Enzyme Activation; D005690:Galactose / Q000494:pharmacology; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010446:Peptide Fragments / Q000378:metabolism; D010703:Phorbol Esters / Q000494:pharmacology; D010718:Phosphatidylserines / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011479:Protamines / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors*; D051571:Protein Kinase C-alpha; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000378:metabolism; D017384:Sequence Deletion; D013379:Substrate Specificity
chemical_list: D017931:DNA Primers; D010446:Peptide Fragments; D010703:Phorbol Esters; D010718:Phosphatidylserines; D011479:Protamines; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; C496628:PRKCA protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D002118:Calcium; D005690:Galactose