Regulation of PKC alpha activity by C1-C2 domain interactions.
In this study, the role of interdomain interactions involving the C1 and C2 domains in the mechanism of activation of PKC was investigated. Using an in vitro assay containing only purified recombinant proteins and the phorbol ester, 4 beta-12-O-tetradecanoylphorbol-13-acetate (TPA), but lacking lipids, it was found that PKC alpha bound specifically, and with high affinity, to a alpha C1A-C1B fusion protein of the same isozyme. The alpha C1A-C1B domain also potently activated the isozyme in a phorbol ester- and diacylglycerol-dependent manner. The level of this activity was comparable with that resulting from membrane association induced under maximally activating conditions. Furthermore, it was found that alpha C1A-C1B bound to a peptide containing the C2 domain of PKC alpha. The alpha C1A-C1B domain also activated conventional PKC beta I, -beta II, and -gamma isoforms, but not novel PKC delta or -epsilon. PKC delta and -epsilon were each activated by their own C1 domains, whereas PKC alpha, -beta I, -beta II, or -gamma activities were unaffected by the C1 domain of PKC delta and only slightly activated by that of PKC epsilon. PKC zeta activity was unaffected by its own C1 domain and those of the other PKC isozymes. Based on these findings, it is proposed that the activating conformational change in PKC alpha results from the dissociation of intra-molecular interactions between the alpha C1A-C1B domain and the C2 domain. Furthermore, it is shown that PKC alpha forms dimers via inter-molecular interactions between the C1 and C2 domains of two neighboring molecules. These mechanisms may also apply for the activation of the other conventional and novel PKC isozymes.
Slater
Simon J
SJ
Department of Pathology, Cell Biology and Anatomy, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Seiz
Jodie L
JL
Cook
Anthony C
AC
Buzas
Christopher J
CJ
Malinowski
Steve A
SA
Kershner
Jennifer L
JL
Stagliano
Brigid A
BA
Stubbs
Christopher D
CD
D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology; D002460:Cell Line; D004789:Enzyme Activation; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D011493:Protein Kinase C / Q000737:chemistry / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D018411:Spodoptera; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection
D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D013755:Tetradecanoylphorbol Acetate