Independence of protein kinase C-delta activity from activation loop phosphorylation: structural basis and altered functions in cells.
Activation loop phosphorylation plays critical regulatory roles for many kinases. Unlike other protein kinase Cs (PKC), PKC-delta does not require phosphorylation of its activation loop (Thr-507) for in vitro activity. We investigated the structural basis for this unusual capacity and its relevance to PKC-delta function in intact cells. Mutational analysis demonstrated that activity without Thr-507 phosphorylation depends on 20 residues N-terminal to the kinase domain and a pair of phenylalanines (Phe-500/Phe-527) unique to PKC-delta in/near the activation loop. Molecular modeling demonstrated that these elements stabilize the activation loop by forming a hydrophobic chain of interactions from the C-lobe to activation loop to N-terminal (helical) extension. In cells PKC-delta mediates both apoptosis and transcription regulation. We found that the T507A mutant of the PKC-delta kinase domain resembled the corresponding wild type in mediating apoptosis in transfected HEK293T cells. But the T507A mutant was completely defective in AP-1 and NF-kappaB reporter assays. A novel assay in which the kinase domain of PKC-delta and its substrate (a fusion protein of PKC substrate peptide with green fluorescent protein) were co-targeted to lipid rafts revealed a major substrate-selective defect of the T507A mutant in phosphorylating the substrate in cells. In vitro analysis showed strong product inhibition on the T507A mutant with particular substrates whose characteristics suggest it contributes to the substrate selective defect of the PKC-delta T507A mutant in cells. Thus, activation loop phosphorylation of PKC-delta may regulate its function in cells in a novel way.
Liu
Yin
Y
Experimental Immunology Branch, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA.
Belkina
Natalya V
NV
Graham
Caroline
C
Shaw
Stephen
S
D017209:Apoptosis; D002478:Cells, Cultured; D004789:Enzyme Activation; D006801:Humans; D007668:Kidney / Q000201:enzymology; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics*; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D010649:Phenylalanine / Q000737:chemistry / Q000235:genetics; D010766:Phosphorylation; D051745:Protein Kinase C-delta / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014162:Transfection
D016328:NF-kappa B; D018808:Transcription Factor AP-1; D010649:Phenylalanine; D051745:Protein Kinase C-delta