Characterization of a protein kinase C-delta (PKC-delta) ATP binding mutant. An inactive enzyme that competitively inhibits wild type PKC-delta enzymatic activity.
To investigate the function of protein kinase C (PKC)-delta, we mutated its ATP binding site by converting the invariant lysine in the catalytic domain (amino acid 376) to an arginine. Expression vectors containing wild type and mutant PKC-delta cDNAs were generated either with or without an influenza virus hemagglutinin epitope tag. After expression in 32D cells by transfection, the PKC-delta ATP binding mutant (PKC-delta K376R) was not able to phosphorylate itself or the PKC-delta pseudosubstrate region-derived substrate, indicating that PKC-delta K376R was an inactive enzyme. PKC activity was inhibited by 67% in 32D cells coexpressing both PKC-delta wild type (PKC-delta WT) and PKC-delta K376R when compared to 32D cells expressing only PKC-delta WT. Mixture of PKC-delta WT and PKC-delta K376R kinase sources in vitro also reduced the enzymatic activity of PKC-delta WT. These results suggest that PKC-delta K376R competes with PKC-delta WT and inhibits PKC-delta WT phosphorylation of its in vitro substrate. While PKC-delta WT overexpressed in 32D cells demonstrated 12-O-tetradecanoylphorbol-13-acetate (TPA)-dependent translocation from the cytosolic to the membrane fraction, PKC-delta K376R was exclusively localized in the membrane fraction even prior to TPA stimulation. Unlike PKC-delta WT which was phosphorylated on tyrosine residue(s) only after TPA treatment, PKC-delta K376R was constitutively phosphorylated on tyrosine residue(s). Although exposure of PKC-delta WT transfectants to TPA induced 32D monocytic differentiation, the 32D/PKC-delta K376R transfectants were resistant to TPA-induced differentiation. Thus, expression of active PKC-delta is required to mediate 32D monocytic differentiation in response to TPA stimulation.
Li
W
W
Laboratory of Cellular and Molecular Biology, National Cancer Institute, Bethesda, Maryland 20892, USA.
Yu
J C
JC
Shin
D Y
DY
Pierce
J H
JH
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D002454:Cell Differentiation; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D003001:Cloning, Molecular; D017931:DNA Primers; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009000:Monocytes / Q000166:cytology; D009154:Mutation; D010766:Phosphorylation; D011485:Protein Binding; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D051745:Protein Kinase C-delta; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism
D017931:DNA Primers; D007527:Isoenzymes; D014443:Tyrosine; D000255:Adenosine Triphosphate; C497259:Prkcd protein, mouse; D011493:Protein Kinase C; D051745:Protein Kinase C-delta