Characterization of a Protein Kinase C-δ (PKC-δ) ATP Binding Mutant

To investigate the function of protein kinase C (PKC)-δ, 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-δ cDNAs were generated either with or without an influenza virus hemagglutinin epitope tag. After expression in 32D cells by transfection, the PKC-δ ATP binding mutant (PKC-δK376R) was not able to phosphorylate itself or the PKC-δ pseudosubstrate region-derived substrate, indicating that PKC-δK376R was an inactive enzyme. PKC activity was inhibited by 67% in 32D cells coexpressing both PKC-δ wild type (PKC-δWT) and PKC-δK376R when compared to 32D cells expressing only PKC-δWT. Mixture of PKC-δWT and PKC-δK376R kinase sources in vitro also reduced the enzymatic activity of PKC-δWT. These results suggest that PKC-δK376R competes with PKC-δWT and inhibits PKC-δWT phosphorylation of its in vitro substrate. While PKC-δWT overexpressed in 32D cells demonstrated 12-O-tetradecanoylphorbol-13-acetate (TPA)-dependent translocation from the cytosolic to the membrane fraction, PKC-δK376R was exclusively localized in the membrane fraction even prior to TPA stimulation. Unlike PKC-δWT which was phosphorylated on tyrosine residue(s) only after TPA treatment, PKC-δK376R was constitutively phosphorylated on tyrosine residue(s). Although exposure of PKC-δWT transfectants to TPA induced 32D monocytic differentiation, the 32D/PKC-δK376R transfectants were resistant to TPA-induced differentiation. Thus, expression of active PKC-δ is required to mediate 32D monocytic differentiation in response to TPA stimulation. To investigate the function of protein kinase C (PKC)-δ, 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-δ cDNAs were generated either with or without an influenza virus hemagglutinin epitope tag. After expression in 32D cells by transfection, the PKC-δ ATP binding mutant (PKC-δK376R) was not able to phosphorylate itself or the PKC-δ pseudosubstrate region-derived substrate, indicating that PKC-δK376R was an inactive enzyme. PKC activity was inhibited by 67% in 32D cells coexpressing both PKC-δ wild type (PKC-δWT) and PKC-δK376R when compared to 32D cells expressing only PKC-δWT. Mixture of PKC-δWT and PKC-δK376R kinase sources in vitro also reduced the enzymatic activity of PKC-δWT. These results suggest that PKC-δK376R competes with PKC-δWT and inhibits PKC-δWT phosphorylation of its in vitro substrate. While PKC-δWT overexpressed in 32D cells demonstrated 12-O-tetradecanoylphorbol-13-acetate (TPA)-dependent translocation from the cytosolic to the membrane fraction, PKC-δK376R was exclusively localized in the membrane fraction even prior to TPA stimulation. Unlike PKC-δWT which was phosphorylated on tyrosine residue(s) only after TPA treatment, PKC-δK376R was constitutively phosphorylated on tyrosine residue(s). Although exposure of PKC-δWT transfectants to TPA induced 32D monocytic differentiation, the 32D/PKC-δK376R transfectants were resistant to TPA-induced differentiation. Thus, expression of active PKC-δ is required to mediate 32D monocytic differentiation in response to TPA stimulation.