Role of hydrophobic residues in the C1b domain of protein kinase C delta on ligand and phospholipid interactions.
The C1 domains of conventional and novel protein kinase C (PKC) isoforms bind diacylglycerol and phorbol esters with high affinity. Highly conserved hydrophobic residues at or near the rim of the binding cleft in the second cysteine-rich domain of PKC-delta (PKC-deltaC1b) were mutated to probe their roles in ligand recognition and lipid interaction. [(3)H]Phorbol 12,13-dibutyrate (PDBu) binding was carried out both in the presence and absence of phospholipids to determine the contribution of lipid association to the ligand affinity. Lipid dependence was determined as a function of lipid concentration and composition. The binding properties of a high affinity branched diacylglycerol with lipophilicity similar to PDBu were compared with those of PDBu to identify residues important for ligand selectivity. As expected, Leu-20 and Leu-24 strongly influenced binding. Substitution of either by aspartic acid abolished binding in either the presence or absence of phosphatidylserine. Mutation of Leu-20 to Arg or of Leu-24 to Lys caused a dramatic (340- and 250-fold, respectively) reduction in PDBu binding in the presence of lipid but only a modest reduction in the weaker binding of PDBu observed in the absence of lipid, suggesting that the main effect was on C1 domain -phospholipid interactions. Mutation of Leu-20 to Lys or of Trp-22 to Lys had modest (3-fold) effects and mutation of Phe-13 to Tyr or Lys was without effect. Binding of the branched diacylglycerol was less dependent on phospholipid and was more sensitive to mutation of Trp-22 to Tyr or Lys, especially in the presence of phospholipid, than was PDBu. In terms of specific PKC isoforms, our results suggest that the presence of Arg-20 in PKC-zeta may contribute to its lack of phorbol ester binding activity. More generally, the results emphasize the interplay between the C1 domain, ligand, and phospholipid in the ternary binding complex.
Molecular Mechanisms of Tumor Promotion Section, Laboratory of Cellular Carcinogenesis and Tumor Promotion and Laboratory of Medicinal Chemistry, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA.
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Wang
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Fang
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Nacro
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Marquez
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Wang
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Blumberg
D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000201:enzymology; D005982:Glutathione Transferase / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D007930:Leucine / Q000737:chemistry; D008024:Ligands; D050356:Lipid Metabolism; D008239:Lysine / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010703:Phorbol Esters / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism*; D011485:Protein Binding; D020033:Protein Isoforms; D011493:Protein Kinase C / Q000737:chemistry* / Q000378:metabolism*; D051745:Protein Kinase C-delta; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014443:Tyrosine / Q000737:chemistry
D007527:Isoenzymes; D008024:Ligands; D010703:Phorbol Esters; D010743:Phospholipids; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D014443:Tyrosine; D001120:Arginine; D005982:Glutathione Transferase; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; D007930:Leucine; D008239:Lysine; D003545:Cysteine