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 bi nd diacylglycerol and phorbol esters with high affinity. Highly conserved h ydrophobic residues at or near the rim of the binding cleft in the second c ysteine-rich domain of PKC-delta (PKC-delta C1b) were mutated to probe thei r roles in ligand recognition and lipid interaction. [H-3]Phorbol 12,13-dib utyrate (PDBu) binding was carried out both in the presence and absence of phospholipids to determine the contribution of lipid association to the lig and affinity. Lipid dependence was determined as a function of lipid concen tration 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 phosph atidylserine, Mutation of Leu-20 to Arg or of Leu-24 to Lys caused a dramat ic (340- and 250-fold, respectively) reduction in PDBu binding in the prese nce of lipid but only a modest reduction in the weaker binding of PDBu obse rved in the absence of lipid, suggesting that the main effect was on C1 dom ain -phospholipid interactions. Mutation of Leu-20 to Lys or of Trp-22 to L ys had modest (S-fold) effects and mutation of Phe-13 to Tyr or Lys was wit hout effect. Binding of the branched diacylglycerol was less dependent on p hospholipid and was more sensitive to mutation of Trp-22 to Tyr or Lys, esp ecially in the presence of phospholipid, than was PDBu, In terms of specifi c 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 general ly, the results emphasize the interplay between the C1 domain, ligand, and phospholipid in the ternary binding complex.