Abstract
The PKC isozymes represent the most prominent family of signaling proteins mediating response to the ubiquitous second messenger diacylglycerol. Among them, PKCθ is critically involved in T-cell activation. Whereas all the other conventional and novel PKC isoforms have twin C1 domains with potent binding activity for phorbol esters, in PKCθ only the C1b domain possesses potent binding activity, with little or no activity reported for the C1a domain. In order to better understand the structural basis accounting for the very weak ligand binding of the PKCθ C1a domain, we assessed the effect on ligand binding of twelve amino acid residues which differed between the C1a and C1b domains of PKCθ. Mutation of Pro9 of the C1a domain of PKCθ to the corresponding Lys9 found in C1b restored in vitro binding activity for [3H]phorbol 12,13-dibutyrate to 3.6 nM, whereas none of the other residues had substantial effect. Interestingly, the converse mutation in the C1b domain of Lys9 to Pro9 only diminished binding affinity to 11.7 nM, compared to 254 nM in the unmutated C1a. In confocal experiments, deletion of the C1b domain from full length PKCθ diminished, whereas deletion of the C1a domain enhanced 5-fold (at 100 nM PMA) the translocation to the plasma membrane. We conclude that the Pro168 residue in the C1a domain of full length PKCθ plays a critical role in the ligand and membrane binding, while exchanging the residue (Lys240) at the same position in C1b domain of full length PKCθ only modestly reduced the membrane interaction.