Identification of the Activator Binding Residues in the Second Cysteine-Rich Regulatory Domain of Protein Kinase C Theta (PKCθ)



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Protein kinase C theta (PKCθ) is a serine threonine kinase, which is predominantly expressed in the T-cells and is selectively translocated in the immunological synapse upon activation. Active PKCθ initiates the downstream activation of the immunological responses against the intruders. Selective PKCθ inhibition may manage autoimmune disorders. PKCθ belongs to novel class of PKCs. Diacylglycerol (DAG) and phorbol esters are the common activators, which bind to the C1 domains of the novel and conventional PKCs. Design of PKCθ selective inhibitors targeting its activator binding C1 domain, requires the knowledge of C1 domain structure and the activator binding residues. PKCθ C1 domain consists of twin cysteine-rich subdomains, C1A and C1B, of which C1B plays a major role in the activation followed by the membrane anchoring PKC. To this end we determined the crystal structure of PKCθC1B subdomain at 1.63 Å, which showed a similar overall structure to that of PKCC1B, except that the orientation of the Trp-253 residue is towards the membrane and the width of the activator pocket opening is narrower. The homologous Trp-252 in PKCC1B is oriented away from the membrane with wider activator pocket opening. Using this structure, five possible activator binding residues were identified though the overlaying of the crystal structure, and alignment of the sequences of PKCθC1B and PKCC1B, followed by molecular docking of a library of DAG and phorbol ester analogs into PKCθC1B as receptor. To determine the role of these residues, Y239A, T243A, W253G, L255G and Q258G mutants in isolated PKCθ C1B domain were designed, expressed and purified from E. coli and their binding affinity (Kd or Ki) for phorbol 12, 13-dibutyrate (PDBu) and Sn-1,2-dioctanoylglycerol (DOG) were measured by radioactive PDBu binding assay. All the mutants showed significantly reduced binding affinity for both PDBu and DOG. Among all the mutants, Q258G showed highest reductions in activator binding affinity than the wild type. The extent of reductions in the binding affinity for θC1B mutants of Y239A, W253A and L255G were different however was much lesser than the homologous mutations in C1B. All the five mutants of full length PKCθ were expressed in HEK293 cells and showed reduced phorbol 12-myristate 13-acetate (TPA) and DOG induced membrane translocation compared to wild type. These results provide insights into the PKC C1B activator binding domain, which will aid in future design of PKCθ selective inhibitors.



Protein kinase C, Activation, Tryptophan, Crystal structures, Membrane translocation, Phorbol ester, Diacylglycerol, Phospholipids, Activator binding residues