Identification of the activator binding residues in protein kinase C theta C1B domain

Abstract

PKCtheta is a Ser/ Thr kinase, which is critically involved in immunity. Diacylglycerol (DAG) and phorbol esters (PE) bind to C1A and C1B domain to activate nPKC and cPKC. Selective PKCtheta inhibition may manage autoimmune disorders. Design of PKCtheta inhibitor targeting C1 domain requires the knowledge of C1 domain structure and its activator binding residues. In this direction we solved crystal structure of PKCtheta C1B and identified possible activator binding residues by molecular docking of a library of DAG and PE analogs into PKCtheta C1B as receptor. Five residues were identified, which are capable of binding to DAG/ PE analogs. To determine the role of these residues in activator binding Y239A, T243A, W253G, L255G and Q258 in isolated C1B domain were designed, expressed and purified from E. coli and their binding affinity (Kd/Ki) for Phorbol 12, 13‐Dibutyrate (PDBu) and Dioctanoylglycerol (DOG) were measured by radioactive PDBu binding assay. All the mutants showed significantly reduced binding affinity for both PDBu and DOG whereas Q258 showed the maximum reduction than the wild type (WT). Same mutations in full length PKCtheta were expressed in HEK293 cells and showed reduced Phorbol 12‐Myristate 13‐Acetate and DOG induced membrane translocation compared to WT. Our studies indicated that these residues are important for activator binding and will aid in future design of the PKCtheta inhibitor.