Catalytic Domain Crystal Structure of Protein Kinase C-θ (PKCθ)

• Published in: The Journal of biological chemistry Vol. 279; no. 48; pp. 50401 - 50409
• Main Authors: Xu, Zhang-Bao, Chaudhary, Divya, Olland, Stephane, Wolfrom, Scott, Czerwinski, Robert, Malakian, Karl, Lin, Laura, Stahl, Mark L., Joseph-McCarthy, Diane, Benander, Christina, Fitz, Lori, Greco, Rita, Somers, William S., Mosyak, Lidia
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 26.11.2004
• Subjects: Amino Acid Sequence; Binding Sites; Crystallography, X-Ray; Humans; Isoenzymes - antagonists & inhibitors; Isoenzymes - chemistry; Isoenzymes - metabolism; Kinetics; Molecular Sequence Data; Peptide Fragments - metabolism; Protein Kinase C - antagonists & inhibitors; Protein Kinase C - chemistry; Protein Kinase C - metabolism; Protein Kinase C-theta; Protein Structure, Tertiary; Sequence Alignment; Staurosporine - metabolism; Substrate Specificity
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M409216200

A member of the novel protein kinase C (PKC) subfamily, PKCθ, is an essential component of the T cell synapse and is required for optimal T cell activation and interleukin-2 production. Selective involvement of PKCθ in TCR signaling makes this enzyme an attractive therapeutic target in T cell-mediated disease processes. In this report we describe the crystal structure of the catalytic domain of PKCθ at 2.0-Å resolution. Human recombinant PKCθ kinase domain was expressed in bacteria as catalytically active phosphorylated enzyme and co-crystallized with its subnanomolar, ATP site inhibitor staurosporine. The structure follows the classic bilobal kinase fold and shows the enzyme in its active conformation and phosphorylated state. Inhibitory interactions between conserved features of staurosporine and the ATP-binding cleft are accompanied by closing of the glycine-rich loop, which also maintains an inhibitory arrangement by blocking the phosphate recognition subsite. The two major phosphorylation sites, Thr-538 in the activation loop and Ser-695 in the hydrophobic motif, are both occupied in the structure, playing key roles in stabilizing active conformation of the enzyme and indicative of PKCθ autocatalytic phosphorylation and activation during bacterial expression. The PKCθ-staurosporine complex represents the first kinase domain crystal structure of any PKC isotypes to be determined and as such should provide valuable insight into PKC specificity and into rational drug design strategies for PKCθ selective leads.