Characterization of Protein Kinase C θ Activation Loop Autophosphorylation and the Kinase Domain Catalytic Mechanism
| Autor: | Lin Liu, Ron Kriz, Rita Greco, Mark Stahl, Karl Malakian, Divya Chaudhary, Robert M. Czerwinski, Laura Lin, Scott Wolfrom, Ann Aulabaugh, Stephane Olland, Ying Huang |
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| Rok vydání: | 2005 |
| Předmět: |
Threonine
Molecular Sequence Data Biology Mitogen-activated protein kinase kinase Binding Competitive Biochemistry Catalysis Substrate Specificity MAP2K7 Adenosine Triphosphate Catalytic Domain Serine ASK1 Amino Acid Sequence c-Raf Phosphorylation Kinase activity Protein Kinase C MAP kinase kinase kinase Osmolar Concentration Autophosphorylation Phosphoproteins Recombinant Proteins Cell biology Adenosine Diphosphate Enzyme Activation Isoenzymes Kinetics Protein Kinase C-theta Mutagenesis Site-Directed Cyclin-dependent kinase 9 |
| Zdroj: | Biochemistry. 44:9563-9573 |
| ISSN: | 1520-4995 0006-2960 |
| Popis: | Protein kinase C theta (PKCtheta), a member of the Ca(2+)-independent novel subfamily of PKCs, is required for T-cell receptor (TCR) signaling and IL2 production. PKCtheta-deficient mice have impaired Th2 responses in a murine ova-induced asthma model, while Th1 responses are normal. As an essential component of the TCR signaling complex, PKCtheta is a unique T-cell therapeutic target in the specific treatment of T-cell-mediated diseases. We report here the PKCtheta autophosphorylation characteristics and elucidation of the catalytic mechanism of the PKCtheta kinase domain using steady-state kinetics. Key phosphorylated residues of the active PKCtheta kinase domain expressed in Escherichia coli were characterized, and mutational analysis of the kinase domain was performed to establish the autophosphorylation and kinase activity relationships. Initial velocity, product inhibition, and dead-end inhibition studies provided assignments of the kinetic mechanism of PCKtheta(362)(-)(706) as ordered, wherein ATP binds kinase first and ADP is released last. Effects of solvent viscosity and ATPgammaS on PKCtheta catalysis demonstrated product release is partially rate limiting. Our studies provide important mechanistic insights into kinase activity and phosphorylation-mediated regulation of the novel PKC isoform, PKCtheta. These results should aid the design and discovery of PKCtheta antagonists as therapeutics for modulating T-cell-mediated immune and respiratory diseases. |
| Databáze: | OpenAIRE |
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