Document Detail

Identification of the activator-binding residues in the second cysteine-rich regulatory domain of protein kinase Cθ (PKCθ).
MedLine Citation:
PMID:  23289588     Owner:  NLM     Status:  MEDLINE    
PKC (protein kinase C) θ is predominantly expressed in T-cells and is critically involved in immunity. Design of PKCθ-selective molecules to manage autoimmune disorders by targeting its activator-binding C1 domain requires the knowledge of its structure and the activator-binding residues. The C1 domain consists of twin C1 domains, C1A and C1B, of which C1B plays a critical role in the membrane translocation and activation of PKCθ. In the present study we determined the crystal structure of PKCθC1B to 1.63 Å (1 Å=0.1 nm) resolution, which showed that Trp(253) at the rim of the activator-binding pocket was orientated towards the membrane, whereas in PKCδC1B the homologous tryptophan residue was orientated away from the membrane. This particular orientation of Trp(253) affects the size of the activator-binding pocket and the membrane affinity. To further probe the structural constraints on activator-binding, five residues lining the activator-binding site were mutated (Y239A, T243A, W253G, L255G and Q258G) and the binding affinities of the PKCθC1B mutants were measured. These mutants showed reduced binding affinities for phorbol ester [PDBu (phorbol 12,13-dibutyrate)] and diacylglycerol [DOG (sn-1,2-dioctanoylglycerol), SAG (sn-1-stearoyl 2-arachidonyl glycerol)]. All five full-length PKCθ mutants exhibited reduced phorbol-ester-induced membrane translocation compared with the wild-type. These results provide insights into the PKCθ activator-binding domain, which will aid in future design of PKCθ-selective molecules.
Ghazi M Rahman; Sreejesh Shanker; Nancy E Lewin; Noemi Kedei; Colin S Hill; B V Venkataram Prasad; Peter M Blumberg; Joydip Das
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Intramural; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  The Biochemical journal     Volume:  451     ISSN:  1470-8728     ISO Abbreviation:  Biochem. J.     Publication Date:  2013 Apr 
Date Detail:
Created Date:  2013-03-15     Completed Date:  2013-05-02     Revised Date:  2013-08-08    
Medline Journal Info:
Nlm Unique ID:  2984726R     Medline TA:  Biochem J     Country:  England    
Other Details:
Languages:  eng     Pagination:  33-44     Citation Subset:  IM    
Department of Pharmacological & Pharmaceutical Sciences, College of Pharmacy, University of Houston, Houston, TX 77204, USA.
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MeSH Terms
Amino Acid Substitution
Binding Sites
Enzyme Activators / chemistry*,  metabolism
Isoenzymes / chemistry,  genetics,  metabolism*
Mutation, Missense
Protein Kinase C / chemistry,  genetics,  metabolism*
Protein Structure, Tertiary
Protein Transport
Tryptophan / chemistry,  genetics,  metabolism
Grant Support
Reg. No./Substance:
0/Enzyme Activators; 0/Isoenzymes; 73-22-3/Tryptophan; EC 2.7.1.-/Prkcq protein, mouse; EC Kinase C
Erratum In:
Biochem J. 2013 Jul 1;453(1):153

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