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Crystallographic analysis of protein kinase c


Protein kinase C (PKC) is a multi-domain serine/threonine kinase that is activated by diacylglycerol (DAG), a short-lived derivative of phosphatidylinositol, 4,5-biphosphate (PIP2) and phorbol esters. The kinase activity of PKC is regulated by its N-terminal region that contains the binding sites for DAG and phorbol esters (C1 domain) and Ca2+ and phospholipids (C2 domain). The PKC family includes several isozymes which define two distinct groups; the Ca2+-dependent (cPKCs) and the Ca2+-independent (nPKCs). The inability of nPKCs to be regulated by Ca2+ has been attributed to the absence of a classical C2 domain. We have recently expressed large quantities of a C2-like domain of a novel PKC (nPKC) in E. coli . We have successfully obtained crystals of this domain suitable for X-ray analysis. We therefore intend to determine the structure of this domain. Moreover, there is recent evidence that this domain influences the interaction between PKC and one of its substrates (Dekker & Parker, data unpublished). Once the crystal structure of this domain has been determined, we will pursue co-crystallization trials of PKC bound to interacting peptides derived from the substrate. Individual domain structures provide an insight into the binding sites for activating ligands of PKC. However, they do not address the overall architecture of the full length molecule or the specific domain:domain interactions. Such interactions may ultimately define the mechanism of regulation of full length PKC. In parallel to our efforts with the C2-like domain, we intend to prepare constructs of the entire catalytic and regulatory domains and to produce such constructs in E. coli . Our current success with the C2-like domain, has encouraged these efforts. Moreover, full length novel PKC isozymes have become available to us from our collaborators (Dr. Peter Parker, ICRF) and attempts to obtain high quality crystals of the full length isozymes have been initiated. To conclude, the solution of the structure of a PKC C2-like domain will provide an additional component of the PKC structure. Moreover, the co-crystallisation trials with the substrate peptides will provide information on residues involved in the PKC:substrate interaction. Our efforts will also extend to studying constructs of consecutive PKC domains in addition to the full length protein with the hope that obtaining even a moderate resolution crystal structure of the full length molecule will provide an insight to the overall architecture and regulation of an essential part of the cell signalling machinery.

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Imperial Cancer Research Fund
44 lincoln's inn fields
WC2A 3PX London
United Kingdom

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