The long-term objective of this research is to elucidate the molecular mechanism of protein kinase C's regulation. Isozymes of this ubiquitous family of proteins transduce the plethora of signals that promote lipid hydrolysis. The proposed research is aimed at elucidating how lipid regulates the structure and function of this key signal transducer. A combination of biochemical, biophysical, molecular biological, and molecular modeling approaches are proposed in order to analyze the contribution of specify protein determinants in 1] targeting protein kinase C to acidic membranes, 2] inducing specific binding to diacylglycerol and phosphatidylserine, and 3] promoting release of the autoinhibitory pseudosubstrate from the active site allowing catalysis. In addition, the regulatory role of phosphorylation will be addressed.
Four specific aims are described below:
The first aim addresses the question: how do diacylglycerol and phosphatidylserine, in concert, induce the high-affinity membrane interaction that is required for pseudosubstrate release and activation? Experiments will address whether diacylglycerol and phosphatidylserine binding sites interact allosterically, whether phosphatidylserine is required to structure the diacylglycerol binding site, whether phorbol esters regulate protein kinase C by the same mechanism as diacylglycerol, and whether atypical protein kinase Cs display the same regulation by diacylglycerol and phosphatidylserine as the other isozymes. Second, the hypothesis that all isozymes of protein kinase C bind to acidic membranes by a common mechanism that involves recognition of acidic lipids by a conserved domain will be tested. The possibility that this domain is already structured for acidic lipid-recognition by the Ca2+- independent isozymes, but requires binding of Ca2+ in order to be structured in the conventional isozymes will be explored.
The third aim tests the role of specific residues in controlling the pseudosubstrate:active site interactions, and in regulating interfacial contacts between the catalytic and regulatory domains. Lastly, the role of phosphorylation on the structural stability, lipid interaction, and function of protein kinase C will be addressed. Experiments are proposed to determine whether the intramolecular autophosphorylation prolongs or inhibits the activated state of protein kinase C, and to test the hypothesis that phosphorylation by another kinase converts protein kinase C from an inactive precursor to a form that is activatable by lipid. Uncontrolled activation of protein kinase C results in malignant transformation; by understanding the enzyme's regulation, new insights into the prevention and treatment of cancer and other diseases resulting from faulty signalling will be possible.
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