The long range goal of this project has been to understand the sequence of events leading from the initial interaction of phorbol esters (PEs) with a cell to later effects of these tumor promoters on expression of specific cell products. PE-stimulated production of Interleukin 2 (IL2) has been studied as a model system. Past work has identified the first step in this pathway as binding of PEs to specific receptors and established that the receptor is Protein Kinase C (PKC). Since IL2 production requires a transcriptional event, means of transmitting the signal from PKC to the nucleus have been sought. Evidence does not support translocation of PKC to nuclei so it has seemed likely that an early PKC-catalyzed phosphorylation event is required to generate a mediator involved in nuclear signalling. Recent studies by our lab and others have raised many questions about how PKC is activated and therefore where in the cell the activation and subsequent mediator generation might occur. Since these questions are of major importance in understanding the early steps in a signal transduction cascade, they will form the focus for this grant. The nature of the requirements for PKC activation will be studied in Aim I. Short chain phospholipids (PCs and PSs) which activate PKC at concentrations near their cmcs will be used as a model to investigate possible requirements for substrate aggregation on the lipid surface, dimerization or higher order structure of PKC, and the importance of the physical state and dynamic properties of the lipid. The roles of calcium and diacylglycerol or PEs in the association of PKC or substrate with phospholipid as distinct from activation of the enzymatic activity will be studied with fluorescene, nmr spectroscopy and other techniques. The possibility that similar states of the lipid are required in long chain systems will be examined by studies on the effects of phospholipid transition temperature (Tm), phase separations, and lipid packing on PKC activity.
In Aim II the effects of phosphorylation on PKC activity and subcellular localization will be examined. Kinases that affect PKC activity and association with subcellular organelles will be identified.
In Aim III, proteins synthesized and/or phosphorylated in response to PE will be identified and the subcellular localization/translocation of these proteins will be investigated.
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