The long-term goal of my research is to understand the mechanism underlying the regulation of synaptic plasticity in the brain. Specifically, I am interested in signal transduction which involves protein kinase C (PKC) activation by lipid second messengers generated through phospholipases. In this proposal, we plan to study the effect of cis-fatty acids (CFA) on the activation of PKC activity using clonal neuroblastoma cells and to further investigate CFA-mediated signal transduction in rat hippocampus. It has been known that CFA can activate brain PKC in vitro in the absence of Ca2+, phospholipids and diacylglycerol (DAG). Recently, we shoed that CFA activates type III (alpha) PKC synergistically with DAG. These studies suggest that there exists another signaling pathway for PKC activation, in addition to the receptor-mediated polyphophoinositide breakdown/DAG formation pathway. Physiologically, CFAs such as arachidonic acid and oleic acid have been shown to enhance long-term potentiation and to modulate ion channels in the hippocampus, and these effects have been attributed specifically to the cis-fatty acid activation of PKC. However, the second messenger role of CFA in the activation of PKC has not yet been established. This is mostly due to lack of definitive evidence for direct CFA activation of PKC in whole cells. We will therefore first test whether CFA directly activates PKC in intact neuroblastoma N1E-115 cells by monitoring CFA-induced protein phosphorylation. Specifically, we will study the phosphorylation of 40/38 kDa doublet protein to examine the significance of CFA effect by setting criteria that should be fulfilled if the phosphorylation is mediated by PKC. We will next study the effect of CFA on PKC activation in the hippocampus, where we have identified two (44 and 47 kDa) CFA- sensitive phosphoproteins. In addition, phosphorylation of these proteins is stimulated synergistically by exogenously applied CFA and DAG. We wish to examine whether endogenously generated CFAs by receptor activation, particularly glutamate receptors, can stimulate hippocampal PKC by measuring receptor-induced lipid metabolism and protein phosphorylation within the hippocampus. We will then characterize these in vivo PKC substrates, which are responsive to signals that involve free fatty acid generation.
