The long-term goal of the proposed research is to understand the molecular mechanisms and biological function of protein kinase C (PKC). PKC has been in the spotlight since the discovery 25 years ago that it is activated by the lipid second messenger, diacylglycerol. Despite PKC's enduring stage presence and tremendous advances in understanding the enzymology and regulation of this key protein, understanding the function of PKC in biology is still under intense pursuit. In the preceding funding period we focused on understanding the molecular mechanisms of PKC as a first step in understanding how this key protein functions in the cell. The goal of this proposal is to both continue elucidating the molecular mechanisms of PKC regulation and to use this knowledge to address the cellular function of PKC. Specific Aims are: 1. Mechanism of Protein Kinase C's Membrane Interaction. The goal of this Specific Aim is to understand how the C1 and C2 domains target and retain PKC on the membrane. These studies continue our long-standing research in understanding the molecular mechanisms of how lipid mediators regulate PKC. Biophysical and cellular studies will be coupled to gain greater insight into the mechanism of translocation of PKC in vivo. 2. Regulation of Protein Kinase C by Dephosphorylation. In the previous funding period, we made major advances in understanding how phosphorylation controls PKC. As part of this work, it became apparent that dephosphorylation, rather than phosphorylation, may be the agonist-dependent regulator of the phosphorylation state of PKC. The goal of this Specific Aim is to understand the mechanism and role of dephosphorylation of PKC in regulating its function. 3. Protein Kinase C Function In Vivo. Using chemical genetics and novel fluorescence technologies, we will test the hypothesis that a major role of PKC is to activate cellular phosphatase activity. This hypothcsis is based on evidence in the previous funding period that PKC activation increases phosphatase activity in cells. Upregulation of phosphatase activity could account for the pleiotropic effects of PKC activation.