Protein kinase C (PKC) has served as the prototype of lipid-regulated enzymes, especially in the case of the phosphatidylinositol (PI) cycle whereby PI-derived diacylglycerol (DAG) serves as the direct activator of PKC. Given the rapid turnover of the PI cycle (within 90 seconds of receptor engagement), this paradigm of PKC activation has by necessity focused on the very acute phase of PKC regulation. Importantly, this paradigm does not address other mechanisms of DAG generation, most notably through phospholipase D (PLD), it does not distinguish mechanisms for regulating activation of specific isoenzymes, and it focuses only on the plasma membrane as a site for PKC action. Extensive results (obtained during the past 5 years) as well as ongoing results have generated exciting information on novel mechanisms and functions of PKC involving translocation of PKC to a novel juxtanuclear/pericentriolar compartment. This novel process requires sustained activation of PKC (30-60 min) in response to ligands of G-protein-coupled receptors (and phorbol esters), and it requires continuous activation of PKC as well as PLD (unlike the acute translocation of PKC to the PM). Functionally, receptor activation leads not only to the translocation of the receptor itself to this juxtanuclear compartment (which has features of a previously-identified but not well-appreciated component of slow recycling endosomes), but also the co-translocation of many other plasma membrane/recycling receptors, channels, other proteins and lipids. Moreover, very recent evidence is beginning to point to active signaling in this novel compartment. Based on these observations, we hypothesize that receptor-induced sustained activation of PKCa/ssII induces the formation of this novel pericentriolar endocytic compartment in a PKC and PLD- dependent manner. This mechanism plays a key role in receptor sequestration and in the regulation of cross- sequestration of other receptors and PM proteins, and may initiate novel signaling in this compartment. This hypothesis will be investigated by pursuing the following specific aims 1) Define and establish ligand- induced novel translocation of receptors and formation of this novel compartment, 'the pericentrion';2) Define roles of receptor-induced and PKC/PLD-mediated pericentrion formation in sequestration of recycling components, their fate, and their function;and 3) Define roles of receptor-induced and PKC/PLD-mediated pericentrion formation in signaling. The proposed and ongoing studies are defining a novel compartment, novel functions of receptors in regulating endocytic trafficking, and the possibility of a novel PKC/PLD signaling compartment. If correct, this novel regulated process may be involved in multiple physiologic and pathophysiologic processes that depend on availability of key proteins and/or lipids at the PM, including regulation of smooth muscles and endothelial cells, both of great importance to vascular biology.
Membrane receptors are critical proteins that serve as targets for many natural compounds (e.g. serotonin, adrenaline, dopamine, and angiotensin) as well as pharmacologic agents important for blood pressure regulation, blood clotting, gastric ulcers and stomach acidity, treatment of depression, and other diseases). We have discovered that sustained stimulation of some of these receptors can result in profound changes in how the cell handles the distribution and function of many other receptors and key proteins. We are defining the significance and mechanisms by which this process occurs and is regulated. In particular we focus on a key signaling protein known as protein kinase C (PKC) and its activating lipids.
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