Abnormalities in pancreatic Beta-cell(B-cell) insulin secretion mechanisms can lead to serious disorders, such as diabetes. Metabolic regulation of insulin exocytosis is only partly understood. Proximal signaling events have been relatively well defined, but less is known about the molecular events necessary to regulate insulin release via exocytosis. Increased B-cell metabolism evokes a series of ion channel activities leading to a rapid rise in cytosolic (Ca2+)i, that has long been presumed to be a major secondary signal to stimulate insulin release. However, it is now clear that additional secondary signals are necessary (including GTP elevation of cytosolic long chain fatty acyl moieties, and certain protein kinase activities), and that (Ca2+)i might be merely a cofactor for exocytosis to proceed. Nonetheless, to date, a connection between secondary signals emanating from B-cell metabolism and the B-cell's exocytotic machinery has yet to be made. Currently, protein kinase activities are thought to be the best candidates for making such a connection, however the appropriate protein kinas substrates that control insulin exocytosis have yet to be identified. A long-term objective of the proposed research is to better define the molecular mechanism underlying regulation of insulin exocytosis. In the past period of funding, in order to gain a """"""""handle"""""""" on the B-cell's exocytotic machinery, the investigators focused on the B-granule associated GTP-binding protein, rab3A. It was found in B-cells that the rab3A effector protein was calmodulin, which associated with rab3a in a GTP- and Ca2+-dependent manner. This provides a means where calmodulin can be recruited to a B-granule, then subsequently transferred to other calmodulin binding proteins (e.g. CaMK-II, calceneurin etc.) as local cytosolic (Ca2+)i increases for their specific activation on a B-granule and/or site of exocytosis. The PI has generated experimental evidenc to support such a hypothesis, which in turn has led the PI to become very interested in Ca2+-dependent phosphorylation of b -granule membrane proteins, as a direct connection between proximal signals (i.e. elevated cytosolic (Ca2+)i, long chain fatty acyl moieties and /or protein kinase activation) and regulation of the B-cell's exocytotic machinery at the molecular level. In preliminary experiments the PI observed five B-granule membrane proteins that get phosphorylated in a Ca2+ -dependent manner (pp138, pp52, pp42, pp26 and pp19), and three that are directly phosphorylated in a fatty acid-induced PKC-mediated manner (pp105, pp36 and pp14). This current proposal is focused o identifying these B-granule phosphoproteins by purification and mass spectrometry microsequence analysis, and their subsequent cloning. It is then intended to characterize the regulated phosphorylation of these B-granule membrane proteins as being key for metabolic control of insulin exocytosis at the molecular level. New insight into the control of insulin secretion should be gained from these studies that may eventually lead to novel diabetes therapies.
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