Catecholamines (CA) are important in regulating a number of central and peripheral nervous system functions. For this reason understanding their fundamental neurobiological properties has been of great interest over the past three decades. An important property of these cells is their capacity to precisely regulate neurotransmitter level in proportion to the degree to which they are stimulated, thus enabling them to meet secretory demands. The phosphorylation and activation of tyrosine hydroxylase (TH), the rate-limiting and controlling enzyme in the CA synthetic pathway, is believed to be the principal mechanism of this regulatory process. Research in this laboratory over the past thirteen years has addressed the role of phosphorylation of TH in catecholamine regulation using an isolated cell model of synthesis regulation, bovine adrenal chromaffin cells. These studies showed that TH phosphorylation is physiologically regulated and that phosphorylation occurs on multiple TH sites; the four sites on TH that are phosphorylated in situ are ser8, ser19, ser31 and ser40. More recently, correlation of the phosphorylation of these phosphorylation sites with the regulation of catecholamine biosynthesis in response to acetylcholine, showed that it is the phosphorylation of ser19, ser31 and ser40 that is pivotal for regulation related to cell depolarization. In vitro studies indicate that a calcium and calmodulin-activated protein kinase (CaM-KII) and extracellular receptor activated protein kinase phosphorylate TH on these sites. The studies proposed for the next period of this grant will determine the cellular and molecular mechanisms that regulate TH activity in response to TH phosphorylation by CaM-KII. The specific goals of the proposed research are to: 1) use a cell permeable polypeptide inhibitor of CaM-KII to determine if CaM-KII is responsible for the TH phosphorylation and activation. 2) determine whether TH, 14-3-3 and CaM-KII exist as a macromolecular complex that facilitates the rapid CaM-KII activation of TH, 3) determine why ser40 phosphorylation of TH by protein kinase C and CaM-KII does not activate TH, (the hypothesis that CA binding to TH subunits blocks TH phosphorylation by these protein kinases will be tested).
