The overall goal is to determine how the ubiquitous intracellular Ca2+ binding calmodulin (CaM) can regulate a wide variety of target proteins in a specific manner. The objective is to elucidate mechanisms that govern regulation of a proposed CaM kinase (CaMK) cascade that was discovered in part, as a outcome of the previous work.
The first aim i s to use the novel reagents, and current knowledge of mammalian CaMKI and IV to determine the mechanisms involved in their regulation by activating CaMK kinases (CaMKK) and examine the physiological relevance of this cascade in mammalian cells. The investigators have cloned and expressed several members of the CaMK family from 4 species (humans, rats, A. nidulans and C. elegans). In mammals the investigators have CaMKI and IV, CaMKK alpha and beta. Whereas the KKs both phosphorylate a single Thr residue located in the activation loops of CaMKI an IV, the regulation of CaMKIV is more complicated than CaMKI. In addition CaMKI is a nuclear enzyme whereas CaMKI is not. The investigators will address the sequence of events required to activate CaMKIV and to enter the nucleus as well as the mechanism of activation and subcellular localization of the 2CaMKKs. These experiments will be carried out using recombinant proteins as well as in Jurkat T cells. They have developed an assay in which CaMKIV is used to drive Ga14-CREB system via phosphorylation of CREB on SER-133 in Jurkact cells. This assay requires an increase in intracellular Ca2+ and phosphorylation of Thr-20 on CamKIV by a CaMKK. They will examine which steps occur in cytoplasm versus nucleus. CaMKK beta contains putative SH3 binding sites in the N-terminus and bipartite nuclear localization sequence/14.3.3 binding site that overlaps the C-terminus of the putative CaM binding site. The investigators will determine the relevance of these domains in vitro and in Jurkat cells.
The second aim i s to evaluate mechanisms involved in activation of enzymes that are homologs to CaMKI/IV and CaMKK in A. nidulans and C. elegans and how these cascades might influence well cycle progression, development or differentiation in these genetically tractable organisms. They will examine the properties of these kinases, determine if they are essential in A. nidulans and, if so, what biological pathway they control. Similar genetic studies in C. elegans will reveal in what cells the CaMKK is expressed and how expression changes during development. Finally, antisense RNA will be used to deplete the CaMKK mRNA to test the functional significance of the enzyme. These complementary approaches will yield exciting new insights into structure/function relationships between CaM and physiologically important CaMKs in these organisms.
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