DESCRIPTION: Numerous studies have demonstrated that mitogen-activated protein kinase (MAPK) cascades link environmental signals to various cellular responses. Recent work by the applicant has focused on searching for MAP kinase substrates and has led to the identification of a potentially new class of protein-serine/threonine kinases that are associated with, and apparently activated by, specific MAP kinases. This class includes at least two closely related molecules, termed Msk1 and Msk2, and at least one other more distant member that also appears to be a kinase, i.e., Mapkip 108. Based on preliminary studies, the applicant proposes a model of Msk regulation wherein inactive Msk exists in a complex with inactive ERK (mitogen-regulated) and p38 (stress-activated) MAP kinases in the cell. In response to certain extracellular signals, the MAP kinases (ERK and p38, but not JNK) phosphorylates and activates the bound Msk. Thus, the overall goal of these studies is to assess the significance of these new MAP kinase substrates in regulating cell proliferation and stress responses. To this end, the following five Specific Aims are proposed: 1) To establish the mechanism of Msk activation by developing a Msk assay and ascertaining how ERK and p38 activation influences Msk (principally Msk2) phosphorylation and kinase activity. 2) To investigate the relevance of Msk regulation by ERK or p38 for the cellular response to mitogens, oncogenes, developmental cues, or stresses. These studies will use over-expressed or mutated Msk2 and analyses of cell behavior such as foci formation, actin reorganization and sensitivity to oncogene transformation. 3) To identify the molecular targets of Msk via an assessment of the subcellular localization of Msk together with approaches such as the use of a peptide library for kinase specificity determinations and the yeast two hybrid system for examining protein-protein interactions. 4) To map the interactions between ERK and Msk using approaches such at the two-hybrid system. 5) To clone Mapkip108 using conventional techniques.