Cytoplasmic and receptor tyrosine kinases, Ras small G proteins, heterotrimeric G proteins, and tumor promoting phorbol esters are among the major regulators of various signaling systems that modulate cell growth, differentiation and development. Inappropriate regulation can result in a variety of diseases due to improper differentiation/development or malignant transformation. One of these signaling systems, often referred to as the MAP kinase pathway, is composed of a regulated serine/threonine kinase module, downstream of the c-Ras protooncogene. Presented simply, this module consists of the protooncogene c-Raf - greater than the dual-specificity kinase MEK - greater than the mitogen-activated kinase, ERK. An ERK-activated kinase, RSK, lies downstream of the ERKs. ERK and RSK translocate to the nucleus in response to mitogens, providing a mechanism for signal transduction from the cytoplasm to nucleus. The research proposed in this application addresses issues regarding the regulation of ERK/RSK activation and downstream signaling. The first objective is to characterize novel inputs into the ERK pathway, downstream of Ras, Raf and MEK, that play an important role in the activation kinetics of ERK and RSK, and subsequent nuclear signaling. An activity that is inactivated upon mitogen stimulation and that negatively regulates the ERKs has been previously identified. Experiments are proposed to biochemically or molecularly identify ERK phosphatases or noncatalytic inhibitors that exhibit the properties of this inhibitor. Candidate molecules will also be examined. The second objective of this proposal is to thoroughly characterize the regulation and biological function of RSK, about which little is known. Experiments proposed to characterize RSK include: structure/function of analysis, identification and mutational analysis of novel, ERK-mediated, and auto phosphorylation sites, characterization of dominant-negative RSK alleles and either effects of downstream targets, the mechanism of RSK nuclear translocation, the regulation of gene expression by RSK, and the role of RSK in regulating cell proliferation. The third major objective of this proposal characterizes nuclear signal transduction by ERK and RSK. The focus of this aim will be on the characterization of c-Fos phosphorylation by ERK and RSK, and the subsequent recruitment of a novel Fos kinase activity. Experiments described are aimed at identifying the Fos kinase using biochemical or molecular approaches. Candidate molecules will also be examined.
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