Nerve growth factor (NGF) acts to direct the survival and differentiation of specific populations of neurons in the periphery and in the central nervous system. The cellular mechanisms by which NGF elicits changes in these neurons has not been resolved. This proposal is focused on defining the specific biochemical steps by which the binding of NGF at the cell surface is transduced, ultimately resulting in changes in gene expression and cellular morphology. In the previous grant period we have identified several enzymes which participate in a signal transduction pathway, termed the MAP kinase cascade. NGF stimulates the serial activation of the protein kinases which comprise this pathway and mediate the transmission of signals from the membrane into the nucleus. This application is directed toward understanding how the most proximal member of this cascade, B-raf, is activated and how the cell can selectively employ this molecule to regulate the flow of information to the interior of the cell. A second aim of this proposal is to investigate the mechanism through which B-raf activates the next member of the cascade, MEK. MEK is phosphorylated and activated by B-raf, however, how this is achieved is unclear and it is of particular interest from a mechanistic viewpoint. MEK in turn activates the MAP kinases and we propose to investigate the biological consequences of their action by inhibiting their actions by introduction of mutant MAP kinase molecules into NGF sensitive cells. The mutant MAP kinases act to block the activation of the normal enzymes, allowing dissection of their biochemical targets and identification of cellular processes which they critically regulate.
