IBN 97-28317 LANDRETH The neurotrophins are growth factors that play critical roles in the development of the nervous system through their ability to sustain specific subpopulations of neurons and direct their morphological and biochemical differentiation. The prototypic neurotrophin, nerve growth factor (NGF), acts through a signaling pathway termed the "MAP kinase (MAPK) cascade". The MAPK cascadeincludes a group of enzymes known as serine/threonine kinases (and referred to as members of the "raf" family), as well as other enymes (kinases) that are serially activated. The present application is focused on understanding how the kinases which comprise this cascade convey signals (the presence of growth factors outside the cell) from the membrane to target areas in the cytoplasm and nucleus which are essential for neuronal differentiation. In the previous grant period we have demonstrated that NGF treatment of a line of cultured cells, known as "PC12 cells" resulted in the activation of two raf family members, B-raf and c- raf. However, only B-raf was mechanistically linked to the activation of the MAPK cascade. We have found that the capacity to drive the MAPK cascade is correlated with the ability of B-raf, but not c-raf, to form a stable complex with the heat shock protein, HSP90. We hypothesize that HSP90 is a component of a molecular scaffold which serves to physically organize the MAPK cascade into a functional module. The first specific aim of this proposal is to establish the identity of the G-proteins (a class of protein in cell membranes which is involved in the transmission of chemical messages from the outside to the inside of a cell) which initiate activation of the MAPK cascade in PC12 cells. The involvement of newly discovered G-proteins in this process will be investigated. The action of the second member of the MAPK cascade, B-raf, will be explored through expression (activation of a gene in cultured cells) of a dominant neg ative B-raf molecule which blocks the action of the endogenous B-raf. These experiments will allow determination of the roles of these molecules in mediating NGF action. We will also investigate whether the MAPK cascade is physically organized as a signaling module. Finally, the PC12 cells are a widely used neuronal model, however, a variety of data suggest that signaling events in these cells may not accurately reflect those occurring in authentic neurons. We propose to investigate the cellular mechanisms subserving the activation of the MAPK cascade in these primary neurons. 1
