The family of 14-3-3 proteins were recently found to associate with many proteins involved in signal transduction and tumorigenesis. These 14-3-3- associated proteins include members of mitogen-activated protein kinase kinase kinases, such as Raf-1; a novel serine/threonine kinase, Bcr, and its oncogenic derivative, Bcr-Abl; and the primary transforming protein of polyomavirus, the middle t antigen. Compelling evidence has been presented that indicates the importance of 14-3-3 in cell growth control and tumorigenesis. However, the biochemical mechanism of the interaction and the precise function of 14-3-3 in the signaling pathways remain obscure. Our recent solution of the 3-dimensional structure of 14-3-3zeta has suggested a model of 14-3-3 interaction that involves a conserved amphipathic groove and a dimerization interface. We propose that binding to an amphipathic helix represents a general mechanism for the interaction of 14-3-3 with diverse cellular proteins, reminiscent of the mechanism proposed for calmodulin, another highly conserved protein that can bind a range of regulatory proteins. The conserved dimer interface of 14-3-3 may allow homo- and heterodimer formation which facilitates or stabilizes the functional association of two bound target proteins. These structural properties lead to a second hypothesis: that 14-3-3 may function as a molecular scaffold or anchor that tethers signaling molecules into an active complex or a productive pathway. Such an organization may contribute to the specificity of signaling pathways. Guided by the crystal structure, the current research will test these hypotheses to determine the biological and molecular basis of 14-3-3 function. The three main goals are: (l) To identify structural determinants of 14-3-3 responsible for ligand-binding, which is important for establishing cellular functions of 14-3-3. (2) To determine how 14-3-3 modulates signaling proteins. Experiments are designed to test whether 14-3-3 functions as a molecular scaffold or anchor to promote and stabilize the functional association of signaling molecules, or as a chaperone-like protein to alter the conformation of the associated proteins. (3) To determine the role of 14- 3-3 in Raf-mediated signaling, and MT- and Bcr-Abl-mediated oncogenesis. Disrupting the interaction of 14-3-3 with associated proteins by overexpressing 14-3-3 mutants is expected to reveal the specific contribution of 14-3-3 to these processes. These experiments will lead to a better understanding of the role of 14-3- 3 in signal transduction, which will provide critical insights into the biochemical basis of cell growth regulation and oncogenesis.
