Apoptosis, or programmed cell death, is an active process that is fundamental to the development and homeostasis of multicellular organisms. For example, in the immune system, apoptosis is responsible for the elimination of potentially autoreactive lymphocytes during their development and for limiting clonal expansion during an immune response. Over the last few years, significant advances have been made in understanding the molecular mechanism of the execution phase of apoptosis. Central to the cell death machinery are a family of cysteine proteases, called caspases, which become activated in a proteolytic cascade and cleave specific substrates. The identity of these substrates, and their role in apoptosis is just begining to be determined. As with other cellular fate decisions, such as growth and differentiation, it is likely that protein phosphorylation/ dephosphorylation plays an important role in regulating apoptosis. The serine/threonine kinase Mst1, a mammalian homologue of the budding yeast Ste20 kinase, is cleaved by caspase-mediated proteolysis in response to apoptotic stimuli. Furthermore, overexpression of Mst1 induces morphological changes characteristic of apoptosis in human B lymphoma cells. Our major goals in this proposal are to study Mst1 regulation, identify Mst1 substrates, and test the hypothesis that Mst1 functions in a positive feedback pathway to amplify the apoptotic response. In particular, we will use a combination of in vivo and in vitro techniques to identify the caspase responsible for Mst1 cleavage and determine whether cleavage results in activation of Mst1. We will also employ transfection approaches to determine whether Mst1, like its homologue Ste20, functions in a mitogen-activated protein kinase cascade. Finally, we will characterize the apoptotic changes induced by Mst1 overexpression and design dominant negative mutants of Mst1 that might block apoptosis. In addition to providing insight into the role of Mst1 during apoptosis it is anticipated that these studies will provide valuable information concerning the role of phosphorylation/ dephosphorylation mechanisms in the regulation of programmed cell death. This may contribute to our understanding of the pathogenesis of human diseases, such as congenital and aquired immunodeficiencies, viral infection, leukemias and lymphomas, where alterations in cell survival have been implicated.
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