KINASES AND CONTROL OF CELL-TYPE SPECIALIZATION

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Protein kinases are known to play a pivotal role in regulatory cascades important to the control of animal cell proliferation and specialization. The medical relevance of such regulatory networks is best appreciated by considering the transformed state where cells have lost aspects of their differentiated phenotype and proliferate outside or normal regulatory constraints. Protein kinases are_among the "oncogenes" that have been identified as collaborative agents responsible for transformation associated with various cancers. The yeast, S. cerevisiae is a model system for the study of molecular mechanisms controlling cell-type specialization. Our objective is to define the role of two protein kinases, the STE7 and STEll gene products, in cell specialization. We will investigate: (1) factors that modulate the timing and/or cell-type specificity of protein kinase activity (2) structural alterations in protein kinases that can modify their control, specificity and function and (3) components in control circuits that interact with protein kinases as substrates or as regulators. The principles uncovered here should be applicable to pathways operating in multicellular eukaryotes. The STE gene products will be expressed in E. coli for the purpose of purification and evaluation of kinase activity and enzymology. The E. coli expressed material will also be used to elicit antisera in rabbits. The STE specific antibodies will be used as reagents to determine cell-type specificity of STE gene product activity, modification or localization. In vitro mutagenesis will be applied to create structural alterations in the STE gene products that may prevent function or otherwise modify specificity of function in vivo. Pseudoreversion studies utilizing the in vitro generated STE mutant alleles will be applied to identify other components of the mating regulatory pathway.