One fundamental biological question is how cellular responses to discrete regulatory signals are recognized and integrated to precisely manipulate gene expression. This problem impacts directly on our understanding of the control of development and differentiation in higher organisms. The process of sporulation in the yeast Saccharomyces cerevisiae is an ideal model system for investigation of the control of developmental switches in eukaryotes. This simple developmental program relies on multiple regulatory influences which interact to promote cell-type-specific induction of meiosis and ascus formation (collectively referred to as sporulation). The MCK1 gene governs the decision to exit the mitotic cell cycle and enter the sporulation pathway, and plays a positive role in mediating centromere behavior during mitosis. The MCK1p gene product has been implicated as a member of a new class of protein kinase with the novel ability to phosphorylate target proteins on serine, threonine and tyrosine residues. Indeed, protein phosphorylation is a commonly exploited mechanism of signal transduction in eukaryotic, developmentally regulated systems. Therefore, elucidation of the mechanism of action of protein kinases is of paramount importance to understanding how an organism controls differentiation, growth and development. Furthermore, the association of many tyrosine kinases with transforming and oncogenic potential contributes added incentive. This proposal describes studies intended to elucidate the mechanism by which MCK1 controls meiotic-specific gene expression through identification and characterization of its substrates, targets and regulators. MCK1p mediates entry into meiosis in response to cell-type and starvation signals by controlling derepression of the meiotic activator gene IME1. Thus, phosphorylation of a target of MCK1p transduces the signal(s) required for initiation of IME1 gene expression. We propose to: (l) Identify and characterize putative substrates of MCK1p by a combination of three approaches: (a) isolation of mutant and dosage-dependent suppressors of an mck1 deficiency; (b) identification of proteins that physically interact with MCK1p; and (c) identification of the MCK1-Response Site in the IME1 promoter and factors that interact with this site. (2) Explore the regulatory influences that modulate MCK1p by analyzing kinase activity and substrate specificity under conditions known to affect entry into meiosis; and (3) Conduct in vitro kinase activity and substrate specificity analyses aimed at characterizing this unique class of protein kinase.