9318376 Herskowitz Growth of normal cells is often regulated by growth factors, either positive growth factors, which stimulate proliferation, or negative growth factors, which inhibit proliferation. The budding yeast, saccharomyces cerevisiae, provides an excellent opportunity to study the molecular mechanism of action of negative growth factors. The yeast mating factors(a-factor and a-factor) are peptides secreted by each of the mating types, a and a. They cause cells of opposite mating type to arrest in the G1 phase of the cell cycle and to differentiate by inducing expression of a variety contains a seven-membrane-spanning receptor and several protein kinases, which include MAP kinases. Cell-cycle arrest ensues because activity of the regulatory kinase,p34 CDC28, is inhibited. A gene, FAR1, has been recently identified whose protein plays a key role in arresting the cell cycle in G1 in response to the negative growth factors. Genetic studies led to the hypothesis that FAR1 protein inhibits one of the G1 cyclins, CLN2. Recent biochemical studies show that FAR1 protein associates with the CDC28 and CLN2 proteins and that the ability to form this complex requires that FAR1 be phosphorylated by the MAP kinase, FUS3 protein. These studies demonstrate that FAR1 is the link between the signal transduction pathway and the machinery that is involved in regulating the cell-cycle. This research is employing a combination of molecular genetic and biochemical techniques to address the following aims: (a) Determine how phosphorylation of FAR1 by FUS3 (and by p34CDC28-CLN2 ) controls activity of FAR1. Phosphorylation sites shall be identified by biochemical methods; mutant sites shall be constructed or otherwise identified to test the role of these sites in vivo and in vitro. (b) Determine how FAR1 inhibits activity of the p34CDC28-CLN2 complex. Immune complexes containing CLN2 and CDC28 shall be isolated and assayed for function in the presence of FAR1 protein; mutant FAR1 proteins (inactive or constitutively active) shall be identified and analyzed to dertemine their ability to inhibit CLN2/CDC28 kinase activity in vitro and in vivo. (c) Explore the role of FAR1 in the mating process of yeast. Mutants of FAR1 defective in a second function of FAR1 shall be isolated and their mating defect characterized. (d) Search for homologues of FAR1 in other organisms. Homologues will be identified by functional rescue of yeast far1 mutants and by nucleic acid hybridization. %%% These studies are relevant to understanding how cell growth is regulated by the external environment, a fundamental problem in cell and developmental biology with relevance to cancer. ***
