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Regulation of the Saccharomyces cerevisiae transcriptional activator STE12 by phosphorylation

University of British Columbia

The transcriptional activator STE12 controls different developmental processes of the yeast Saccharomyces cerevisiae. Different environmental signals represented by mating pheromone in haploids and nitrogen starvation in diploids initiate various signal transduction pathways, which activate the common target STE12 in the nucleus. Phosphorylation of STE12 has previously been demonstrated to accompany an increase in STE12 activity in response to mating pheromone. In this study, the phosphorylation state of STE12 was examined in cells growing in various physiological conditions. In haploid yeast, STE12 was found to be heavily phosphorylated in the absence of mating pheromone. Upon pheromone treatment of haploids, STE12 acquires two additional phosphorylations and activates expression of genes necessary for mating. The pheromone-dependent phosphorylations require an intact pheromone response pathway and nuclear localization of STE12. However, the appearance of these phosphorylations does not require the DNA binding ability of STE12, suggesting they are not a consequence of transcriptional activation. Seastar MAPK can phosphorylate recombinant STE12 specifically on most of the sites which are phosphorylated in vivo. Using this approach, I localized most of the phosphorylation sites on STE12 between amino acids 216-473, and 2 phosphorylations within the C-terminal 215 amino acids. By analyzing phosphorylation of recombinant STE12 proteins I identified phosphorylation sites at threonine525, serine261 and serine226. Phosphorylation of serine261 specifically inhibits STE12 in haploid cells and in diploid cells under normal growth conditions. Upon nitrogen starvation, serine261 becomes dephosphorylated and expression of nitrogen-starvation responsive genes is enhanced. Phosphorylation of serine226 may control cell cycle dependent regulation of pheromoneresponsive genes. An alanine mutation at serine226 increases induction of the pheromone responsive gene, FUS1 and cells carrying this mutation recover at a slower rate from pheromone-induced Gl cell cycle arrest. Phosphorylation at serine226 is dependent on the activity of the cdk CDC28 and causes instability of STE12 at Gl/S boundary of the cell division cycle. These experiments demonstrate that STE12 activity is influenced by multiple signals through specific phosphorylations.

Thesis/Dissertation

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2009-03-30

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http://hdl.handle.net/2429/6643

Biochemistry and Molecular Biology

University of British Columbia

UBCV

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