Characterization of the basal and pheromone-stimulated phosphorylation states of Ste12p

The Saccharomyces cerevisiae transcription factor Ste12p is required for basal and activated expression of pheromone-responsive genes, and for invasive growth in haploid cells. In diploid yeast, Ste12p is implicated in pseudohyphal development. The ability of Ste12p to effect these various responses in three different cell types must require stringent regulation of its transcriptional activation function and interaction with additional transcription factors. We have examined the phosphorylation state of Ste12p in untreated and pheromone-treated haploid cells, and found eight constitutively phosphorylated peptides. Phosphorylation at the constitutive sites does not require the protein kinases of the pheromone-response pathway. Treatment of haploid yeast with mating pheromone causes the appearance of novel relatively minor phosphorylations on Ste12p. Brief [35S]methionine labeling reveals novel pheromone-dependent, electrophoretically slower migrating Ste12p species. Similarly, the sole difference we observe in tryptic phosphopeptides generated from Ste12p from pheromone-treated and untreated cells is the transient appearance of two novel minor hydrophobic phosphopeptides. The pheromone-dependent phosphorylation of Ste12p requires an intact pheromone-response pathway and localization of Ste12p to the nucleus, but does not require the Ste12p DNA-binding domain. We conclude from these experiments that the pheromone-response pathway induces the formation of specific hyperphosphorylation on Ste12p, which can only be detected as apparently minor modifications in vivo. We argue that, if Ste12p is regulated by direct pheromone-responsive phosphorylation, then that phosphorylation must be represented by the two novel phosphopeptides. However, we cannot exclude the possibility that pheromone-responsive transcription is controlled by direct phosphorylation of a target other than Ste12p.