Phosphorylation of Gal4p at S699
is required for gal induction, but not for activation in yeast lacking
the inhibitor Gal80p. We have suggested that Gal4p phosphorylation is
mediated by GTF complex kinases, and that phospho-S699 regulates
interaction of Gal4p with Gal80p. Both RNA Pol II holoenzyme kinases,
Kin28p and Srb10p, phosphorylate Gal4p at S699 in vitro . In
vivo all Gal4p phosphorylation is eliminated at the n.p. temp. in
kin28 ts yeast, whereas phospho-S699 is specifically
eliminated in srb10 cells. In contrast to WT yeast, galactose
induction is identical in srb10 yeast expressing WT GAL4
or GAL4 S699A. Therefore, although Kin28p and Srb10p may be
partially redundant for Gal4p phosphorylation, phospho-S699 is mediated
predominately by Srb10p. Gal4p phosphorylation appears to be regulated
by a signaling mechanism which functions independently of GAL3 in
response to fermentable carbon. In gal80 yeast phosphorylation is
stimulated when cells are shifted from non-fermentable to fermentable
carbon-containing medium. Additionally, the LTA response, typical of
gal3 yeast, is eliminated by the GAL4 S699A mutation.
Basal GAL transcription in WT yeast is elevated by the presence
of any fermentable source of carbon; elevated basal transcription is not
observed in yeast expressing GAL4 S699A. Genetic analysis
indicates that the GAL3 -independent signal includes RAS2
and mating pheromone-response pathway components. These results suggest
that Gal4p activity is modulated by a holoenzyme cdk-mediated
phosphorylation, which may be controlled by a signaling mechanism that
overlaps those regulating response to pheromone and nitrogen
availability.
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