TOR signaling modulates Cdk8-dependent GAL gene expression in Saccharomyces cerevisiae

Abstract

Cdk8 of the RNA Polymerase II mediator complex regulates genes by phosphorylating sequence specific transcription factors. Despite conserved importance for eukaryotic transcriptional regulation, the signals regulating Cdk8 are unknown. Full induction of the yeast GAL genes requires phosphorylation of Gal4 by Cdk8, and we exploited this requirement for growth of gal3 yeast on galactose to identify mutants affecting Cdk8 activity. Several mutants from the screen produced defects in TOR signaling. A mutant designated gal four throttle (gft) 1, gft1, was identified as an allele of hom3, encoding aspartokinase. Defects in gft1/ hom3 caused hypersensitivity to rapamycin, and constitutive nuclear localization of Gat1. Furthermore, mutations of tor1 or tco89, encoding TORC1 components, also prevented GAL expression in gal3 yeast, and tco89 was determined to be allelic to gft7. Disruption of cdc55, encoding a subunit of PP2A regulated by TOR signaling, suppressed the effect of gft1/ hom3, gft7/ tco89, and tor1 mutations on GAL expression in gal3 yeast, but not of cdk8/ srb10 disruptions or Gal4 S699A mutation. Mutations of gft1/ hom3 and tor1 did not affect kinase activity of Cdk8 in vitro, but caused loss of Gal4 phosphorylation in vivo. These observations demonstrate that TOR signaling regulates GAL induction through the activity of PP2A/ Cdc55, and are consistent with the contention that Cdk8-dependent phosphorylation of Gal4 S699 is opposed by PP2A/ Cdc55 dephosphorylation. These results provide insight into how induction of transcription by a specific inducer can be modulated by global nutritional signals through regulation of Cdk8-dependent phosphorylation.