Negative regulation of Gcn4 and Msn2 transcription factors by Srb10 cyclin-dependent kinase

The budding yeast transcriptional activator Gcn4 is rapidly degraded in an SCFCdc4-dependent manner in vivo. Upon fractionation of yeast extracts to i dentify factors that mediate Gcn4 ubiquitination, we found that Srb10 phosp horylates Gcn4 and thereby marks it for recognition by SCFCdc4 ubiquitin li gase. Srb10 is a physiological regulator of Gcn4 stability because both pho sphorylation and turnover of Gcn4 are diminished in srb10 mutants. Gcn4 is almost completely stabilized in srb10 Delta pho85 Delta cells, or upon muta tion of all Srb10 phosphorylation sites within Gcn4, suggesting that the Ph o85 and Srb10 cyclin-dependent kinases (CDKs) conspire to limit the accumul ation of Gcn4. The multistress response transcriptional regulator Msn2 is a lso a substrate for Srb10 and is hyperphosphorylated in an Srb10-dependent manner upon heat-stress-induced translocation into the nucleus. Whereas Msn 2 is cytoplasmic in resting wild-type cells, its nuclear exclusion is parti ally compromised in srb10 mutant cells. Srb10 has been shown to repress a s ubset of genes in vivo, and has been proposed to inhibit transcription via phosphorylation of the C-terminal domain of RNA polymerase II. We propose t hat Srb10 also inhibits gene expression by promoting the rapid degradation or nuclear export of specific transcription factors. Simultaneous down-regu lation of both transcriptional regulatory proteins and RNA polymerase may e nhance the potency and specificity of transcriptional inhibition by Srb10.