Cdk8 Regulates Stability of the Transcription Factor Phd1 To Control Pseudohyphal Differentiation of Saccharomyces cerevisiae

Abstract

The yeast Saccharomyces differentiates into filamentous pseudohyphae when exposed to a poor source of nitrogen in a process involving a collection of transcription factors regulated by nutrient signaling pathways. Phd1 is important for this process in that it regulates expression of most other transcription factors involved in differentiation and can induce filamentation on its own when overproduced. In this article, we show that Phd1 is an unstable protein whose degradation is initiated through phosphorylation by Cdk8 of the RNA polymerase II mediator subcomplex. Phd1 is stabilized by cdk8 disruption, and the naturally filamenting ⌺1278b strain was found to have a sequence polymorphism that eliminates a Cdk8 phosphorylation site, which both stabilizes the protein and contributes to enhanced differentiation. In nitrogen-starved cells, PHD1 expression is upregulated and the Phd1 protein becomes stabilized, which causes its accumulation during differentiation. PHD1 expression is partially dependent upon Ste12, which was also previously shown to be destabilized by Cdk8-dependent phosphorylations, but to a significantly smaller extent than Phd1. These observations demonstrate the central role that Cdk8 plays in initiation of differentiation. Cdk8 activity is inhibited in cells shifted to limiting nutrient conditions, and we argue that this effect drives the initiation of differentiation through stabilization of multiple transcription factors, including Phd1, that cause activation of genes necessary for filamentous response.

Fine-Tuning of Histone H3 Lys4 Methylation During Pseudohyphal Differentiation by the CDK Submodule of RNA Polymerase II

“…None of the strains containing SSN8 displayed significant enhancement of flocculation over the duration of the time course ( Figure 1B and Figure S1B). However, deleting SSN8 resulted in increased flocculation rates, which is consistent with earlier reports documenting this phenotype (Nelson et al 2003;Raithatha et al 2012). Surprisingly, the histone H3 Lys4 methylation regulators JHD2 and SET1 influenced flocculation in the SSN8 mutant.…”

“…Since its identification, the Ssn8p/Ssn3p (Cnc1p/Cdk8p) protein complex has been characterized as both an activator and a repressor of gene transcription, with many molecular targets identified including pol II CTD, other mediator subunits, and transcription factors (Hengartner et al 1995;Chi et al 2001;Nelson et al 2003;Raithatha et al 2012;Nemet et al 2014). Microarray studies of yeast lacking the SSN3 kinase showed that 40% of the 173 genes that were upregulated were also increased during diauxic shift (Holstege et al 1998).…”

“…It is also possible that deleting SSN8 and thereby eliminating Ssn3p kinase activity has broad impacts on many pseudohyphal regulators. For example, phosphorylation of Ste12p, Gcn4p, Msn2p, and Phd1p stimulates their proteasome-dependent degradation (Chi et al 2001;Nelson et al 2003;Raithatha et al 2012). All of these targets are known to stimulate FLO11 transcription, providing a mechanism in which SSN8 may negatively coregulate multiple transcription factors that all function in the same biological process.…”

Analysis of the Candida albicans Phosphoproteome

“…P sites within the global transcriptional coregulatory Mediator complex were found both in the large-scale data set and in a targeted follow-up study. Cdk8 is the sole kinase component of Mediator, and subsequent in vitro assays identified P sites within Mediator that were phosphorylated by Cdk8 in vitro at sites consistent with the previously described Cdk8 target motif (phospho-S/T-P) (29)(30)(31)(32)(33). This data set gives a first in-depth analysis of the C. albicans phosphoproteome and is the largest fungal phosphopeptide data set at this time.…”

Deletion of the MED13 and CDK8 subunits of the Mediator improves the phenotype of a long-lived respiratory deficient mutant of Podospora anserina

“…In fungi, the roles of MED13 and of the kinase module have been studied most extensively in the yeasts Saccharomyces cerevisiae (Chi et al, 2001;Gonzalez et al, 2014;Krasley et al, 2006;Nelson et al, 2003;Raithatha et al, 2012;Shahi et al, 2010;Singh et al, 2006;van de Peppel et al, 2005), Schizosaccharomyces pombe (Linder et al, 2008;Zhu et al, 2006), and in human and plant pathogens such as Candida albicans (Lindsay et al, 2014;Uwamahoro et al, 2012;Zhang et al, 2013) and Cryptococcus neoformans (Wang et al, 2011). These studies revealed that the kinase module regulates hundreds of genes associated with carbon source utilization, stress responses, morphogenesis, filamentation and adhesion (Blankenship et al, 2010;Bluhm and Woloshuk, 2006;Chang et al, 2004;Cooper et al, 1999;Holstege et al, 1998;Larschan and Winston, 2005;Linder et al, 2008;Samuelsen et al, 2003;Uwamahoro et al, 2012;Wang et al, 2011;Wheeler et al, 2008;Zhou et al, 2010).…”