Yeast Gcn4p Stabilization Is Initiated by the Dissociation of the Nuclear Pho85p/Pcl5p Complex

Abstract

Protein stability of the c-jun-like yeast bZIP transcriptional activator Gcn4p is exclusively controlled in the yeast nucleus. Phosphorylation by the nuclear Pho85p cyclin-dependent protein kinase, a functional homolog of mammalian Cdk5, initiates the Gcn4p degradation pathway in complex with the cyclin Pcl5p. We show that the initial step in Gcn4p stabilization is the dissociation of the Pho85p/Pcl5p complex. Pcl7p, another nuclear and constantly present cyclin, is required for Gcn4p stabilization and is able to associate to Pho85p independently of the activity of the Gcn4p degradation pathway. In addition, the nuclear cyclin-dependent Pho85p kinase inhibitor Pho81p is required for Gcn4p stabilization. Pho81p only interacts with Pcl5p when Gcn4p is rapidly degraded but constitutively interacts with Pcl7p. Our data suggest that Pcl7p and Pho81p are antagonists of the Pho85p/Pcl5p complex formation in a yet unknown way, which are specifically required for Gcn4p stabilization. We suggest that dissociation of the Pho85p/Pcl5p complex as initial step in Gcn4p stabilization is a prerequisite for a shift of equilibrium to an increased amount of the Pho85p/Pcl7p complexes and subsequently results in decreased Gcn4p phosphorylation and therefore increased stability of the transcription factor.

Integration of General Amino Acid Control and Target of Rapamycin (TOR) Regulatory Pathways in Nitrogen Assimilation in Yeast

“…Currently, we understand only the general mechanistic schemes for how Gcn4p can delineate between core and stress-specific gene transcription. Gcn4p activation of transcription is largely thought to be a consequence of its increased concentration in the cell, which involves translational control induced by eIF2␣ phosphorylation and its turnover, triggered by Pho85p/Pcl5p (55,62,63). Elevated levels of Gcn4p lead to its enhanced binding at the GCREs in the promoters of its target genes, which then serves to recruit different co-activators that facilitate association with RNA polymerase II (64).…”

“…The coordination of Gcn4p and Gln3p in GABA catabolism will be addressed further below. Also part of the Gcn4p activation core is Pcl5p, a cyclin partner for the Pho85p protein kinase that is required for phosphorylation and subsequent feedback degradation of Gcn4p (55,56). Gcn4p induces the expression of two protein kinase genes, PKP2 and YDL025C (RTK1).…”

Regulation of Amino Acid, Nucleotide, and Phosphate Metabolism in Saccharomyces cerevisiae

“…Some of these responses impinge directly on Gcn2 (Cherkasova et al 2010;Zaborske et al 2009Zaborske et al , 2010 and some function independently, apparently in parallel. Notably, Gcn4 stability is increased under amino acid starvation (Kornitzer et al 1994;Shemer et al 2002;Bomeke et al 2006;Aviram et al 2008;Streckfuss-Bomeke et al 2009). …”

Sumoylation of transcription factor Gcn4 facilitates its Srb10-mediated clearance from promoters in yeast

“…Two pathways are known to result in Gcn4 degradation through ubiquitin-mediated proteolysis, each involving a different cyclin-dependent kinase (CDK): (1) Pho85, which phosphorylates Gcn4 when amino acid levels are restored (Shemer et al 2002;Bomeke et al 2006), thereby marking it for ubiquitination and subsequent degradation through the 26S proteasome (see Fig. 2D,E; Kornitzer et al 1994;Meimoun et al 2000); and (2) Srb10, a component of the RNAP II Mediator complex that is recruited to activated promoters, including those activated by Gcn4 (Qiu et al 2004).…”