Phosphate-Activated Cyclin-Dependent Kinase Stabilizes G₁ Cyclin To Trigger Cell Cycle Entry

Abstract

G 1 cyclins, in association with a cyclin-dependent kinase (CDK), are universal activators of the transcriptional G 1 -S machinery during entry into the cell cycle. Regulation of cyclin degradation is crucial for coordinating progression through the cell cycle, but the mechanisms that modulate cyclin stability to control cell cycle entry are still unknown. Here, we show that a lack of phosphate downregulates Cln3 cyclin and leads to G 1 arrest in Saccharomyces cerevisiae. The stability of Cln3 protein is diminished in strains with low activity of Pho85, a phosphate-sensing CDK. Cln3 is an in vitro substrate of Pho85, and both proteins interact in vivo. More interestingly, cells that carry a CLN3 allele encoding aspartic acid substitutions at the sites of Pho85 phosphorylation maintain high levels of Cln3 independently of Pho85 activity. Moreover, these cells do not properly arrest in G 1 in the absence of phosphate and they die prematurely. Finally, the activity of Pho85 is essential for accumulating Cln3 and for reentering the cell cycle after phosphate refeeding. Taken together, our data indicate that Cln3 is a molecular target of the Pho85 kinase that is required to modulate cell cycle entry in response to environmental changes in nutrient availability.

Defects in Phosphate Acquisition and Storage Influence Virulence of Cryptococcus neoformans

“…This pho⌬⌬⌬ triple mutant also displayed resistance to bivalent cations, reduced formation of capsule and melanin, and an increased proportion of enlarged cells. Cell enlargement may indicate a problem with cell cycle control and cell division because phosphate regulation is linked with the cell cycle in yeast (54,55). We anticipated that phosphate uptake was important for virulence because candidate components of the phosphate highaffinity uptake system were upregulated during macrophage interaction (9,10).…”

Less is more: Nutrient limitation induces cross-talk of nutrient sensing pathways with NAD+ homeostasis and contributes to longevity

“…Pi sensing also affects the cell cycle: Cln3 (G 1 cyclin) is a target for phosphorylation by Pho80-Pho85 (Menoyo et al, 2013). Under Pi starvation, Pho80-Pho85 is inactive and Cln3 is dephosphorylated, which leads to proteasomal degradation and cell cycle arrest.…”

Multiple Pho85-Dependent Mechanisms Control G1 Cyclin Abundance in Response to Nutrient Stress

“…First, the degron sites in the PEST region are phosphorylated by Cln3-Cdk1 via cis autophosphorylation (5). Second, the flanking two sites of the PEST region are phosphorylated by Pho80-Pho85 to prevent the degradation of Cln3 (3). And yet, as proposed by Truman et al (7), Pho85 must also be able to phosphorylate the degron sites within the PEST region when activated by the cyclins Pcl2 and Clg1.…”

“…In the case of Cln3, Cdk1-mediated phosphorylation of certain sites within its PEST domain provides epitopes for its SCF Cdc4/Grr1 -dependent ubiquitinylation and subsequent proteasome-dependent proteolysis (5). However, Menoyo et al (3) demonstrate that two S/TP sites flanking the PEST region that are phosphorylated by the Pho80-Pho85 complex have the opposite effect: stabilizing Cln3 and countering its Cdk1-driven degradation.…”

“…A lack of phosphate causes inhibition of the kinase by the Pho81-IP 7 complex, leading to dephosphorylation of Pho4 and its retention in the nucleus, permitting the expression of phosphate acquisition genes. By analyzing the S/TP motifs in the PEST region of Cln3, Menoyo et al (3) found that two flanking sites in the cluster of 9 S/TP motifs contain a hydrophobic residue at position ϩ3 from the phosphorylation site, which is a particular substrate recognition element for Pho85 but not for Cdk1. Using mutational analysis, it was established that phosphorylation of these two residues by the Pho80-Pho85 complex made the Cdk1-driven degradation of Cln3 less efficient.…”