Phosphorylation of Cdc5 regulates its accumulation

<p>Abstract</p> <p>Background</p> <p>Cdc5 (polo kinase/Plk1) is a highly conserved key regulator of the <it>S. cerevisiae </it>cell cycle from S-phase until cytokinesis. However, much of the regulatory mechanisms that govern Cdc5 remain to be determined. Cdc5 is phosphorylated on up to 10 sites during mitosis. In this study, we investigated the function of phosphorylation site T23, the only full consensus Cdk1 (Cdc28) phosphorylation site present.</p> <p>Findings</p> <p><it>Cdc5^T23A</it>introduces a degron that reduces its cellular amount to undetectable levels, which are nevertheless sufficient for normal cell proliferation. The degron acts <it>in cis </it>and is reversed by N-terminal GFP-tagging. Cdk1 kinase activity is required to maintain Cdc5 levels during G2. This, Cdk1 inhibited, Cdc5 degradation is APC/C^Cdh1independent and requires new protein synthesis. Cdc5^T23Eis hyperactive, and reduces the levels of Cdc5 (<it>in trans</it>) and drastically reduces Clb2 levels.</p> <p>Conclusions</p> <p>Phosphorylation of Cdc5 by Cdk1 is required to maintain Cdc5 levels during G2. However, phosphorylation of T23 (probably by Cdk1) caps Cdc5 and other <it>CLB2 </it>cluster protein accumulation, preventing potential protein toxicity, which may arise from their overexpression or from APC/C^Cdh1inactivation.</p

Cdk1 and SUMO Regulate Swe1 Stability

The Swe1/Wee1 kinase phosphorylates and inhibits Cdk1-Clb2 and is a major mitotic switch. Swe1 levels are controlled by ubiquitin mediated degradation, which is regulated by interactions with various mitotic kinases. We have recently reported that Swe1 levels are capable of sensing the progress of the cell cycle by measuring the levels of Cdk1-Clb2, Cdc5 and Hsl1. We report here a novel mechanism that regulates the levels of Swe1. We show that S.cerevisiae Swe1 is modified by Smt3/SUMO on residue K594 in a Cdk1 dependant manner. A degradation of the swe1K594R mutant that cannot be modified by Smt3 is considerably delayed in comparison to wild type Swe1. Swe1K594R cells express elevated levels of Swe1 protein and demonstrate higher levels of Swe1 activity as manifested by Cdk1-Y19 phosphorylation. Interestingly this mutant is not targeted, like wild type Swe1, to the bud neck where Swe1 degradation takes place. We show that Swe1 is SUMOylated by the Siz1 SUMO ligase, and consequently siz1Δ cells express elevated levels of Swe1 protein and activity. Finally we show that swe1K594R cells are sensitive to osmotic stress, which is in line with their compromised regulation of Swe1 degradation

The polyHIS Tract of Yeast AMPK Coordinates Carbon Metabolism with Iron Availability

Energy status in all eukaryotic cells is sensed by AMP-kinases. We have previously found that the poly-histidine tract at the N-terminus of S. cerevisiae AMPK (Snf1) inhibits its function in the presence of glucose via a pH-regulated mechanism. We show here that in the absence of glucose, the poly-histidine tract has a second function, linking together carbon and iron metabolism. Under conditions of iron deprivation, when different iron-intense cellular systems compete for this scarce resource, Snf1 is inhibited. The inhibition is via an interaction of the poly-histidine tract with the low-iron transcription factor Aft1. Aft1 inhibition of Snf1 occurs in the nucleus at the nuclear membrane, and only inhibits nuclear Snf1, without affecting cytosolic Snf1 activities. Thus, the temporal and spatial regulation of Snf1 activity enables a differential response to iron depending upon the type of carbon source. The linkage of nuclear Snf1 activity to iron sufficiency ensures that sufficient clusters are available to support respiratory enzymatic activity and tests mitochondrial competency prior to activation of nuclear Snf1

Cross-Talk between Carbon Metabolism and the DNA Damage Response in S. cerevisiae

Yeast cells with DNA damage avoid respiration, presumably because products of oxidative metabolism can be harmful to DNA. We show that DNA damage inhibits the activity of the Snf1 (AMP-activated) protein kinase (AMPK), which activates expression of genes required for respiration. Glucose and DNA damage upregulate SUMOylation of Snf1, catalyzed by the SUMO E3 ligase Mms21, which inhibits SNF1 activity. The DNA damage checkpoint kinases Mec1/ATR and Tel1/ATM, as well as the nutrient-sensing protein kinase A (PKA), regulate Mms21 activity toward Snf1. Mec1 and Tel1 are required for two SNF1-regulated processes—glucose sensing and ADH2 gene expression—even without exogenous genotoxic stress. Our results imply that inhibition of Snf1 by SUMOylation is a mechanism by which cells lower their respiration in response to DNA damage. This raises the possibility that activation of DNA damage checkpoint mechanisms could contribute to aerobic fermentation (Warburg effect), a hallmark of cancer cells

Phosphorylation and dephosphorylation regulate APC/C^Cdh1 substrate degradation

<p>The Anaphase Promoting Complex/Cyclosome (APC/C) ubiquitin ligase activated by its G1 specific adaptor protein Cdh1 is a major regulator of the cell cycle. The APC/C^Cdh1 mediates degradation of dozens of proteins, however, the kinetics and requirements for their degradation are largely unknown. We demonstrate that overexpression of the constitutive active <i>CDH1</i>^<i>m11</i> mutant that is not inhibited by phosphorylation results in mitotic exit in the absence of the FEAR and MEN pathways, and DNA re-replication in the absence of Cdc7 activity. This mode of mitotic exit also reveals additional requirements for APC/C^Cdh1 substrate degradation, which for some substrates such as Pds1 or Clb5 is dephosphorylation, but for others such as Cdc5 is phosphorylation.</p