Kinase activity-dependent nuclear export opposes stress-induced nuclear accumulation and retention of Hog1 mitogen-activated protein kinase in the budding yeast Saccharomyces cerevisiae

Budding yeast adjusts to increases in external osmolarity via a specific mitogen-activated protein kinase signal pathway, the high-osmolarity glycerol response (HOG) pathway. Studies with a functional Hog1-green fluorescent protein (GFP) fusion reveal that even under nonstress conditions the mitogen-activated protein kinase Hog1 cycles between cytoplasmic and nuclear compartments. The basal distribution of the protein seems independent of its activator, Pbs2, and independent of its phosphorylation status. Upon osmotic challenge, the Hog1-GFP fusion becomes rapidly concentrated in the nucleus from which it is reexported after return to an iso-osmotic environment or after adaptation to high osmolarity. The preconditions and kinetics of increased nuclear localization correlate with those found for the dual phosphorylation of Hog1-GFP. The duration of Hog1 nuclear residence is modulated by the presence of the general stress activators Msn2 and Msn4. Reexport of Hog1 to the cytoplasm does not require de novo protein synthesis but depends on Hog1 kinase activity. Thus, at least three different mechanisms contribute to the intracellular distribution pattern of Hog1: phosphorylation-dependent nuclear accumulation, retention by nuclear targets, and a kinase-induced export.     

Delayed Turnover of Unphosphorylated Ssk1 during Carbon Stress Activates the Yeast Hog1 Map Kinase Pathway

In Saccharomyces cerevisiae, the Hog1 mitogen-activated protein kinase (MAPK) pathway coordinates the adaptation to osmotic stress and was recently reported to respond to acute changes in glucose levels. Similarly as in osmotic stress, glucose starvation leads to a transient accumulation of Hog1 in the nucleus. However, the kinetics and the mechanism of Hog1 activation are different for these stress conditions. During osmotic shock the activation of Hog1 can be transduced by either the Sho1 or the Sln1/Ypd1/Ssk1 branch. During glucose starvation the phosphorylation of Hog1 is slower and is completely dependent on Ssk1, but independent of Sho1. To characterize the mechanism of activation of Hog1 during carbon stress, we examined the turnover of Ssk1 protein levels upon glucose starvation in the presence of cycloheximide and monitored protein levels by western blotting. Our data demonstrate that unphosphorylated Ssk1 was quickly degraded during exponential growth and after osmotic stress but remained remarkably stable during glucose limitation. We conclude that glucose starvation induces a delay in the turnover of unphosphorylated Ssk1, which is sufficient to activate the Hog1 MAPK pathway. Although unphosphorylated Ssk1 is known to be degraded by the proteasome, its stabilization is apparently not due to changes in cellular localization or decrease in ubiquitination levels during glucose limitation.     

酿酒酵母促分裂原蛋白激酶Hog1p 介导的渗透胁迫反应调控机制

 高渗透性甘油促分裂原激酶信号转导途径（high osmolarity glycerol mitogen activated protein kinase signaling transduction pathway,HOG-MAPK）是调控酿酒酵母对外界高渗透压胁迫环境应答的主要途径,促分裂原蛋白激酶Hog1p（MAPK Hog1p）是其中的关键性作用因子.在高渗透压刺激时,MAPK Hog1p接受信号被特异性激活并进入核内,调控相关胁迫应答基因的表达,并介导该时期细胞周期的阻滞,从而增强细胞对外界不利环境的适应能力.对胁迫条件下酿酒酵母中MAPK Hog1p作用机制的进一步研究,有利于更深入地了解哺乳动物体内逆境激发促分裂原蛋白激酶途径的功能和调控机制.     

Ptc1, a type 2C Ser/Thr phosphatase, inactivates the HOG pathway by dephosphorylating the mitogen-activated protein kinase Hog1

The HOG (high-osmolarity glycerol) mitogen-activated protein kinase (MAPK) pathway regulates the osmotic stress response in the yeast Saccharomyces cerevisiae. Three type 2C Ser/Thr phosphatases (PTCs), Ptc1, Ptc2, and Ptc3, have been isolated as negative regulators of this pathway. Previously, multicopy expression of PTC1 and PTC3 was shown to suppress lethality of the sln1Delta strain due to hyperactivation of the HOG pathway. In this work, we show that PTC2 also suppresses sln1Delta lethality. Furthermore, the phosphatase activity of these PTCs was needed for suppression, as mutation of a conserved Asp residue, likely to coordinate a metal ion, inactivated PTCs. Further analysis of Ptc1 function in vivo showed that it inactivates the MAPK, Hog1, but not the MEK, Pbs2. In the wild type, Hog1 kinase activity increased transiently, approximately 12-fold in response to osmotic stress, while overexpression of PTC1 limited activation to approximately 3-fold. In contrast, overexpression of PTC1 did not inhibit phosphorylation of Hog1 Tyr in the phosphorylation lip, suggesting that Ptc1 does not act on Pbs2. Deletion of PTC1 also strongly affected Hog1, leading to high basal Hog1 activity and sustained Hog1 activity in response to osmotic stress, the latter being consistent with a role for Ptc1 in adaptation. In vitro, Ptc1 but not the metal binding site mutant, Ptc1D58N, inactivated Hog1 by dephosphorylating the phosphothreonine but not the phosphotyrosine residue in the phosphorylation lip. Consistent with its role as a negative regulator of Hog1, which accumulates in the nucleus upon activation, Ptc1 was found in both the nucleus and the cytoplasm. Thus, one function of Ptc1 is to inactivate Hog1.     

The Stress-activated Protein Kinase Hog1 Mediates S Phase Delay in Response to Osmostress

Control of cell cycle progression by stress-activated protein kinases (SAPKs) is essential for cell adaptation to extracellular stimuli. Exposure of yeast to osmostress activates the Hog1 SAPK, which modulates cell cycle progression at G1 and G2 by the phosphorylation of elements of the cell cycle machinery, such as Sic1 and Hsl1, and by down-regulation of G1 and G2 cyclins. Here, we show that upon stress, Hog1 also modulates S phase progression. The control of S phase is independent of the S phase DNA damage checkpoint and of the previously characterized Hog1 cell cycle targets Sic1 and Hsl1. Hog1 uses at least two distinct mechanisms in its control over S phase progression. At early S phase, the SAPK prevents firing of replication origins by delaying the accumulation of the S phase cyclins Clb5 and Clb6. In addition, Hog1 prevents S phase progression when activated later in S phase or cells containing a genetic bypass for cyclin-dependent kinase activity. Hog1 interacts with components of the replication complex and delays phosphorylation of the Dpb2 subunit of the DNA polymerase. The two mechanisms of Hog1 action lead to delayed firing of origins and prolonged replication, respectively. The Hog1-dependent delay of replication could be important to allow Hog1 to induce gene expression before replication.     

Phosphorylation of Hsl1 by Hog1 leads to a G2 arrest essential for cell survival at high osmolarity

Control of cell cycle progression by stress-activated protein kinases (SAPKs) is essential for cell adaptation to extracellular stimuli. Exposure of yeast to osmostress leads to activation of the Hog1 SAPK, which controls cell cycle at G1 by the targeting of Sic1. Here, we show that survival to osmostress also requires regulation of G2 progression. Activated Hog1 interacts and directly phosphorylates a residue within the Hsl7-docking site of the Hsl1 checkpoint kinase, which results in delocalization of Hsl7 from the septin ring and leads to Swe1 accumulation. Upon Hog1 activation, cells containing a nonphosphorylatable Hsl1 by Hog1 are unable to promote Hsl7 delocalization, fail to arrest at G2 and become sensitive to osmostress. Together, we present a novel mechanism that regulates the Hsl1-Hsl7 complex to integrate stress signals to mediate cell cycle arrest and, demonstrate that a single MAPK coordinately modulates different cell cycle checkpoints to improve cell survival upon stress.     

The immunosuppressant FK506 uncovers a positive regulatory cross-talk between the Hog1p and Gcn2p pathways

The immunosuppressant Tacrolimus (FK506) has increased the survival rates of organ transplantation. FK506 exerts its immunosuppressive effect by inhibition of the protein phosphatase calcineurin in activated T-cells. Unfortunately, FK506 therapy is associated with undesired non-therapeutic effects involving targets other than calcineurin. To identify these targets we have addressed FK506 cellular toxicity in budding yeast. We show that FK506 increased cell sensitivity upon osmotic challenge independently of calcineurin and the FK506-binding proteins Fpr1p, -2p, -3p, and -4p. FK506 also induced strong amino acid starvation and activation of the general control (GCN) pathway. Tryptophan prototrophy or excess tryptophan overcame FK506 toxicity, showing that tryptophan deprivation mediated this effect. Mutation of the GCN3 and -4 genes partially alleviated FK506 toxicity, suggesting that activation of the GCN pathway by FK506 was also involved in osmotic tolerance. FK506 enhanced osmotic stress-dependent Hog1p kinase phosphorylation that was not accompanied by induction of a Hog1p-dependent reporter. Interestingly, deletion of the GCN2 gene suppressed FK506-dependent Hog1p hyperphosphorylation and restored Hog1p-dependent reporter activity. Conversely, deletion of the HOG1 gene impaired FK506-dependent activation of Gcn2p kinase and translation of a GCN4-LacZ reporter, highlighting functional cross-talk between the Gcn2p and Hog1p protein kinases. Taken together, these data demonstrate that both FK506-induced amino acid starvation and activation of the GCN pathway contribute to cell sensitivity to osmotic stress and reveal a positive regulatory loop between the Hog1p and Gcn2p pathways. Given the conserved nature of Gcn2p and Hog1p pathways, this mechanism of FK506 toxicity could be relevant to the non-therapeutic effects of FK506 therapy.     

Hog1 mediates cell-cycle arrest in G1 phase by the dual targeting of Sic1

Activation of stress-activated protein kinases (SAPKs) is essential for proper cell adaptation to extracellular stimuli. The exposure of yeast cells to high osmolarity, or mutations that lead to activation of the Hog1 SAPK, result in cell-cycle arrest. The mechanisms by which Hog1 and SAPKs in general regulate cell-cycle progression are not completely understood. Here we show that Hog1 regulates cell cycle progression at the G1 phase by a dual mechanism that involves downregulation of cyclin expression and direct targeting of the CDK-inhibitor protein Sic1. Hog1 interacts physically with Sic1 in vivo and in vitro, and phosphorylates a single residue at the carboxyl terminus of Sic1, which, in combination with the downregulation of cyclin expression, results in Sic1 stabilization and inhibition of cell-cycle progression. Cells lacking Sic1 or containing a Sic1 allele mutated in the Hog1 phosphorylation site are unable to arrest at G1 phase after Hog1 activation, and become sensitive to osmostress. Together, our data indicate that the Sic1 CDK-inhibitor is the molecular target for the SAPK Hog1 that is required to modulate cell-cycle progression in response to stress.     

Rck1 up-regulates Hog1 activity by down-regulating Slt2 activity in Saccharomyces cerevisiae

We previously reported that the over-expression of KDX1 up-regulates RCK1 gene expression. To further understand the function of Rck1, microarray analysis was performed using a RCK1 over-expressing strain. Based on microarray and Northern blot analyses, we determined that the expression of KDX1 was down-regulated when RCK1 was over-expressed. Furthermore, we determined that phosphorylated forms of Slt2 and Mkk2 were down-regulated by the over-expression of RCK1. Ptp2, a phosphatase that is regulated by the Slt2 MAP kinase pathway, was down-regulated by the over-expression of RCK1. Ptp2 is a negative regulator of Hog1; thus, the phosphorylated form of Hog1 was up-regulated by RCK1 over-expression. A point mutation of lysine 152 to arginine resulted in a failure to up-regulate Hog1 and the subsequent down-regulation of CTT1, which is a Hog1 pathway target gene. Furthermore, using microarray and Northern blot analyses, we determined that genes that are regulated by Msn2/Msn4 were up-regulated by Rck1 and that this was the result of Hog1 activation by RCK1 over-expression. Together, our results suggest that Rck1 inhibits Slt2 MAP kinase pathway activity and then Ptp2, which subsequently activates Hog1.