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.     

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.     

Rad3 and Sty1 function in Schizosaccharomyces pombe: an integrated response to DNA damage and environmental stress?

In Schizosaccharomyces pombe , the Ataxia Telangiectasia-mutated (Atm)/Atm and Rad 3 Related (Atr) homologue Rad3 is an essential regulator of the response to DNA damage and stalled replication forks. Rad3 activates the downstream kinases Chk1 and Cds1. These kinases in turn inhibit cell cycle progression by mediating Cdc2 phosphorylation. Studies in both yeast and mammalian cells suggest additional roles for Rad3 in regulating cellular responses to environmental stress. In S. pombe , cellular responses to various environmental stresses are regulated primarily through the stress-activated MAP kinase p38 homologue Sty1. An important function of Sty1 is to drive cells rapidly through mitosis by facilitating the accumulation of Cdc25. Interestingly, Sty1 is activated simultaneously with Rad3 following exposure to UV radiation or ionizing radiation (IR). Similarly, exposure to environmental stresses induces the expression of rad3 ⁺, cds1 ⁺ and other checkpoint regulator genes. It is currently unclear how the pathways regulated by Sty1 and Rad3 and their opposing effects on mitosis are integrated. Recent studies suggest that Sty1 and Rad3 function together to regulate the expression of several stress response genes following exposure to IR. In this review, we discuss current knowledge on the interaction of Rad3/Atm and Sty1/p38 in regulating cellular responses to environmental stress and DNA damage.     

A conserved stress-activated protein kinase regulates a core stress response in the human pathogen Candida albicans

Previous work has implicated the Hog1 stress-activated protein kinase (SAPK) in osmotic and oxidative stress responses in the human pathogen Candida albicans. In this study, we have characterized the role of Hog1 in mediating these and other stress responses in C. albicans. We provide evidence that a SAPK-dependent core stress response exists in this pathogen. The Hog1 SAPK is phosphorylated and it accumulates in the nucleus in response to diverse stress conditions. In addition, we have identified Hog1-regulated genes that are induced in response to stress conditions that activate Hog1. These analyses reveal both activator and repressor functions for the Hog1 SAPK. Our results also demonstrate that stress cross-protection, a classical hallmark of the core stress response, occurs in C. albicans between stresses that activate the Hog1 SAPK. Importantly, we find that the core stress response in C. albicans has adapted to the environmental niche of this human pathogen. This niche specificity is reflected by the specific environmental conditions that drive the Hog1-regulated core stress response in C. albicans and by differences in the molecular circuitry that control this response.     

A functional role for p38 MAPK in modulating mitotic transit in the absence of stress

Although p38 MAPK is known to be activated in response to various environmental stresses and to have inhibitory roles in cell proliferation and tumor progression, its role in cell cycle progression in the absence of stress is unknown in most cell types. In the case of G(2)/M cell cycle control, p38 activation has been shown to trigger a rapid G(2)/M cell cycle checkpoint after DNA damage stress and a spindle checkpoint after microtubule disruption. In the course of our studies, we observed that p38 became actively phosphorylated, and its kinase activity increased transiently during G(2)/M cell cycle transition. Using an immunocytochemistry approach, the active form of p38 was found at the centrosome from late G(2) throughout mitosis, which suggests functional relevance for active p38 protein during mitotic entry. A closer examination reveals that p38 inhibition by pharmacologic inhibitors significantly accelerated the timing of mitotic entry. In addition, long term exposure of the inhibitor enhanced Cdc2 activity. These results indicate that p38 activity during G(2)/M may be involved in a mechanism for fine tuning the initiation of mitosis and perhaps transit of mitosis. Consistent with our previous findings, Cdc25B was phosphorylated on serine 309 at the centrosome during G(2)/M when p38 was active at this site; Cdc25B phosphorylation inhibits Cdc25B activity, and this phosphorylation was found to be p38-dependent. Taken together, our findings suggest that p38 regulates the timing of mitotic entry via modulation of Cdc25B activity under normal nonstress conditions.     

Activation of Srk1 by the mitogen-activated protein kinase Sty1/Spc1 precedes its dissociation from the kinase and signals its degradation

Control of cell cycle progression by stress-activated protein kinases (SAPKs) is essential for cell adaptation to extracellular stimuli. The Schizosaccharomyces pombe SAPK Sty1/Spc1 orchestrates general changes in gene expression in response to diverse forms of cytotoxic stress. Here we show that Sty1/Spc1 is bound to its target, the Srk1 kinase, when the signaling pathway is inactive. In response to stress, Sty1/Spc1 phosphorylates Srk1 at threonine 463 of the regulatory domain, inducing both activation of Srk1 kinase, which negatively regulates cell cycle progression by inhibiting Cdc25, and dissociation of Srk1 from the SAPK, which leads to Srk1 degradation by the proteasome.     

The protein kinase PKR is required for p38 MAPK activation and the innate immune response to bacterial endotoxin

Protein kinase RNA-regulated (PKR) is an established component of innate antiviral immunity. Recently, PKR has been shown to be essential for signal transduction in other situations of cellular stress. The relationship between PKR and the stress-activated protein kinases (SAPKs), such as p38 mitogen-activated protein kinase (MAPK), is not clear. Using embryonic fibroblasts from PKR wild-type and null mice, we established a requirement for PKR in the activation of SAPKs by double-stranded RNA, lipopolysaccharide (LPS) and proinflammatory cytokines. This does not reflect a global failure to activate SAPKs in the PKR-null background as these kinases are activated normally by anisomycin and other physicochemical stress. Activation of p38 MAPK was restored in immortalized PKR-null cells by reconstitution with human PKR. We also show that LPS induction of interleukin-6 and interleukin-12 mRNA is defective in PKR-null cells, and that production of these cytokines is impaired in PKR-null mice challenged with LPS. Our findings indicate, for the first time, that PKR is required for p38 MAPK signaling and plays a potentially important role in the innate response against bacterial endotoxin.     

Protein kinase cascades activated by stress and inflammatory cytokines

Signal transduction pathways constructed around a core module of three consecutive protein kinases, the most distal being a member of the extracellular signal-regulated kinase (ERK) family, are ubiquitous among eukaryotes. Recent work has defined two cascades activated preferentially by the inflammatory cytokines TNF-α and IL-1-β, as well as by a wide variety of cellular stresses such as UV and ionizing radiation, hyperosmolarity, heat stress, oxidative stress, etc. One pathway converges on the ERK subfamily known as the ‘stress activated’ protein kinases (SAPKs, also termed Jun N-terminal kinases, JNKs), whereas the second pathway recruits the p38 kinases. Upstream inputs are diverse, and include small GTPases (primarily Rac and Cdc42; secondarily Ras) acting through mammalian homologs of the yeast Ste20 kinase, other kinase subfamilies (e.g. GC kinase) and ceramide, a putative second messenger for certain TNF-α actions. These two cascades signal cell cycle delay, cellular repair or apoptosis in most cells, as well as activation of immune and reticuloendothelial cells.     

Inactivation of the Cdc25 phosphatase by the stress-activated Srk1 kinase in fission yeast

The mechanisms by which environmental stress regulates cell cycle progression are poorly understood. In fission yeast, we show that Srk1 kinase, which associates with the stress-activated p38/Sty1 MAP kinase, regulates the onset of mitosis by inhibiting the Cdc25 phosphatase. Srk1 is periodically active in G2, and its overexpression causes cell cycle arrest in late G2 phase, whereas cells lacking srk1 enter mitosis prematurely. We find that Srk1 interacts with and phosphorylates Cdc25 at the same sites phosphorylated by the Chk1 and Cds1 (Chk2) kinases and that this phosphorylation is necessary for Srk1 to delay mitotic entry. Phosphorylation by Srk1 causes Cdc25 to bind to Rad24, a 14-3-3 protein family member, and accumulation of Cdc25 in the cytoplasm. However, Srk1 does not regulate Cdc25 in response to replication arrest or DNA damage but, rather, during a normal cell cycle and in response to nongenotoxic environmental stress.     

The Srk1 protein kinase is a target for the Sty1 stress-activated MAPK in fission yeast

The fission yeast stress-activated Sty1/Spc1 MAPK pathway responds to a similar range of stresses as do the mammalian p38 and SAPK/JNK MAPK pathways. In addition, sty1(-) cells are sterile and exhibit a G(2) cell cycle delay, indicating additional roles of Sty1 in meiosis and cell cycle progression. To identify novel proteins involved in stress responses, a microarray analysis of the Schizosaccharomyces pombe genome was performed to find genes that are up-regulated following exposure to stress in a Sty1-dependent manner. One such gene identified, srk1(+) (Sty1-regulated kinase 1), encodes a putative serine/threonine kinase homologous to mammalian calmodulin kinases. At the C terminus of Srk1 is a putative MAPK binding motif similar to that in the p38 substrates, MAPK-activated protein kinases 2 and 3. Indeed, we find that Srk1 is present in a complex with the Sty1 MAPK and is directly phosphorylated by Sty1. Furthermore, upon stress, Srk1 translocates from the cytoplasm to the nucleus in a process that is dependent on the Sty1 MAPK. Finally, we show that Srk1 has a role in regulating meiosis in fission yeast; following nitrogen limitation, srk1(-) cells enter meiosis significantly faster than wild-type cells and overexpression of srk1(+) inhibits the nitrogen starvation-induced arrest in G(1).     

The role of stress-kinase p38 in cell cycle blocking delay under osmotic stress

Alteration in osmotic conditions leads to activation of some defensive mechanisms resulting in blocking the cell cycle progression. One of stress kinases, activated after osmotic stress, is p38 MAPK. In the present study the role of p38 both in intra-S-phase and G2/M checkpoint is shown. Besides, p38-dependent degradation of Cdc25A phosphatase after osmotic stress is demonstrated. Expression of stable form of Cdc25A results in a partial abrogation of G2/M block of the cell cycle, but has no effect on DNA synthesis arrest.     

The p57 CDKi integrates stress signals into cell-cycle progression to promote cell survival upon stress

The p57(Kip2) cyclin-dependent kinase inhibitor (CDKi) has been implicated in embryogenesis, stem-cell senescence and pathologies, but little is known of its role in cell cycle control. Here, we show that p57(Kip2) is targeted by the p38 stress-activated protein kinase (SAPK). Phosphorylation of p57(Kip2) at T143 by p38 enhances its association with and inhibition of Cdk2, which results in cell-cycle delay upon stress. Genetic inactivation of the SAPK or the CDKi abolishes cell-cycle delay upon osmostress and results in decreased cell viability. Oxidative stress and ionomycin also induce p38-mediated phosphorylation of p57 and cells lacking p38 or p57 display reduced viability to these stresses. Therefore, cell survival to various stresses depends on p57 phosphorylation by p38 that inhibits CDK activity. Together, these findings provide a novel molecular mechanism by which cells can delay cell cycle progression to maximize cell survival upon stress.