ATR (ataxia telangiectasia mutated- and Rad3-related kinase) is activated by mild hypothermia in mammalian cells and subsequently activates p53

In vitro cultured mammalian cells respond to mild hypothermia (27-33?°C) by attenuating cellular processes and slowing and arresting the cell cycle. The slowing of the cell cycle at the upper range (31-33?°C) and its complete arrest at the lower range (27-28?°C) of mild hypothermia is effected by the activation of p53 and subsequent expression of p21. However, the mechanism by which cold is perceived in mammalian cells with the subsequent activation of p53 has remained undetermined. In the present paper, we report that the exposure of Chinese-hamster ovary-K1 cells to mildly hypothermic conditions activates the ATR (ataxia telangiectasia mutated- and Rad3-related kinase)-p53-p21 signalling pathway and is thus a key pathway involved in p53 activation upon mild hypothermia. In addition, we show that although p38MAPK (p38 mitogen-activated protein kinase) is also involved in activation of p53 upon mild hypothermia, this is probably the result of activation of p38MAPK by ATR. Furthermore, we show that cold-induced changes in cell membrane lipid composition are correlated with the activation of the ATR-p53-p21 pathway. Therefore we provide the first mechanistic detail of cell sensing and signalling upon mild hypothermia in mammalian cells leading to p53 and p21 activation, which is known to lead to cell cycle arrest.     

Schistosoma mansoni excretory-secretory products stimulate a p38 signalling pathway in Biomphalaria glabrata embryonic cells

Following infection with Schistosoma mansoni larvae, haemocytes of resistant Biomphalaria glabrata snails execute a rapid defence during which they migrate towards and encapsulate the parasites. Such immediate and precise responses are thought to depend on signal transduction cascades though the signalling components involved remain largely unknown. It is proposed that mitogen-activated protein kinases may play a role in B. glabrata immune signalling, in particular p38 mitogen-activated protein kinases, which are known to be associated with stress and inflammatory signalling. Using degenerate PCR followed by Rapid Amplification of cDNA Ends a full-length p38 mitogen-activated protein kinase-like cDNA was cloned from both the B. glabrata embryonic (Bge) cell line (Bge-p38) and haemocytes (Bgh-p38). In addition, B. glabrata p38 mitogen-activated protein kinase activation was examined at the protein level in Western blot analyses using an antibody that specifically recognises activated/diphosphorylated p38 mitogen-activated protein kinase. Results showed that Bge cell p38 mitogen-activated protein kinase was activated/phosphorylated following 30 min incubation with anisomycin, an established p38 mitogen-activated protein kinase activator. Furthermore, p38 mitogen-activated protein kinase was also activated after only 5 min exposure to either the beta-glucan polymer laminarin or S. mansoni larval excretory-secretory products. In a comparative study, activated haemocyte p38 mitogen-activated protein kinase could also be detected using the anti-phosphorylated p38 antibody following cell treatment with anisomycin. However, in contrast with Bge cells, haemocyte p38 was not activated by either excretory-secretory products or laminarin treatments, suggesting fundamental differences in the role of p38 mitogen-activated protein kinase in signal transduction pathways between haemocytes and Bge cells.     

Involvement of MAP-kinase cascades in regulation of sodium-butyrate-induced premature senescence

We have studied the role of the stress kinases p38 and JNK1,2 in premature senescence induced by sodium butyrate (NaBut), a histone deacetylase inhibitor, in mouse embryonic fibroblasts transformed by E1A+cHa-Ras oncogenes. It was found that transformants from p38 knockout cells are able to implement NaBut-induced senescence exhibited by cell cycle arrest, inhibition of proliferation, hypertrophic changes associated with mTORC1 activation and SA-β-galactosidase activity. In jnk1,2 knockouts, the NaButinduced senescence program was inhibited. NaBut-induced senescence in p38 knockouts closely correlates with mTORC1 activation shown by inhibiting mTORC1 with rapamycin. In jnk1,2 knockouts, mTORC1 complex is not activated. We believe that JNK1,2 kinases are required for mTORC1 activation and exhibition of premature senescence markers induced by NaBut in E1A+cHa-Ras transformants.     

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.     

Inhibition of centrosome protein assembly leads to p53-dependent exit from the cell cycle

Previous evidence has indicated that an intact centrosome is essential for cell cycle progress and that elimination of the centrosome or depletion of individual centrosome proteins prevents the entry into S phase. To investigate the molecular mechanisms of centrosome-dependent cell cycle progress, we performed RNA silencing experiments of two centrosome-associated proteins, pericentriolar material 1 (PCM-1) and pericentrin, in primary human fibroblasts. We found that cells depleted of PCM-1 or pericentrin show lower levels of markers for S phase and cell proliferation, including cyclin A, Ki-67, proliferating cell nuclear antigen, minichromosome maintenance deficient 3, and phosphorylated retinoblastoma protein. Also, the percentage of cells undergoing DNA replication was reduced by >50%. At the same time, levels of p53 and p21 increased in these cells, and cells were predisposed to undergo senescence. Conversely, depletion of centrosome proteins in cells lacking p53 did not cause any cell cycle arrest. Inhibition of p38 mitogen-activated protein kinase rescued cell cycle activity after centrosome protein depletion, indicating that p53 is activated by the p38 stress pathway.     

Basic Properties of the p38 Signaling Pathway in Response to Hyperosmotic Shock

Some properties of signaling systems, like ultrasensitivity, hysteresis (a form of biochemical memory), and all-or-none responses at a single cell level, are important to understand the regulation of irreversible processes. Xenopus oocytes are a suitable cell model to study these properties. The p38 MAPK (mitogen-activated protein kinase) pathway is activated by different stress stimuli, including osmostress, and regulates multiple biological processes, from immune response to cell cycle. Recently, we have reported that activation of p38 and JNK regulate osmostress-induced apoptosis in Xenopus oocytes and that sustained activation of p38 accelerates cytochrome c release and caspase-3 activation. However, the signaling properties of p38 in response to hyperosmotic shock have not been studied. Here we show, using Xenopus oocytes as a cell model, that hyperosmotic shock activates the p38 signaling pathway with an ultrasensitive and bimodal response in a time-dependent manner, and with low hysteresis. At a single cell level, p38 activation is not well correlated with cytochrome c release 2 h after hyperosmotic shock, but a good correlation is observed at 4 h after treatment. Interestingly, cytochrome c microinjection induces p38 phosphorylation through caspase-3 activation, and caspase inhibition reduces p38 activation induced by osmostress, indicating that a positive feedback loop is engaged by hyperosmotic shock. To know the properties of the stress protein kinases activated by hyperosmotic shock will facilitate the design of computational models to predict cellular responses in human diseases caused by perturbations in fluid osmolarity.     

Oncogenic ras and p53 cooperate to induce cellular senescence

Oncogenic activation of the mitogen-activated protein (MAP) kinase cascade in murine fibroblasts initiates a senescence-like cell cycle arrest that depends on the ARF/p53 tumor suppressor pathway. To investigate whether p53 is sufficient to induce senescence, we introduced a conditional murine p53 allele (p53(val135)) into p53-null mouse embryonic fibroblasts and examined cell proliferation and senescence in cells expressing p53, oncogenic Ras, or both gene products. Conditional p53 activation efficiently induced a reversible cell cycle arrest but was unable to induce features of senescence. In contrast, coexpression of oncogenic ras or activated mek1 with p53 enhanced both p53 levels and activity relative to that observed for p53 alone and produced an irreversible cell cycle arrest that displayed features of cellular senescence. p19(ARF) was required for this effect, since p53(-/-) ARF(-/-) double-null cells were unable to undergo senescence following coexpression of oncogenic Ras and p53. Although the levels of exogenous p53 achieved in ARF-null cells were relatively low, the stabilizing effects of p19(ARF) on p53 could not explain the cooperation between oncogenic Ras and p53 in promoting senescence. Hence, enforced p53 expression without oncogenic ras in p53(-/-) mdm2(-/-) double-null cells produced extremely high p53 levels but did not induce senescence. Taken together, our results indicate that oncogenic activation of the MAP kinase pathway in murine fibroblasts converts p53 into a senescence inducer through both quantitative and qualitative mechanisms.     

A rit GTPase-p38 mitogen-activated protein kinase survival pathway confers resistance to cellular stress

Cells mobilize diverse signaling cascades to protect against stress-mediated injury. Ras family GTPases play a pivotal role in cell fate determination, serving as molecular switches to control the integration of multiple signaling pathways. p38 mitogen-activated protein kinase (MAPK) signaling serves as a critical fulcrum in this process, regulating networks that stimulate cellular apoptosis but also have the capacity to promote cell survival. However, relatively little is known concerning this functional dichotomy, particularly the regulation of p38-dependent survival pathways. Here, we demonstrate that the Rit GTPase promotes cell survival by directing an unexpected p38 MAPK-dependent AKT survival pathway. Following stress exposure, Rit small hairpin RNA interference (shRNAi)-treated cells display increased apoptosis and selective disruption of p38 MAPK signaling, while expression of constitutively activated Rit promotes p38-AKT-dependent cell survival. Rit, but not Ras or Rap GTPases, can associate with, and is critical for, stress-mediated activation of the scaffolded p38-MK2-HSP27-AKT prosurvival signaling complex. Together, our studies establish Rit as a central regulator of a p38 MAPK-dependent signaling cascade that functions as a critical cellular survival mechanism in response to stress.     

Role of p38alpha kinase in activation of premature senescence program in transformed mouse fibroblasts

We investigated the role of p38alpha stress-kinase in regulation of premature senescence program, stimulated by histone deacetylase inhibitor--sodium butyrate (NaB)--after application to rodent transformed cell lines. Investigation was performed on the E1A + cHa-ras transformants selected from mice embryonic fibroblasts null at the p38alpha kinase gene or null fibroblasts at the PPM1D gene, which encoded phosphatase Wip1. Absence of Wip1 led to constitutive activation of p38alpha kinase. It was revealed that after NaB treatment both cell lines completely stopped proliferation due to irreversible cell cycle arrest in G1/S phase. In both cell lines sodium butyrate induced sustained block of prolifaration due to irreversible cell cycle arrest in G1/S phase. Following sodium butyrate treatment cells expressed marker of senescence--beta-galactosidase activity (SA-beta-Gal). Long-term (during several days) NaB treatment of cells led to partial restoration of actin cytoskeleton, focal adhesion contacts and heterochromatin focus formation (SAHF) in the nucleus of senescent cells. Obtained data allow us to suppose that irreversible process of cellular senescence activated by sodium butyrate can occur in the absence of functionally active p38 kinase by means of other ways of cell cycle suppression.     

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.     

Contribution of circulating leukocytes to cytokine production in pancreatic duct obstruction-induced acute pancreatitis in rats

p38 MAPK was originally characterized as a stress-induced kinase, along with JNK. Subsequently, p38 MAPK was found to be activated by stimuli other than cellular stress, such as growth factors and mitogens, like interleukin (IL)-2, IL-7 and IL-3. A notable exception was IL-4, as studies in mast cells showed no activation of p38 MAPK by this cytokine. In this study we show that the regulation of p38 MAPK is cell type dependent. Like other cytokines that signal through the gamma (gamma)(c), IL-4 can activate p38 MAPK in the CT6 T-cell line and BA/F3 pro-B-cells. However, IL-4 was unable to activate p38 MAPK in the murine macrophage cell line, RAW 264.7 and, indeed, prolonged exposure of cells to IL-4 results in suppression of LPS-induced MAPK activation. This result correlates with the well defined inhibitory effect of IL-4 on tumour necrosis factor alpha (TNFalpha) production. In contrast, studies in primary human monocytes showed that prolonged exposure to IL-4 resulted in enhanced activation of LPS-stimulated p38 MAPK; this correlated with an enhanced TNFalpha production. These data highlight the complexity of IL-4 signalling mechanisms, the diversity that can exist in the regulation of a given signalling pathway by a given cytokine and, furthermore, indicate the problems that can arise from extrapolation between different cell systems.     

Phosphorylation of Cdc25C by pp90Rsk Contributes to a G2 Cell Cycle Arrest in Xenopus Cycling Egg Extracts

In unfertilized Xenopus eggs, the p42 mitogen activated protein kinase (p42MAPK) pathway isknown to maintain cell cycle arrest at metaphase of meiosis II. However, constitutive activation ofp42MAPK in post-meiotic, cycling Xenopus egg extracts can lead to either a G2 or M-phase arrestof the cell cycle, depending on the timing of p42MAPK activation. Here, we examined themolecular mechanism by which activation of the p42MAPK pathway during interphase leads to cellcycle arrest in G2. When either a recombinant wild type Cdc25C(WT) or a mutated form ofCdc25C, in which serine 287 was replaced by an alanine (S287A), was added to cycling eggextracts, S287A accelerated entry into M-phase. Furthermore, the addition of S287A overcame theG2 arrest caused by p42MAPK, driving the extract into M-phase. p90Rsk, a kinase that is the targetof p42MAPK, was phosphorylated and activated (pp90Rsk) in the G2-arrested egg extracts, and wasable to phosphorylate WT but not S287A in vitro. 14-3-3 proteins were associated with endogenousCdc25C in G2-arrested extracts. Cdc25C(WT) that had been phosphorylated by pp90Rsk bound 14-3-3?, whereas S287A could not. These data suggest that the link between the p42MAPK signalingpathway and Cdc25C involves the activation of pp90Rsk and its phosphorylation of Cdc25C at S287,causing the binding of 14-3-3 proteins. We propose that the binding of 14-3-3 proteins to pp90Rskphosphorylated-Cdc25C results in a G2 arrest in a manner similar to the cell cycle delays inducedby differentiation signals that occur later in embryonic development.     

Inhibition of centriole duplication by centrobin depletion leads to p38–p53 mediated cell-cycle arrest

Previously, we have identified a novel centrosomal protein centrobin that asymmetrically localizes to the daughter centriole. We found that depletion of centrobin expression inhibited the centriole duplication and impaired cytokinesis. However, the biological significance of centrobin in the cell cycle remains unknown. In the current study, we observed that silencing centrobin significantly inhibited the proliferation of lung cancer cell A549 and prevented the cells from G1 to S transition, whereas the growth rate of lung cancer cell line H1299, a p53-null cell line, was not affected. Furthermore, we demonstrated that the G1–S-phase arrest induced by centrobin knockdown in A549 cells is mediated by the upregulation of cell-cycle regulator p53, which is associated with the activation of cellular stress induced p38 pathway instead of DNA damage induced ATM pathway. Inhibition of p38 activity or downregulation of p38 expression could overcome the cell-cycle arrest caused by centrobin depletion. Taken together, our current findings demonstrated that centrobin plays an important role in the progression of cell cycle, and a tight association between the cell-cycle progression and defective centrosomes caused by depletion of centrobin.     

Activation of AMP-activated Protein Kinase Suppresses Oxidized Low-density Lipoprotein-induced Macrophage Proliferation

Macrophage-derived foam cells play important roles in the progression of atherosclerosis. We reported previously that ERK1/2-dependent granulocyte/macrophage colony-stimulating factor (GM-CSF) expression, leading to p38 MAPK/ Akt signaling, is important for oxidized low density lipoprotein (Ox-LDL)-induced macrophage proliferation. Here, we investigated whether activation of AMP-activated protein kinase (AMPK) could suppress macrophage proliferation. Ox-LDL-induced proliferation of mouse peritoneal macrophages was assessed by [³H]thymidine incorporation and cell counting assays. The proliferation was significantly inhibited by the AMPK activator 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) and restored by dominant-negative AMPKα1, suggesting that AMPK activation suppressed macrophage proliferation. AICAR partially suppressed Ox-LDL-induced ERK1/2 phosphorylation and GM-CSF expression, suggesting that another mechanism is also involved in the AICAR-mediated suppression of macrophage proliferation. AICAR suppressed GM-CSF-induced macrophage proliferation without suppressing p38 MAPK/Akt signaling. GM-CSF suppressed p53 phosphorylation and expression and induced Rb phosphorylation. Overexpression of p53 or p27^kip suppressed GM-CSF-induced macrophage proliferation. AICAR induced cell cycle arrest, increased p53 phosphorylation and expression, and suppressed GM-CSF-induced Rb phosphorylation via AMPK activation. Moreover, AICAR induced p21^cip and p27^kip expression via AMPK activation, and small interfering RNA (siRNA) of p21^cip and p27^kip restored AICAR-mediated suppression of macrophage proliferation. In conclusion, AMPK activation suppressed Ox-LDL-induced macrophage proliferation by suppressing GM-CSF expression and inducing cell cycle arrest. These effects of AMPK activation may represent therapeutic targets for atherosclerosis.     

The stress kinase MRK contributes to regulation of DNA damage checkpoints through a p38gamma-independent pathway

DNA damage induced by ionizing radiation (IR) activates a complex cellular response that includes checkpoints leading to cell cycle arrest. The stress-activated mitogen-activated protein kinase (MAPK) p38gamma has been implicated in the G(2) phase checkpoint induced by IR. We recently discovered MRK as a member of the MAPK kinase kinase family that activates p38gamma. Here we investigated the role of MRK in the checkpoint response to IR. We identified autophosphorylation sites on MRK that are important for its kinase activity. A phosphospecific antibody that recognizes these sites showed that MRK is activated upon IR in a rapid and sustained manner. MRK depletion by RNA interference resulted in defective S and G(2) checkpoints induced by IR that were accompanied by reduced Chk2 phosphorylation and delayed Cdc25A degradation. We also showed that Chk2 is a substrate for MRK in vitro and is phosphorylated at Thr(68) by active MRK in cells. MRK depletion also increased sensitivity to the killing effects of IR. In addition, MRK depletion reduced IR-induced activation of p38gamma but had no effect on p38alpha activation, indicating that MRK is a specific activator of p38gamma after IR. Inhibition of p38gamma by RNA interference, however, did not impair IR-induced checkpoints. Thus, in response to IR MRK controls two independent pathways: the Chk2-Cdc25A pathway leading to cell cycle arrest and the p38gamma MAPK pathway.     

The Extracellular Signal-regulated Kinase–Mitogen-activated Protein Kinase Pathway Phosphorylates and Targets Cdc25A for SCFβ-TrCP-dependent Degradation for Cell Cycle Arrest

The extracellular signal-regulated kinase (ERK) pathway is generally mitogenic, but, upon strong activation, it causes cell cycle arrest by a not-yet fully understood mechanism. In response to genotoxic stress, Chk1 hyperphosphorylates Cdc25A, a positive cell cycle regulator, and targets it for Skp1/Cullin1/F-box protein (SCF)^β-TrCP ubiquitin ligase-dependent degradation, thereby leading to cell cycle arrest. Here, we show that strong ERK activation can also phosphorylate and target Cdc25A for SCF^β-TrCP-dependent degradation. When strongly activated in Xenopus eggs, the ERK pathway induces prominent phosphorylation and SCF^β-TrCP-dependent degradation of Cdc25A. p90rsk, the kinase downstream of ERK, directly phosphorylates Cdc25A on multiple sites, which, interestingly, overlap with Chk1 phosphorylation sites. Furthermore, ERK itself phosphorylates Cdc25A on multiple sites, a major site of which apparently is phosphorylated by cyclin-dependent kinase (Cdk) in Chk1-induced degradation. p90rsk phosphorylation and ERK phosphorylation contribute, roughly equally and additively, to the degradation of Cdc25A, and such Cdc25A degradation occurs during oocyte maturation in which the endogenous ERK pathway is fully activated. Finally, and importantly, ERK-induced Cdc25A degradation can elicit cell cycle arrest in early embryos. These results suggest that strong ERK activation can target Cdc25A for degradation in a manner similar to, but independent of, Chk1 for cell cycle arrest.     

The Germinal Center Kinase GCK-1 Is a Negative Regulator of MAP Kinase Activation and Apoptosis in the C. elegans Germline

The germinal center kinases (GCK) constitute a large, highly conserved family of proteins that has been implicated in a wide variety of cellular processes including cell growth and proliferation, polarity, migration, and stress responses. Although diverse, these functions have been attributed to an evolutionarily conserved role for GCKs in the activation of ERK, JNK, and p38 MAP kinase pathways. In addition, multiple GCKs from different species promote apoptotic cell death. In contrast to these paradigms, we found that a C. elegans GCK, GCK-1, functions to inhibit MAP kinase activation and apoptosis in the C. elegans germline. In the absence of GCK-1, a specific MAP kinase isoform is ectopically activated and oocytes undergo abnormal development. Moreover, GCK-1- deficient animals display a significant increase in germ cell death. Our results suggest that individual germinal center kinases act in mechanistically distinct ways and that these functions are likely to depend on organ- and developmental-specific contexts.