Cytoplasmic localization of Wis1 MAPKK by nuclear export signal is important for nuclear targeting of Spc1/Sty1 MAPK in fission yeast

Mitogen-activated protein kinase (MAPK) cascade is a ubiquitous signaling module that transmits extracellular stimuli through the cytoplasm to the nucleus; in response to activating stimuli, MAPKs translocate into the nucleus. Mammalian MEK MAPK kinases (MAPKKs) have in their N termini an MAPK-docking site and a nuclear export signal (NES) sequence, which are known to play critical roles in maintaining ERK MAPKs in the cytoplasm of unstimulated cells. Herein, we show that the Wis1 MAPKK of the stress-activated Spc1 MAPK cascade in fission yeast also has a MAPK-docking site and an NES sequence in its N-terminal domain. Unexpectedly, an inactivating mutation to the NES of chromosomal wis1(+) does not affect the subcellular localization of Spc1 MAPK, whereas this NES mutation disturbs the cytoplasmic localization of Wis1. However, when Wis1 is targeted to the nucleus by fusing to a nuclear localization signal sequence, stress-induced nuclear translocation of Spc1 is abrogated, indicating that cytoplasmic Wis1 is required for nuclear transport of Spc1 upon stress. Moreover, we have observed that a fraction of Wis1 translocates into the nucleus in response to stress. These results suggest that cytoplasmic localization of Wis1 MAPKK by its NES is important for stress signaling to the nucleus.     

Mcs4 mitotic catastrophe suppressor regulates the fission yeast cell cycle through the Wik1-Wis1-Spc1 kinase cascade.

Spc1 in Schizosaccharomyces pombe is a member of the stress-activated protein kinase family, an evolutionary conserved subfamily of mitogen-activated protein kinases (MAPKs). Spc1 is activated by a MAPK kinase homologue, Wis1, and negatively regulated by Pyp1 and Pyp2 tyrosine phosphatases. Mutations in the spc1+ and wis1+ genes cause a G2 cell cycle delay that is exacerbated during stress. Herein, we describe two upstream regulators of the Wis1-Spc1 cascade. wik1+ (Wis1 kinase) was identified from its homology to budding yeast SSK2, which encodes a MAPKK kinase that regulates the HOG1 osmosensing pathway. Delta wik1 cells are impaired in stress-induced activation of Spc1 and show a G2 cell cycle delay and osmosensitive growth. Moreover, overproduction of a constitutively active form of Wik1 induces hyperactivation of Spc1 in wis1(+)-dependent manner, suggesting that Wik1 regulates Spc1 through activation of Wis1. A mutation of mcs4+ (mitotic catastrophe suppressor) was originally isolated as a suppressor of the mitotic catastrophe phenotype of a cdc2-3w wee1-50 double mutant. We have found that mcs4- cells are defective at activation of Spc1 in response to various forms of stress. Epistasis analysis has placed Mcs4-upstream of Wik1 in the Spc1 activation cascade. These results indicate that Mcs4 is part of a sensor system for multiple environmental signals that modulates the timing of entry into mitosis by regulating the Wik1-Wis1-Spc1 kinase cascade. Inactivation of the sensor system delays the onset of mitosis and rescues lethal premature mitosis in cdc2-3w wee1-50 cells.     

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 Win1 Mitotic Regulator Is a Component of the Fission Yeast Stress-activated Sty1 MAPK Pathway

The fission yeast Sty1 mitogen-activated protein (MAP) kinase (MAPK) and its activator the Wis1 MAP kinase kinase (MAPKK) are required for cell cycle control, initiation of sexual differentiation, and protection against cellular stress. Like the mammalian JNK/SAPK and p38/CSBP1 MAPKs, Sty1 is activated by a range of environmental insults including osmotic stress, hydrogen peroxide, UV light, menadione, heat shock, and the protein synthesis inhibitor anisomycin. We have recently identified two upstream regulators of the Wis1 MAPKK, namely the Wak1 MAPKKK and the Mcs4 response regulator. Cells lacking Mcs4 or Wak1, however, are able to proliferate under stressful conditions and undergo sexual differentiation, suggesting that additional pathway(s) control the Wis1 MAPKK. We now show that this additional signal information is provided, at least in part, by the Win1 mitotic regulator. We show that Wak1 and Win1 coordinately control activation of Sty1 in response to multiple environmental stresses, but that Wak1 and Win1 perform distinct roles in the control of Sty1 under poor nutritional conditions. Our results suggest that the stress-activated Sty1 MAPK integrates information from multiple signaling pathways.     

A Highlights from MBoC Selection: Response regulator–mediated MAPKKK heteromer promotes stress signaling to the Spc1 MAPK in fission yeast

The Spc1 mitogen-activated protein kinase (MAPK) cascade in fission yeast is activated by two MAPK kinase kinase (MAPKKK) paralogues, Wis4 and Win1, in response to multiple forms of environmental stress. Previous studies identified Mcs4, a “response regulator” protein that associates with the MAPKKKs and receives peroxide stress signals by phosphorelay from the Mak2/Mak3 sensor histidine kinases. Here we show that Mcs4 has an unexpected, phosphorelay-independent function in promoting heteromer association between the Wis4 and Win1 MAPKKKs. Only one of the MAPKKKs in the heteromer complex needs to be catalytically active, but disturbing the integrity of the complex by mutations to Mcs4, Wis4, or Win1 results in reduced MAPKKK–MAPKK interaction and, consequently, compromised MAPK activation. The physical interaction among Mcs4, Wis4, and Win1 is constitutive and not responsive to stress stimuli. Therefore the Mcs4–MAPKKK heteromer complex might serve as a stable platform/scaffold for signaling proteins that convey input and output of different stress signals. The Wis4–Win1 complex discovered in fission yeast demonstrates that heteromer-mediated mechanisms are not limited to mammalian MAPKKKs.     

The Schizosaccharomyces pombe Cdc7 protein kinase required for septum formation is a client protein of Cdc37

Cdc37 is an essential molecular chaperone found in fungi and metazoa whose main specificity is for certain protein kinases. Cdc37 can act as an Hsp90 cochaperone or alone; in yeasts, the interaction with Hsp90 is weak and appears not to be essential for Cdc37 function. Numerous genetic interactions between Cdc37 and likely client proteins have been observed in yeasts, but biochemical confirmation has been reported in only a few cases. We and others have generated and characterized temperature-sensitive cdc37 alleles in S. pombe and have used them to investigate the cellular roles of Cdc37: previous work has shown that mitotic Cdc2 is a major client. In this paper, we describe a screen for mutations synthetically lethal with a cdc37ts mutant with the aim of identifying genes encoding further client proteins of Cdc37. Ten such strains were isolated, and genomic libraries were screened for rescuing plasmids. In one case, a truncated cdc7 gene was identified. Further experiments showed that the mutation in this strain was indeed in cdc7. Cdc7 is a protein kinase required for septum initiation, and we show that its kinase activity is greatly reduced when Cdc37 function is impaired. Cdc7 normally locates to the spindle pole body during mitosis, and this appears to be unaffected in the cdc37ts mutant. Other evidence suggests that, in addition to mitosis and septum initiation, Cdc37 may also be required for septum cleavage.     

Cdc37 has distinct roles in protein kinase quality control that protect nascent chains from degradation and promote posttranslational maturation

Cdc37 is a molecular chaperone that functions with Hsp90 to promote protein kinase folding. Analysis of 65 Saccharomyces cerevisiae protein kinases ( approximately 50% of the kinome) in a cdc37 mutant strain showed that 51 had decreased abundance compared with levels in the wild-type strain. Several lipid kinases also accumulated in reduced amounts in the cdc37 mutant strain. Results from our pulse-labeling studies showed that Cdc37 protects nascent kinase chains from rapid degradation shortly after synthesis. This degradation phenotype was suppressed when cdc37 mutant cells were grown at reduced temperatures, although this did not lead to a full restoration of kinase activity. We propose that Cdc37 functions at distinct steps in kinase biogenesis that involves protecting nascent chains from rapid degradation followed by its folding function in association with Hsp90. Our studies demonstrate that Cdc37 has a general role in kinome biogenesis.     

Mitogen-activated protein kinase signaling in plants under abiotic stress

Mitogen-activated protein kinase cascade is evolutionarily conserved signal transduction module involved in transducing extracellular signals to the nucleus for appropriate cellular adjustment. This cascade consists essentially of three components, a MAPK kinase kinase (MAPKKK), a MAPK kinase (MAPKK) and a MAPK connected to each other by the event of phosphorylation. These kinases play various roles in intra- and extra-cellular signaling in plants by transferring the information from sensors to responses. Signaling through MAP kinase cascade can lead to cellular responses including cell division, differentiation as well as responses to various stresses. MAPK signaling has also been associated with hormonal responses. In plants, MAP kinases are represented by multigene families and are involved in efficient transmission of specific stimuli and also involved in the regulation of the antioxidant defense system in response to stress signaling. In the current review we summarize and investigate the participation of MAPKs as possible mediators of various abiotic stresses in plants.Key words: abiotic stress, cross talk, mitogen-activated protein kinases, heat map, MAPK signaling, signal transduction, stress signaling     

Mitogen-activated protein kinases: A new therapeutic target in cardiac pathology

Eukaryotic cells respond to different external stimuli by activation of mechanisms of cell signaling. One of the major systems participating in the transduction of signal from the cell membrane to nuclear and other intracellular targets is the highly conserved mitogen-activated protein kinase (MAPK) superfamily. The members of MAPK family are involved in the regulation of a large variety of cellular processes such as cell growth, differentiation, development, cell cycle, death and survival. Several MAPK subfamilies, each with apparently unique signaling pathway, have been identified in the mammalian myocardium. These cascades differ in their upstream activation sequence and in downstream substrate specifity. Each pathway follows the same conserved three-kinase module consisting of MAPK, MAPK kinase (MAPKK, MKK or MEK), and MAPK kinase kinase (MAPKKK, MEKK). The major groups of MAPKs found in cardiac tissue include the extracellular signal-regulated kinases (ERKs), the stress-activated/c-Jun NH2-terminal kinases (SAPK/JNKs), p38-MAPK, and ERK5/big MAPK 1 (BMK1). The ERKs are strongly activated by mitogenic and growth factors and by physical stress, whereas SAPK/JNKs and p38-MAPK can be activated by various cell stresses, such as hyperosmotic shock, metabolic stress or protein synthesis inhibitors, UV radiation, heat shock, cytokines, and ischemia. Activation of MAPKs family plays a key role in the pathogenesis of various processes in the heart, e.g. myocardial hypertrophy and its transition to heart failure, in ischemic and reperfusion injury, as well in the cardioprotection conferred by ischemia- or pharmacologically-induced preconditioning. The following approaches are currently utilized to elucidate the role of MAPKs in the myocardium: (i) studies of the effects of myocardial processes on the activity of these kinases; (ii) pharmacological modulations of MAPKs activity and evaluation of their impact on the (patho)physiological processes in the heart; (iii) gene targeting or expression of constitutively active and dominant-negative forms of enzymes (adenovirus-mediated gene transfer).This review is focused on the regulatory role of MAPKs in the myocardium, with particular regard to their involvement in pathophysiological processes, such as myocardial hypertrophy and heart failure, ischemia/reperfusion injury, as well as in the mechanisms of cardioprotection. In addition, it summarizes current information on pharmacological modulations of MAPKs activity and their impact on the cardiac response to pathophysiological processes.     

Role of the Caenorhabditis elegans Shc adaptor protein in the c-Jun N-terminal kinase signaling pathway

Mitogen-activated protein kinases (MAPKs) are integral to the mechanisms by which cells respond to physiological stimuli and a wide variety of environmental stresses. In Caenorhabditis elegans, the stress response is controlled by a c-Jun N-terminal kinase (JNK)-like mitogen-activated protein kinase (MAPK) signaling pathway, which is regulated by MLK-1 MAPK kinase kinase (MAPKKK), MEK-1 MAPK kinase (MAPKK), and KGB-1 JNK-like MAPK. In this study, we identify the shc-1 gene, which encodes a C. elegans homolog of Shc, as a factor that specifically interacts with MEK-1. The shc-1 loss-of-function mutation is defective in activation of KGB-1, resulting in hypersensitivity to heavy metals. A specific tyrosine residue in the NPXY motif of MLK-1 creates a docking site for SHC-1 with the phosphotyrosine binding (PTB) domain. Introduction of a mutation that perturbs binding to the PTB domain or the NPXY motif abolishes the function of SHC-1 or MLK-1, respectively, thereby abolishing the resistance to heavy metal stress. These results suggest that SHC-1 acts as a scaffold to link MAPKKK to MAPKK activation in the KGB-1 MAPK signal transduction pathway.     

Molecular aspects of mechanical stress-induced cardiac hypertrophy

To elucidate the signal transduction pathway from external stimuli to nuclear gene expression in mechanical stress-induced cardiac hypertrophy, we examined the time course of activation of protein kinases such as Raf-1 kinase (Raf-1), mitogen-activated protein kinase kinase (MAPKK), MAP kinases (MAPKs) and 90-kDa ribosomal S6 kinase (p90^rsk) in neonatal rat cardiomyocytes. Mechanical stretch rapidly activated Raf-1 and its maximal activation was observed at 1–2 min after stretch. The activity of MAPKK was also increased by stretch, with a peak at 5 min after stretch. In addition, MAPKs and p90^rsk were maximally activated at 8 min and at 10–30 min after stretch, respectively. Next, the relationship between mechanical stress-induced hypertrophy and the cardiac renin-angiotensin system was investigated. When the stretch-conditioned culture medium was transferred to the culture dish of non-stretched cardiac myocytes, the medium activated MAPK activity slightly but significantly, and the activation was completely blocked by the type 1 angiotensin II receptor antagonist, CV-11974. However, activation of Raf-1 and MAPKs provoked by stretching cardiomyocytes was only partially suppressed by pretreatment with CV-11974. These results suggest that mechanical stress activates the protein kinase cascade of phosphorylation in cardiac myocytes in the order of Raf-1, MAPKK, MAPKs and p90^rsk, and that angiotensin II, which is secreted from stretched myocytes, activates a part of these protein kinases.Abbreviations MAPK mitogen-activated protein kinase - MAPKK MAP kinase kinase - Raf-1 - Raf- 1 kinase p90^rsk, 90 kDa ribosomal S6 kinase; AngII - angiotensin II - MAPKKK MAP kinase kinase kinase - rMAPK recombinant MAPKK fused to gluthathione S transferase - MMAKK recombinant MAPK fused to maltose binding protein - MBP myelin basic protein - ACE angiotensin-converting enzyme     

CK2 controls multiple protein kinases by phosphorylating a kinase-targeting molecular chaperone, Cdc37

Cdc37 is a kinase-associated molecular chaperone whose function in concert with Hsp90 is essential for many signaling protein kinases. Here, we report that mammalian Cdc37 is a pivotal substrate of CK2 (casein kinase II). Purified Cdc37 was phosphorylated in vitro on a conserved serine residue, Ser13, by CK2. Moreover, Ser13 was the unique phosphorylation site of Cdc37 in vivo. Crucially, the CK2 phosphorylation of Cdc37 on Ser13 was essential for the optimal binding activity of Cdc37 toward various kinases examined, including Raf1, Akt, Aurora-B, Cdk4, Src, MOK, MAK, and MRK. In addition, nonphosphorylatable mutants of Cdc37 significantly suppressed the association of Hsp90 with protein kinases, while the Hsp90-binding activity of the mutants was unchanged. The treatment of cells with a specific CK2 inhibitor suppressed the phosphorylation of Cdc37 in vivo and reduced the levels of Cdc37 target kinases. These results unveil a regulatory mechanism of Cdc37, identify a novel molecular link between CK2 and many crucial protein kinases via Cdc37, and reveal the molecular basis for the ability of CK2 to regulate pleiotropic cellular functions.     

The molecular chaperone Cdc37 is required for Ste11 function and pheromone-induced cell cycle arrest

The molecular chaperone Cdc37 is thought to act in part as a targeting subunit of the heat-shock protein 90 (Hsp90) chaperone complex. We demonstrate here that Cdc37 is required for activity of the kinase Ste11 in budding yeast. A cdc37 mutant strain is defective in Ste11-mediated pheromone signaling and in accumulation and functional maturation of the constitutively active Ste11 version Ste11DeltaN. Moreover, Cdc37, Ste11DeltaN and Hsp90 coprecipitate pairwise. Thus, Hsp90 and Cdc37 may transiently associate with Ste11 to promote proper folding and/or association with additional regulatory factors. Our results establish Ste11 as the first endogenous Cdc37 client protein in yeast.     

Candida albicans Cdc37 interacts with the Crk1 kinase and is required for Crk1 production

Crk1, a Cdc2-related protein kinase from the human pathogenic fungus Candida albicans, plays an important role in hyphal development and virulence. To address its regulatory mechanisms, we searched for Crk1 interacting proteins by two-hybrid screening. A CDC37 ortholog (CaCDC37) was cloned from the screening with the Crk1 kinase domain as the bait. The CaCdc37 interacted preferentially with the kinase domain of Crk1 (Crk1N) as shown by two-hybrid and immunoprecipitation experiments. CaCDC37 could complement a cdc37 thermosensitive mutant (cdc37-34) of Saccharomyces cerevisiae. Importantly, Crk1 protein was hardly detectable in the cdc37-34 mutant at restrictive temperature. However, upon expression of CaCdc37 in the cdc37 mutant, Crk1 protein was detected even at restrictive temperature. Our data suggested that CaCdc37 was required for the production of Crk1 kinase. Like Cdc37 proteins of S. cerevisiae and higher eukaryotes, CaCdc37 might function as a molecular chaperone that stabilized Crk1 and other protein kinases in C. albicans. In support of this, CaSTI1 was identified from a two-hybrid screen with the full-length Crk1 as the bait. CaSti1 showed two-hybrid interactions with both Crk1 and the CaCdc37.