CPMK2, an SLT2-homologous mitogen-activated protein (MAP) kinase,is essential for pathogenesis of Claviceps purpurea on rye: evidence for a second conserved pathogenesis-related MAP kinase cascade in phytopathogenic fungi

Cpmk2, encoding a mitogen-activated protein (MAP) kinase from the ascomycete Claviceps purpurea, is an orthologue of SLT2 from Saccharomyces cerevisiae, the first isolated from a biotrophic, non-appressorium-forming pathogen. Deletion mutants obtained by a gene replacement approach show impaired vegetative properties (no conidiation) and a significantly reduced virulence, although they retain a limited ability to colonize the host tissue. Increased sensitivity to protoplasting enzymes indicates that the cell wall structure of the mutants may be altered. As the phenotypes of these mutants are similar to those observed in strains of the rice pathogen, Magnaporthe grisea, that have been deprived of their MAP kinase gene mps1, the ability of cpmk2 to complement the defects of delta mps1 was investigated. Interestingly, the C. purpurea gene, under the control of its own promoter, was able to complement the M. grisea mutant phenotype: transformants were able to sporulate and form infection hyphae on onion epidermis and were fully pathogenic on barley leaves. This indicates that, despite the differences in infection strategies, which include host and organ specificity, mode of penetration and colonization of host tissue, CPMK2/MPS1 defines a second MAP kinase cascade (after the Fus3p/PMK1 cascade) essential for fungal pathogenicity.     

Counteractive control of polarized morphogenesis during mating by mitogen-activated protein kinase Fus3 and G1 cyclin-dependent kinase

Cell polarization in response to external cues is critical to many eukaryotic cells. During pheromone-induced mating in Saccharomyces cerevisiae, the mitogen-activated protein kinase (MAPK) Fus3 induces polarization of the actin cytoskeleton toward a landmark generated by the pheromone receptor. Here, we analyze the role of Fus3 activation and cell cycle arrest in mating morphogenesis. The MAPK scaffold Ste5 is initially recruited to the plasma membrane in random patches that polarize before shmoo emergence. Polarized localization of Ste5 is important for shmooing. In fus3 mutants, Ste5 is recruited to significantly more of the plasma membrane, whereas recruitment of Bni1 formin, Cdc24 guanine exchange factor, and Ste20 p21-activated protein kinase are inhibited. In contrast, polarized recruitment still occurs in a far1 mutant that is also defective in G1 arrest. Remarkably, loss of Cln2 or Cdc28 cyclin-dependent kinase restores polarized localization of Bni1, Ste5, and Ste20 to a fus3 mutant. These and other findings suggest Fus3 induces polarized growth in G1 phase cells by down-regulating Ste5 recruitment and by inhibiting Cln/Cdc28 kinase, which prevents basal recruitment of Ste5, Cdc42-mediated asymmetry, and mating morphogenesis.     

Atf1 is a target of the mitogen-activated protein kinase Pmk1 and regulates cell integrity in fission yeast

In fission yeast, knockout of the calcineurin gene resulted in hypersensitivity to Cl(-), and the overexpression of pmp1(+) encoding a dual-specificity phosphatase for Pmk1 mitogen-activated protein kinase (MAPK) or the knockout of the components of the Pmk1 pathway complemented the Cl(-) hypersensitivity of calcineurin deletion. Here, we showed that the overexpression of ptc1(+) and ptc3(+), both encoding type 2C protein phosphatase (PP2C), previously known to inactivate the Wis1-Spc1-Atf1 stress-activated MAPK signaling pathway, suppressed the Cl(-) hypersensitivity of calcineurin deletion. We also demonstrated that the mRNA levels of these two PP2Cs and pyp2(+), another negative regulator of Spc1, are dependent on Pmk1. Notably, the deletion of Atf1, but not that of Spc1, displayed hypersensitivity to the cell wall-damaging agents and also suppressed the Cl(-) hypersensitivity of calcineurin deletion, both of which are characteristic phenotypes shared by the mutation of the components of the Pmk1 MAPK pathway. Moreover, micafungin treatment induced Pmk1 hyperactivation that resulted in Atf1 hyperphosphorylation. Together, our results suggest that PP2C is involved in a negative feedback loop of the Pmk1 signaling, and results also demonstrate that Atf1 is a key component of the cell integrity signaling downstream of Pmk1 MAPK.     

The Arabidopsis mitogen-activated protein kinase kinase MKK3 is upstream of group C mitogen-activated protein kinases and participates in pathogen signaling

Although the Arabidopsis thaliana genome contains genes encoding 20 mitogen-activated protein kinases (MAPKs) and 10 MAPK kinases (MAPKKs), most of them are still functionally uncharacterized. In this work, we analyzed the function of the group B MAPK kinase, MKK3. Transgenic ProMKK3:GUS lines showed basal expression in vascular tissues that was strongly induced by Pseudomonas syringae pv tomato strain DC3000 (Pst DC3000) infection but not by abiotic stresses. The growth of virulent Pst DC3000 was increased in mkk3 knockout plants and decreased in MKK3-overexpressing plants. Moreover, MKK3 overexpression lines showed increased expression of several PR genes. By yeast two-hybrid analysis, coimmunoprecipitation, and protein kinase assays, MKK3 was revealed to be an upstream activator of the group C MAPKs MPK1, MPK2, MPK7, and MPK14. Flagellin-derived flg22 peptide strongly activated MPK6 but resulted in poor activation of MPK7. By contrast, MPK6 and MPK7 were both activated by H(2)O(2), but only MPK7 activation was enhanced by MKK3. In agreement with the notion that MKK3 regulates the expression of PR genes, ProPR1:GUS expression was strongly enhanced by coexpression of MKK3-MPK7. Our results reveal that the MKK3 pathway plays a role in pathogen defense and further underscore the importance and complexity of MAPK signaling in plant stress responses.     

Evidence for a role of mitogen-activated protein kinase 3/mitogen-activated protein kinase in the development of testicular ischemia-reperfusion injury

Mitogen-activated protein kinase (MAPK) 3/MAPK1 (also known as ERK1/ERK2) plays an important role in the signal transduction pathways. To our knowledge, however, its role in the development of testicular ischemia-reperfusion injury has not yet been investigated. Therefore, we studied the pattern of MAPK3/MAPK1 activation in a experimental model of testicular ischemia-reperfusion injury. We also investigated MAPK8 to understand whether an association exists between these two MAPKs. Adult male Sprague-Dawley rats were subjected to 1 h of testicular ischemia followed by 24 h of reperfusion or to a sham testicular ischemia-reperfusion. Animals were randomized to receive PD98059, which is an inhibitor of MAPK3/MAPK1 (10 mg/kg i.p. administered immediately after detorsion), or its vehicle. The time course of MAPK3/MAPK1, MAPK8, and tumor necrosis factor (TNF; also known as TNF alpha) expression and a histological examination in both the ischemic-reperfused testis and the contralateral one were performed. In both testes, MAPK3/MAPK1 and MAPK8 expression appeared following 10 min of reperfusion and reached their highest activation after 30 min. The MAPK levels slowly decreased, and no significant expression of either kinase was observed following 2 h of reperfusion. Expression of TNF was evident after 1 h of reperfusion and reached its maximum increase after 3 h. PD98059 blunted MAPK3/MAPK1 and MAPK8, reduced TNF expression, and improved the testicular damage caused by ischemia-reperfusion injury in both testes. These data emphasize that MAPK3/MAPK1 has a role in testicular damage and that its blockade might have a future therapeutic role for the management of patients with unilateral testicular torsion.     

Rho2 is a target of the farnesyltransferase Cpp1 and acts upstream of Pmk1 mitogen-activated protein kinase signaling in fission yeast

We have previously demonstrated that knockout of the calcineurin gene or inhibition of calcineurin activity by immunosuppressants resulted in hypersensitivity to Cl- in fission yeast. We also demonstrated that knockout of the components of the Pmk1 mitogen-activated protein kinase (MAPK) pathway, such as Pmk1 or Pek1 complemented the hypersensitivity to Cl-. Using this interaction between calcineurin and Pmk1 MAPK, here we developed a genetic screen that aims to identify new regulators of the Pmk1 signaling and isolated vic (viable in the presence of immunosuppressant and chloride ion) mutants. One of the mutants, vic1-1, carried a missense mutation in the cpp1+ gene encoding a beta subunit of the protein farnesyltransferase, which caused an amino acid substitution of aspartate 155 of Cpp1 to asparagine (Cpp1(D155N)). Analysis of the mutant strain revealed that Rho2 is a novel target of Cpp1. Moreover, Cpp1 and Rho2 act upstream of Pck2-Pmk1 MAPK signaling pathway, thereby resulting in the vic phenotype upon their mutations. Interestingly, compared with other substrates of Cpp1, defects of Rho2 function were more phenotypically manifested by the Cpp1(D155N) mutation. Together, our results demonstrate that Cpp1 is a key component of the Pck2-Pmk1 signaling through the spatial control of the small GTPase Rho2.     

The MAP kinase Pmk1 and protein kinase A are required for rotenone resistance in the fission yeast, Schizosaccharomyces pombe

Rotenone is a widely used pesticide that induces Parkinson’s disease-like symptoms in rats and death of dopaminergic neurons in culture. Although rotenone is a potent inhibitor of complex I of the mitochondrial electron transport chain, it can induce death of dopaminergic neurons independently of complex I inhibition. Here we describe effects of rotenone in the fission yeast, Schizosaccharomyces pombe, which lacks complex I and carries out rotenone-insensitive cellular respiration. We show that rotenone induces generation of reactive oxygen species (ROS) as well as fragmentation of mitochondrial networks in treated S. pombe cells. While rotenone is only modestly inhibitory to growth of wild type S. pombe cells, it is strongly inhibitory to growth of mutants lacking the ERK-type MAP kinase, Pmk1, or protein kinase A (PKA). In contrast, cells lacking the p38 MAP kinase, Spc1, exhibit modest resistance to rotenone. Consistent with these findings, we provide evidence that Pmk1 and PKA, but not Spc1, are required for clearance of ROS in rotenone treated S. pombe cells. Our results demonstrate the usefulness of S. pombe for elucidating complex I-independent molecular targets of rotenone as well as mechanisms conferring resistance to the toxin.     

Glycogen synthase kinase 3 beta is a natural activator of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 1 (MEKK1)

Glycogen synthase kinase 3beta (GSK3 beta) is implicated in many biological events, including embryonic development, cell differentiation, apoptosis, and insulin response. GSK3 beta has now been shown to induce activation of the mitogen-activated protein kinase kinase kinase MEKK1 and thereby to promote signaling by the stress-activated protein kinase pathway. GSK3 beta-binding protein blocked the activation of MEKK1 by GSK3 beta in human embryonic kidney 293 (HEK293) cells. Furthermore, co-immunoprecipitation analysis revealed a physical association between endogenous GSK3 beta and MEKK1 in HEK293 cells. Overexpression of axin1, a GSK3 beta-regulated scaffolding protein, did not affect the physical interaction between GSK3 beta and MEKK1 in transfected HEK293 cells. Exposure of cells to insulin inhibited the activation of MEKK1 by GSK3 beta, and this inhibitory effect of insulin was abolished by the phosphatidylinositol 3-kinase inhibitor wortmannin. Furthermore, MEKK1 activity under either basal or UV- or tumor necrosis factor alpha-stimulated conditions was reduced in embryonic fibroblasts derived from GSK3 beta knockout mice compared with that in such cells from wild-type mice. Ectopic expression of GSK3 beta increased both basal and tumor necrosis factor alpha-stimulated activities of MEKK1 in GSK3 beta(-/-) cells. Together, these observations suggest that GSK3 beta functions as a natural activator of MEKK1.     

The NPK1 mitogen-activated protein kinase kinase kinase contains a functional nuclear localization signal at the binding site for the NACK1 kinesin-like protein

The tobacco mitogen-activated protein kinase kinase kinase NPK1 localizes to the equatorial region of phragmoplasts by interacting with kinesin-like protein NACK1. This leads to activation of NPK1 kinase at late M phase, which is necessary for cell plate formation. Until now, its localization during interphase has not been reported. We investigated the subcellular localization of NPK1 in tobacco-cultured BY-2 cells at interphase using indirect immunofluorescence microscopy and fusion to green fluorescent protein (GFP). Fluorescence of anti-NPK1 antibodies and GFP-fused NPK1 were detected only in the nuclei of BY-2 cells at interphase. Examination of the amino acid sequence of NPK1 showed that at the carboxyl-terminal region in the regulatory domain, which contains the binding site of NACK1, NPK1 contained a cluster of basic amino acids that resemble a bipartite nuclear localization signal (NLS). Amino acid substitution mutations in the critical residues in putative NLS caused a marked reduction in nuclear localization of NPK1 in BY-2 cells, indicating that this sequence is functional in tobacco BY-2 cells. We also found that the 64-amino acid sequence at the carboxyl terminus that contains NLS sequence is essential for interaction with NACK1, and that mutations in the NLS sequence prevented NPK1 from interacting with NACK1. Thus, the amino acid sequence at the carboxyl-terminal region of NPK1 has dual functions for nuclear localization during interphase and binding NACK1 in M phase.     

The MEKK1-MKK1/MKK2-MPK4 kinase cascade negatively regulates immunity mediated by a mitogen-activated protein kinase kinase kinase in Arabidopsis

In Arabidopsis thaliana, the MEKK1-MKK1/MKK2-MPK4 mitogen-activated protein (MAP) kinase cascade represses cell death and immune responses. In mekk1, mkk1 mkk2, and mpk4 mutants, programmed cell death and defense responses are constitutively activated, but the mechanism by which MEKK1, MKK1/MKK2, and MPK4 negatively regulate cell death and immunity was unknown. From a screen for suppressors of mkk1 mkk2, we found that mutations in suppressor of mkk1 mkk2 1 (summ1) suppress the cell death and defense responses not only in mkk1 mkk2 but also in mekk1 and mpk4. SUMM1 encodes the MAP kinase kinase kinase MEKK2. It interacts with MPK4 and is phosphorylated by MPK4 in vitro. Overexpression of SUMM1 activates cell death and defense responses that are dependent on the nucleotide binding-leucine-rich repeat protein SUMM2. Taken together, our data suggest that the MEKK1-MKK1/MKK2-MPK4 kinase cascade negatively regulates MEKK2 and activation of MEKK2 triggers SUMM2-mediated immune responses.     

Phosphatidylinositol 3-kinase is required for integrin-stimulated AKT and Raf-1/mitogen-activated protein kinase pathway activation.

Cell attachment to fibronectin stimulates the integrin-dependent interaction of p85-associated phosphatidylinositol (PI) 3-kinase with integrin-dependent focal adhesion kinase (FAK) as well as activation of the Ras/mitogen-activated protein (MAP) kinase pathway. However, it is not known if this PI 3-kinase-FAK interaction increases the synthesis of the 3-phosphorylated phosphoinositides (3-PPIs) or what role, if any, is played by activated PI 3-kinase in integrin signaling. We demonstrate here the integrin-dependent accumulation of the PI 3-kinase products, PI 3,4-bisphosphate [PI(3,4)P2] and PI(3,4,5)P3, as well as activation of AKT kinase, a serine/threonine kinase that can be stimulated by binding of PI(3,4)P2. The PI 3-kinase inhibitors wortmannin and LY294002 significantly decreased the integrin-induced accumulation of the 3-PPIs and activation of AKT kinase, without having significant effects on the levels of PI(4,5)P2 or tyrosine phosphorylation of paxillin. These inhibitors also reduced cell adhesion/spreading onto fibronectin but had no effect on attachment to polylysine. Interestingly, integrin-mediated Erk-2, Mek-1, and Raf-1 activation, but not Ras-GTP loading, was inhibited at least 80% by wortmannin and LY294002. In support of the pharmacologic results, fibronectin activation of Erk-2 and AKT kinases was completely inhibited by overexpression of a dominant interfering p85 subunit of PI 3-kinase. We conclude that integrin-mediated adhesion to fibronectin results in the accumulation of the PI 3-kinase products PI(3,4)P2 and PI(3,4,5)P3 as well as the PI 3-kinase-dependent activation of the kinases Raf-1, Mek-1, Erk-2, and AKT and that PI 3-kinase may function upstream of Raf-1 but downstream of Ras in integrin activation of Erk-2 MAP and AKT kinases.     

A tobacco protein kinase, NPK2, has a domain homologous to a domain found in activators of mitogen-activated protein kinases (MAPKKs)

A cDNA (cNPK2) that encodes a protein of 518 amino acids was isolated from a library prepared from poly(A)⁺ RNAs of tobacco cells in suspension culture. The N-terminal half of the predicted NPK2 protein is similar in amino acid sequence to the catalytic domains of kinases that activate mitogen-activated protein kinases (designated here MAPKKs) from various animals and to those of yeast homologs of MAPKKs. The N-terminal domain of NPK2 was produced as a fusion protein in Escherichia coli, and the purified fusion protein was found to be capable of autophosphorylation of threonine and serine residues. These results indicate that the N-terminal domain of NPK2 has activity of a serine/threonine protein kinase. Southern blot analysis showed that genomic DNAs from various plant species, including Arabidopsis thaliana and sweet potato, hybridized strongly with cNPK2, indicating that these plants also have genes that are closely related to the gene for NPK2. The structural similarity between the catalytic domain of NPK2 and those of MAPKKs and their homologs suggests that tobacco NPK2 corresponds to MAPKKs of other organisms. Given the existence of plant homologs of an MAP kinase and tobacco NPK1, which is structurally and functionally homologous to one of the activator kinases of yeast homologs of MAPKK (MAPKKKs), it seems likely that a signal transduction pathway mediated by a protein kinase cascade that is analogous to the MAP kinase cascades proposed in yeasts and animals, is also conserved in plants.     

The roles of phosphatidylinositol 3-kinase and protein kinase Czeta for thrombopoietin-induced mitogen-activated protein kinase activation in primary murine megakaryocytes

Thrombopoietin (TPO) stimulates a network of intracellular signaling pathways that displays extensive cross-talk. We have demonstrated previously that the ERK/mitogen-activated protein kinase pathway is important for TPO-induced endomitosis in primary megakaryocytes (MKs). One known pathway by which TPO induces ERK activation is through the association of Shc with the penultimate phosphotyrosine within the TPO receptor, Mpl. However, several investigators found that the membrane-proximal half of the cytoplasmic domain of Mpl is sufficient to activate ERK in vitro and support base-line megakaryopoiesis in vivo. Using BaF3 cells expressing a truncated Mpl (T69Mpl) as a tool to identify non-Shc/Ras-dependent signaling pathways, we describe here novel mechanisms of TPO-induced ERK activation mediated, in part, by phosphoinositide 3-kinase (PI3K). Similar to cells expressing full-length receptor, PI3K was activated by its incorporation into a complex with IRS2 or Gab2. Furthermore, the MEK-phosphorylating activity of protein kinase Czeta (PKCzeta) was also enhanced after TPO stimulation of T69Mpl, contributing to ERK activity. PKCzeta and PI3K also contribute to TPO-induced ERK activation in MKs, confirming their physiological relevance. Like in BaF3 cells, a TPO-induced signaling complex containing p85PI3K is detectable in MKs expressing T61Mpl and is probably responsible for PI3K activation. These data demonstrate a novel role of PI3K and PKCzeta in steady-state megakaryopoiesis.     

Dictyostelium stress-activated protein kinase alpha, a novel stress-activated mitogen-activated protein kinase kinase kinase-like kinase, is important for the proper regulation of the cytoskeleton

Mitogen-activated protein kinase cascades regulate various cellular functions, including growth, cell differentiation, development, and stress responses. We have identified a new Dictyostelium kinase (stress-activated protein kinase [SAPK]alpha), which is related to members of the mixed lineage kinase class of mitogen-activated protein kinase kinases. SAPKalpha is activated by osmotic stress, heat shock, and detachment from the substratum and by a membrane-permeable cGMP analog, a known regulator of stress responses in Dictyostelium. SAPKalpha is important for cellular resistance to stresses, because SAPKalpha null cells exhibit reduced viability in response to osmotic stress. We found that SAPKalpha mutants affect cellular processes requiring proper regulation of the actin cytoskeleton, including cell motility, morphogenesis, cytokinesis, and cell adhesion. Overexpression of SAPKalpha results in highly elevated basal and chemoattractant-stimulated F-actin levels and strong aggregation and developmental defects, including a failure to polarize and chemotax, and abnormal morphogenesis. These phenotypes require a kinase-active SAPKalpha. SAPKalpha null cells exhibit reduced chemoattractant-stimulated F-actin levels, cytokinesis, developmental and adhesion defects, and a motility defect that is less severe than that exhibited by SAPKalpha-overexpressing cells. SAPKalpha colocalizes with F-actin in F-actin-enriched structures, including membrane ruffles and pseudopodia during chemotaxis. Although SAPKalpha is required for these F-actin-mediated processes, it is not detectably activated in response to chemoattractant stimulation.     

The Fus3/Kss1 MAP kinase homolog Amk1 regulates the expression of genes encoding hydrolytic enzymes in Alternaria brassicicola

Mitogen-activated protein (MAP) kinases have been shown to be required for virulence in diverse phytopathogenic fungi. To study its role in pathogenicity, we disrupted the Amk1 MAP kinase gene, a homolog of the Fus3/Kss1 MAP kinases in Saccharomyces cerevisiae, in the necrotrophic Brassica pathogen, Alternaria brassicicola. The amk1 disruption mutants showed null pathogenicity on intact host plants. However, amk1 mutants were able to colonize host plants when they were inoculated on a physically damaged host surface, or when they were inoculated along with nutrient supplements. On intact plants, mutants expressed extremely low amounts of several hydrolytic enzyme genes that were induced over 10-fold in the wild-type during infection. These genes were also dramatically induced in the mutants on wounded plants. These results imply a correlation between virulence and the expression level of specific hydrolytic enzyme genes plus the presence of an unidentified pathway controlling these genes in addition to or in conjunction with the Amk1 pathway.     

The complete pathway for catalytic activation of the mitogen-activated protein kinase, ERK2

The mitogen-activated protein (MAP) kinase ERK2 is an essential signal transduction molecule that mediates extracellular signaling by all polypeptide growth factors. Full activation of ERK2 requires phosphorylation at both a threonine residue (Thr(183)) conserved in most protein kinases as well as a tyrosine residue (Tyr(185)) unique to members of the mitogen-activated protein kinase family. We have characterized the kinetic role of phosphorylation at each site with respect to the overall activation mechanism, providing a complete picture of the reaction steps involved. Phosphorylation at Tyr(185) serves to configure the ATP binding site, while phosphorylation at both residues is required to stabilize binding of the protein substrate, myelin basic protein. Similar control mechanisms are employed to stabilize ATP and myelin basic protein in the phosphoryl group transfer reaction, accounting for the enormous increase in turnover rate. The mechanism of ERK2 activation is kinetically similar to that of the cell cycle control protein, cdk2/cyclinA. Phosphorylation of Tyr(185) in ERK2 and association of cyclinA with cdk2 both serve to stabilize ATP binding. Subsequent phosphorylation of both enzymes on threonine serves to stabilize binding of the phosphoacceptor substrate.