The Fission Yeast Mitotic Regulator win1+ Encodes an MAP Kinase Kinase Kinase That Phosphorylates and Activates Wis1 MAP Kinase Kinase in Response to High Osmolarity

The Schizosaccharomyces pombe win1-1 mutant has a defect in the G2-M transition of the cell cycle. Although the defect is suppressed by wis1+ and wis4+, which are components of a stress-activated MAP kinase pathway that links stress response and cell cycle control, the molecular identity of Win1 has not been known. We show here that win1+ encodes a polypeptide of 1436 residues with an apparent molecular size of 180 kDa and demonstrate that Win1 is a MAP kinase kinase kinase that phosphorylates and activates Wis1. Despite extensive similarities between Win1 and Wis4, the two MAP kinase kinase kinases have distinct functions. Wis4 is able to compensate for loss of Win1 only under unstressed conditions to maintain basal Wis1 activity, but it fails to suppress the osmosignaling defect conferred by win1 mutations. The win1-1 mutation is a spontaneous duplication of 16 nucleotides, which leads to a frameshift and production of a truncated protein lacking the kinase domain. We discuss the cell cycle phenotype of the win1-1 cdc25-22 wee1-50 mutant and its suppression by wis genes.

A p90rsk Mutant Constitutively Interacting with MAP Kinase Uncouples MAP Kinase from p34cdc2/Cyclin B Activation in Xenopus Oocytes

The efficient activation of p90rsk by MAP kinase requires their interaction through a docking site located at the C-terminal end of p90rsk. The MAP kinase p42mpk1 can associate with p90rsk in G2-arrested but not in mature Xenopus oocytes. In contrast, an N-terminally truncated p90rsk mutant named D2 constitutively interacts with p42mpk1. In this report we show that expression of D2 inhibits Xenopus oocyte maturation. The inhibition requires the p42mpk1 docking site. D2 expression uncouples the activation of p42mpk1 and p34cdc2/cyclin B in response to progesterone but does not prevent signaling through p90rsk. Instead, D2 interferes with a p42mpk1-triggered pathway, which regulates the phosphorylation and activation of Plx1, a potential activator of the Cdc25 phosphatase. This new pathway that links the activation of p42mpk1 and Plx1 during oocyte maturation is independent of p34cdc2/cyclin B activity but requires protein synthesis. Using D2, we also provide evidence that the sustained activation of p42mpk1 can trigger nuclear migration in oocytes. Our results indicate that D2 is a useful tool to study MAP kinase function(s) during oocyte maturation. Truncated substrates such as D2, which constitutively interact with MAP kinases, may also be helpful to study signal transduction by MAP kinases in other cellular processes.

Rck2, a member of the calmodulin-protein kinase family, links protein synthesis to high osmolarity MAP kinase signaling in budding yeast

Rck2, a yeast Ser/Thr protein kinase homologous to mammalian calmodulin kinases, requires phosphorylation for activation. We provide evidence that in budding yeast, this step can be executed by the osmostress-activated mitogen-activated protein kinase Hog1. Rck2 phosphorylation was transiently increased during osmostress or in mutants with a hyperactive high osmolarity glycerol (HOG) pathway. This modification depended on catalytically active Hog1 kinase and two putative mitogen-activated protein kinase phosphorylation sites in Rck2. Immunokinase assays showed that Hog1 can directly phosphorylate Rck2 to stimulate its enzymatic activity toward translation elongation factor 2. We demonstrate that Hog1 and Rck2 are necessary for attenuation of protein synthesis in response to osmotic challenge and show that modification of elongation factor 2 induced by osmostress depends on Rck2 and Hog1 in vivo. Therefore, we propose that the transient down-regulation of protein synthesis after osmotic shock is a response not to damage but to an extracellular signal mediated by Hog1 and Rck2.

Requirement of mitogen-activated protein kinase kinase 3 (MKK3) for tumor necrosis factor-induced cytokine expression

The p38 mitogen-activated protein kinase is activated by treatment of cells with cytokines and by exposure to environmental stress. The effects of these stimuli on p38 MAP kinase are mediated by the MAP kinase kinases (MKKs) MKK3, MKK4, and MKK6. We have examined the function of the p38 MAP kinase signaling pathway by investigating the effect of targeted disruption of the Mkk3 gene. Here we report that Mkk3 gene disruption caused a selective defect in the response of fibroblasts to the proinflammatory cytokine tumor necrosis factor, including reduced p38 MAP kinase activation and cytokine expression. These data demonstrate that the MKK3 protein kinase is a critical component of a tumor necrosis factor-stimulated signaling pathway that causes increased expression of inflammatory cytokines.

Activation of Both MAP Kinase and Phosphatidylinositide 3-Kinase by Ras Is Required for Hepatocyte Growth Factor/Scatter Factor–induced Adherens Junction Disassembly

Hepatocyte growth factor/scatter factor (HGF/SF) stimulates the motility of epithelial cells, initially inducing centrifugal spreading of colonies followed by disruption of cell–cell junctions and subsequent cell scattering. In Madin–Darby canine kidney cells, HGF/SF-induced motility involves actin reorganization mediated by Ras, but whether Ras and downstream signals regulate the breakdown of intercellular adhesions has not been established. Both HGF/SF and V12Ras induced the loss of the adherens junction proteins E-cadherin and β-catenin from intercellular junctions during cell spreading, and the HGF/SF response was blocked by dominant-negative N17Ras. Desmosomes and tight junctions were regulated separately from adherens junctions, because they were not disrupted by V12Ras. MAP kinase, phosphatidylinositide 3-kinase (PI 3-kinase), and Rac were required downstream of Ras, because loss of adherens junctions was blocked by the inhibitors PD098059 and LY294002 or by dominant-inhibitory mutants of MAP kinase kinase 1 or Rac1. All of these inhibitors also prevented HGF/SF-induced cell scattering. Interestingly, activated Raf or the activated p110α subunit of PI 3-kinase alone did not induce disruption of adherens junctions. These results indicate that activation of both MAP kinase and PI 3-kinase by Ras is required for adherens junction disassembly and that this is essential for the motile response to HGF/SF.

Activation of NF-κB by nontypeable Hemophilus influenzae is mediated by toll-like receptor 2-TAK1-dependent NIK–IKKα/β–IκBα and MKK3/6–p38 MAP kinase signaling pathways in epithelial cells

Nontypeable Hemophilus influenzae (NTHi) is an important human pathogen in both children and adults. In children, it causes otitis media, the most common childhood infection and the leading cause of conductive hearing loss in the United States. In adults, it causes lower respiratory tract infections in the setting of chronic obstructive pulmonary disease, the fourth leading cause of death in the United States. The molecular mechanisms underlying the pathogenesis of NTHi-induced infections remain undefined, but they may involve activation of NF-κB, a transcriptional activator of multiple host defense genes involved in immune and inflammatory responses. Here, we show that NTHi strongly activates NF-κB in human epithelial cells via two distinct signaling pathways, NF-κB translocation-dependent and -independent pathways. The NF-κB translocation-dependent pathway involves activation of NF-κB inducing kinase (NIK)–IKKα/β complex leading to IκBα phosphorylation and degradation, whereas the NF-κB translocation-independent pathway involves activation of MKK3/6–p38 mitogen-activated protein (MAP) kinase pathway. Bifurcation of NTHi-induced NIK–IKKα/β-IκBα and MKK3/6–p38 MAP kinase pathways may occur at transforming growth factor-β activated kinase 1 (TAK1). Furthermore, we show that toll-like receptor 2 (TLR2) is required for NTHi-induced NF-κB activation. In addition, several key inflammatory mediators including IL-1β, IL-8, and tumor necrosis factor-α are up-regulated by NTHi. Finally, P6, a 16-kDa lipoprotein highly conserved in the outer membrane of all NTHi and H. influenzae type b strains, appears to also activate NF-κB via similar signaling pathways. Taken together, our results demonstrate that NTHi activates NF-κB via TLR2–TAK1-dependent NIK–IKKα/β-IκBα and MKK3/6–p38 MAP kinase signaling pathways. These studies may bring new insights into molecular pathogenesis of NTHi-induced infections and open up new therapeutic targets for these diseases.

Activation of the Raf-1/MAP kinase cascade is not sufficient for Ras transformation of RIE-1 epithelial cells.

The potent transforming activity of membrane-targeted Raf-1 (Raf-CAAX) suggests that Ras transformation is triggered primarily by a Ras-mediated translocation of Raf-1 to the plasma membrane. However, whereas constitutively activated mutants of Ras [H-Ras(61L) and K-Ras4B(12V)] and Raf-1 (DeltaRaf-22W and Raf-CAAX) caused indistinguishable morphologic and growth (in soft agar and nude mice) transformation of NIH 3T3 fibroblasts, only mutant Ras caused morphologic transformation of RIE-1 rat intestinal cells. Furthermore, only mutant Ras-expressing RIE-1 cells formed colonies in soft agar and developed rapid and progressive tumors in nude mice. We also observed that activated Ras, but not Raf-1, caused transformation of IEC-6 rat intestinal and MCF-10A human mammary epithelial cells. Although both Ras- and DeltaRaf-22W-expressing RIE-1 cells showed elevated Raf-1 and mitogen-activated protein (MAP) kinase activities, only Ras-transformed cells produced secreted factors that promoted RIE-1 transformation. Incubation of untransformed RIE-1 cells in the presence of conditioned medium from Ras-expressing, but not DeltaRaf-22W-expressing, cells caused a rapid and stable morphologic transformation that was indistinguishable from the morphology of Ras-transformed RIE-1 cells. Thus, induction of an autocrine growth mechanism may distinguish the transforming actions of Ras and Raf. In summary, our observations demonstrate that oncogenic Ras activation of the Raf/MAP kinase pathway alone is not sufficient for full tumorigenic transformation of RIE-1 epithelial cells. Thus, Raf-independent signaling events are essential for oncogenic Ras transformation of epithelial cells, but not fibroblasts.

Neurotrophins stimulate phosphorylation of synapsin I by MAP kinase and regulate synapsin I-actin interactions.

The ability of neurotrophins to modulate the survival and differentiation of neuronal populations involves the Trk/MAP (mitogen-activated protein kinase) kinase signaling pathway. More recently, neurotrophins have also been shown to regulate synaptic transmission. The synapsins are a family of neuron-specific phosphoproteins that play a role in regulation of neurotransmitter release, in axonal elongation, and in formation and maintenance of synaptic contacts. We report here that synapsin I is a downstream effector for the neurotrophin/Trk/MAP kinase cascade. Using purified components, we show that MAP kinase stoichiometrically phosphorylated synapsin I at three sites (Ser-62, Ser-67, and Ser-549). Phosphorylation of these sites was detected in rat brain homogenates, in cultured cerebrocortical neurons, and in isolated presynaptic terminals. Brain-derived neurotrophic factor and nerve growth factor upregulated phosphorylation of synapsin I at MAP kinase-dependent sites in intact cerebrocortical neurons and PC12 cells, respectively, while KCl- induced depolarization of cultured neurons decreased the phosphorylation state at these sites. MAP kinase-dependent phosphorylation of synapsin I significantly reduced its ability to promote G-actin polymerization and to bundle actin filaments. The results suggest that MAP kinase-dependent phosphorylation of synapsin I may contribute to the modulation of synaptic plasticity by neurotrophins and by other signaling pathways that converge at the level of MAP kinase activation.

Regulation of mitogen-activated protein kinases by a calcium/calmodulin-dependent protein kinase cascade.

Membrane depolarization of NG108 cells gives rapid (< 5 min) activation of Ca2+/calmodulin-dependent protein kinase IV (CaM-KIV), as well as activation of c-Jun N-terminal kinase (JNK). To investigate whether the Ca2+-dependent activation of mitogen-activated protein kinases (ERK, JNK, and p38) might be mediated by the CaM kinase cascade, we have transfected PC12 cells, which lack CaM-KIV, with constitutively active mutants of CaM kinase kinase and/or CaM-KIV (CaM-KKc and CaM-KIVc, respectively). In the absence of depolarization, CaM-KKc transfection had no effect on Elk-dependent transcription of a luciferase reporter gene, whereas CaM-KIVc alone or in combination with CaM-KKc gave 7- to 10-fold and 60- to 80-fold stimulations, respectively, which were blocked by mitogen-activated protein (MAP) kinase phosphatase cotransfection. When epitope-tagged constructs of MAP kinases were co-transfected with CaM-KKc plus CaM-KIVc, the immunoprecipitated MAP kinases were activated 2-fold (ERK-2) and 7- to 10-fold (JNK-1 and p38). The JNK and p38 pathways were further investigated using specific c-Jun or ATF2-dependent transcriptional assays. We found that c-Jun/ATF2-dependent transcriptions were enhanced 7- to 10-fold by CaM-KIVc and 20- to 30-fold by CaM-KKc plus CaM-KIVc. In the case of the Jun-dependent transcription, this effect was not due to direct phosphorylation of c-Jun by activated CaM-KIV, since transcription was blocked by a dominant-negative JNK and by two MAP kinase phosphatases. Mutation of the phosphorylation site (Thr196) in CaM-KIV, which mediates its activation by CaM-KIV kinase, prevented activation of Elk-1, c-Jun, and ATF2 by the CaM kinase cascade. These results establish a new Ca2+-dependent mechanism for regulating MAP kinase pathways and resultant transcription.

Mitogen-activated protein kinase kinase 7 is an activator of the c-Jun NH2-terminal kinase

The c-Jun NH2-terminal kinase (JNK) group of mitogen-activated protein (MAP) kinases is activated by phosphorylation on Thr and Tyr. Here we report the molecular cloning of a new member of the mammalian MAP kinase kinase group (MKK7) that functions as an activator of JNK. In vitro protein kinase assays demonstrate that MKK7 phosphorylates and activates JNK, but not the p38 or extracellular signal-regulated kinase groups of MAP kinase. Expression of MKK7 in cultured cells causes activation of the JNK signal transduction pathway. MKK7 is therefore established to be a novel component of the JNK signal transduction pathway.

Peroxide Sensors for the Fission Yeast Stress-activated Mitogen-activated Protein Kinase Pathway

The Schizosaccharomyces pombe stress-activated Sty1p/Spc1p mitogen-activated protein (MAP) kinase regulates gene expression through the Atf1p and Pap1p transcription factors, homologs of human ATF2 and c-Jun, respectively. Mcs4p, a response regulator protein, acts upstream of Sty1p by binding the Wak1p/Wis4p MAP kinase kinase kinase. We show that phosphorylation of Mcs4p on a conserved aspartic acid residue is required for activation of Sty1p only in response to peroxide stress. Mcs4p acts in a conserved phospho-relay system initiated by two PAS/PAC domain-containing histidine kinases, Mak2p and Mak3p. In the absence of Mak2p or Mak3p, Sty1p fails to phosphorylate the Atf1p transcription factor or induce Atf1p-dependent gene expression. As a consequence, cells lacking Mak2p and Mak3p are sensitive to peroxide attack in the absence of Prr1p, a distinct response regulator protein that functions in association with Pap1p. The Mak1p histidine kinase, which also contains PAS/PAC repeats, does not regulate Sty1p or Atf1p but is partially required for Pap1p- and Prr1p-dependent transcription. We conclude that the transcriptional response to free radical attack is initiated by at least two distinct phospho-relay pathways in fission yeast.

Stress signaling in plants: a mitogen-activated protein kinase pathway is activated by cold and drought.

Yeast and animals use mitogen-activated protein (MAP) kinase cascades to mediate stress and extracellular signals. We have tested whether MAP kinases are involved in mediating environmental stress responses in plants. Using specific peptide antibodies that were raised against different alfalfa MAP kinases, we found exclusive activation of p44MMK4 kinase in drought- and cold-treated plants. p44MMK4 kinase was transiently activated by these treatments and was correlated with a shift in the electrophoretic mobility of the p44MMK4 protein. Although transcript levels of the MMK4 gene accumulated after drought and cold treatment, no changes in p44MMK4 steady state protein levels were observed, indicating a posttranslational activation mechanism. Extreme temperatures, drought, and salt stress are considered to be different forms of osmotic stress. However, high salt concentrations or heat shock did not induce activation of p44MMK4, indicating the existence of distinct mechanisms to mediate different stresses in alfalfa. Stress adaptation in plants is mediated by abscisic acid (ABA)-dependent and ABA-independent processes. Although ABA rapidly induced the transcription of an ABA-inducible marker gene, MMK4 transcript levels did not increase and p44MMK4 kinase was not activated. These data indicate that the MMK4 kinase pathway mediates drought and cold signaling independently of ABA.

A tyrosine kinase profile of prostate carcinoma.

Tyrosine kinases play central roles in the growth and differentiation of normal and tumor cells. In this study, we have analyzed the general tyrosine kinase expression profile of a prostate carcinoma (PCA) xenograft, CWR22. We describe here an improved reverse transcriptase-PCR approach that permits identification of nearly 40 different kinases in a single screening; several of these kinases are newly cloned kinases and some are novel. According to this, there are 11 receptor kinases, 9 nonreceptor kinases, and at least 7 dual kinases expressed in the xenograft tissue. The receptor kinases include erbB2, erbB3, Ret, platelet-derived growth factor receptor, sky, nyk, eph, htk, sek (eph), ddr, and tkt. The nonreceptor kinases are lck, yes, abl, arg, JakI, tyk2, and etk/bmx. Most of the dual kinases are in the mitogen-activating protein (MAP) kinase-kinase (MKK) family, which includes MKK3, MKK4, MEK5, and a novel one. As a complementary approach, we also analyzed by specific reverse transcriptase-PCR primers the expression profile of erbB/epidermal growth factor receptor family receptors in a variety of PCA specimens, cell lines, and benign prostatic hyperplasia. We found that erbB1, -2, and -3 are often coexpressed in prostate tissues, but not in erbB4. The information established here should provide a base line to study the possible growth and oncogenic signals of PCA.

A gene encoding a mitogen-activated protein kinase kinase kinase is induced simultaneously with genes for a mitogen-activated protein kinase and an S6 ribosomal protein kinase by touch, cold, and water stress in Arabidopsis thaliana.

We describe here the cloning and characterization of a cDNA encoding a protein kinase that has high sequence homology to members of the mitogen-activated protein kinase (MAPK) kinase kinase (MAPKKK or MEKK) family; this cDNA is named cATMEKKI (Arabidopsis thaliana MAP kinase or ERK kinase kinase 1). The catalytic domain of the putative ATMEKK1 protein shows approximately 40% identity with the amino acid sequences of the catalytic domains of MAPKKKs (such as Byr2 from Schizosaccharomyces pombe, Ste11 from Saccharomyces cerevisiae, Bck1 from S. cerevisiae, MEKK from mouse, and NPK1 from tobacco). In yeast cells that overexpress ATMEKK1, the protein kinase replaces Ste11 in responding to mating pheromone. In this study, the expression of three protein kinases was examined by Northern blot analyses: ATMEKK1 (structurally related to MAPKKK), ATMPK3 (structurally related to MAPK), and ATPK19 (structurally related to ribosomal S6 kinase). The mRNA levels of these three protein kinases increased markedly and simultaneously in response to touch, cold, and salinity stress. These results suggest that MAP kinase cascades, which are thought to respond to a variety of extracellular signals, are regulated not only at the posttranslational level but also at the transcriptional level in plants and that MAP kinase cascades in plants may function in transducing signals in the presence of environmental stress.

MEKK1 phosphorylates MEK1 and MEK2 but does not cause activation of mitogen-activated protein kinase.

A constitutively active fragment of rat MEK kinase 1 (MEKK1) consisting of only its catalytic domain (MEKK-C) expressed in bacteria quantitatively activates recombinant mitogen-activated protein (MAP) kinase/extracellular signal-regulated protein kinase (ERK) kinases 1 and 2 (MEK1 and MEK2) in vitro. Activation of MEK1 by MEKK-C is accompanied by phosphorylation of S218 and S222, which are also phosphorylated by the protein kinases c-Mos and Raf-1. MEKK1 has been implicated in regulation of a parallel but distinct cascade that leads to phosphorylation of N-terminal sites on c-Jun; thus, its role in the MAP kinase pathway has been questioned. However, in addition to its capacity to phosphorylate MEK1 in vitro, MEKK-C interacts with MEK1 in the two-hybrid system, and expression of mouse MEKK1 or MEKK-C in mammalian cells causes constitutive activation of both MEK1 and MEK2. Neither cotransfected nor endogenous ERK2 is highly activated by MEKK1 compared to its stimulation by epidermal growth factor in spite of significant activation of endogenous MEK. Thus, other as yet undefined mechanisms may be involved in determining information flow through the MAP kinase and related pathways.