Targeted deletion of the mitogen-activated protein kinase kinase 4 gene in the nervous system causes severe brain developmental defects and premature death

The c-Jun NH₂-terminal protein kinase (JNK) is a mitogen-activated protein kinase (MAPK) involved in the regulation of various physiological processes. Its activity is increased upon phosphorylation by the MAPK kinases MKK4 and MKK7. The early embryonic death of mice lacking an mkk4 or mkk7 gene has provided genetic evidence that MKK4 and MKK7 have nonredundant functions in vivo. To elucidate the physiological role of MKK4, we generated a novel mouse model in which the mkk4 gene could be specifically deleted in the brain. At birth, the mutant mice were indistinguishable from their control littermates, but they stopped growing a few days later and died prematurely, displaying severe neurological defects. Decreased JNK activity in the absence of MKK4 correlated with impaired phosphorylation of a subset of physiologically relevant JNK substrates and with altered gene expression. These defects resulted in the misalignment of the Purkinje cells in the cerebellum and delayed radial migration in the cerebral cortex. Together, our data demonstrate for the first time that MKK4 is an essential activator of JNK required for the normal development of the brain.     

Differential regulation of HSP70 expression by the JNK kinases SEK1 and MKK7 in mouse embryonic stem cells treated with cadmium

JNK, a member of the mitogen-activated protein kinases (MAPKs), is activated by the MAPK kinases SEK1 and MKK7 in response to environmental stresses. In the present study, the effects of CdCl2 treatment on MAPK phosphorylation and HSP70 expression were examined in mouse embryonic stem (ES) cells lacking the sek1 gene, the mkk7 gene, or both. Following CdCl2 exposure, the phosphorylation of JNK, p38, and ERK was suppressed in sek1-/- mkk7-/- cells. When sek1-/- or mkk7-/- cells were treated with CdCl2, JNK phosphorylation, but not the phosphorylation of either p38 or ERK, was markedly reduced, while a weak reduction in p38 phosphorylation was observed in sek1-/- cells. Thus, both SEK1 and MKK7 are required for JNK phosphorylation, whereas their role in p38 and ERK phosphorylation could overlap with that of another kinase. We also observed that CdCl2-induced HSP70 expression was abolished in sek1-/- mkk7-/- cells, was reduced in sek1-/- cells, and was enhanced in mkk7-/- cells. Similarly, the phosphorylation of heat shock factor 1 (HSF1) was decreased in sek1-/- mkk7-/- and sek1-/- cells, but was increased in mkk7-/- cells. Transfection with siRNA specific for JNK1, JNK2, p38, ERK1, or ERK2 suppressed CdCl2-induced HSP70 expression. In contrast, silencing of p38 or p38 resulted in further accumulation of HSP70 protein. These results suggest that HSP70 expression is up-regulated by SEK1 and down-regulated by MKK7 through distinct MAPK isoforms in mouse ES cells treated with CdCl2.     

MEK kinase 3 directly activates MKK6 and MKK7, specific activators of the p38 and c-Jun NH2-terminal kinases.

Mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase kinase kinase 3 (MEKK3) activates the c-Jun NH2-terminal kinase (JNK) pathway, although no substrates for MEKK3 have been identified. We have examined the regulation by MEKK3 of MAPK kinase 7 (MKK7) and MKK6, two novel MAPK kinases specific for JNK and p38, respectively. Coexpression of MKK7 with MEKK3 in COS-7 cells enhanced MKK7 autophosphorylation and its ability to activate recombinant JNK1 in vitro. MKK6 autophosphorylation and in vitro activation of p38alpha were also observed following coexpression of MKK6 with MEKK3. MEKK2, a closely related homologue of MEKK3, also activated MKK7 and MKK6 in COS-7 cells. Importantly, immunoprecipitates of either MEKK3 or MEKK2 directly activated recombinant MKK7 and MKK6 in vitro. These data identify MEKK3 as a MAPK kinase kinase specific for MKK7 and MKK6 in the JNK and p38 pathways. We have also examined whether MEKK3 or MEKK2 activates p38 in intact cells using MAPK-activated protein kinase-2 (MAPKAPK2) as an affinity ligand and substrate. Anisomycin, sorbitol, or the expression of MEKK3 in HEK293 cells enhanced MAPKAPK2 phosphorylation, whereas MEKK2 was less effective. Furthermore, MAPKAPK2 phosphorylation induced by MEKK3 or cellular stress was abolished by the p38 inhibitor SB-203580, suggesting that MEKK3 is coupled to p38 activation in intact cells.     

EDR1 Physically Interacts with MKK4/MKK5 and Negatively Regulates a MAP Kinase Cascade to Modulate Plant Innate Immunity

Mitogen-activated protein (MAP) kinase signaling cascades play important roles in the regulation of plant defense. The Raf-like MAP kinase kinase kinase (MAPKKK) EDR1 negatively regulates plant defense responses and cell death. However, how EDR1 functions, and whether it affects the regulation of MAPK cascades, are not well understood. Here, we showed that EDR1 negatively regulates the MKK4/MKK5-MPK3/MPK6 kinase cascade in Arabidopsis. We found that edr1 mutants have highly activated MPK3/MPK6 kinase activity and higher levels of MPK3/MPK6 proteins than wild type. EDR1 physically interacts with MKK4 and MKK5, and this interaction requires the N-terminal domain of EDR1. EDR1 also negatively affects MKK4/MKK5 protein levels. In addition, the mpk3, mkk4 and mkk5 mutations suppress edr1-mediated resistance, and over-expression of MKK4 or MKK5 causes edr1-like resistance and mildew-induced cell death. Taken together, our data indicate that EDR1 physically associates with MKK4/MKK5 and negatively regulates the MAPK cascade to fine-tune plant innate immunity.     

MEKK1, MKK1/MKK2 and MPK4 function together in a mitogen-activated protein kinase cascade to regulate innate immunity in plants

Mitogen-activated protein kinase (MAPK) cascades play important roles in regulating plant innate immune responses. In a genetic screen to search for mutants with constitutive defense responses, we identified multiple alleles of mpk4 and mekk1 that exhibit cell death and constitutive defense responses. Bimolecular fluorescence complementation (BiFC) analysis showed that both MPK4 and MEKK1 interact with MKK1 and MKK2, two closely related MAPK kinases. mkk1 and mkk2 single mutant plants do not have obvious mutant phenotypes. To test whether MKK1 and MKK2 function redundantly, mkk1 mkk2 double mutants were generated. The mkk1 mkk2 double mutant plants die at seedling stage and the seedling-lethality phenotype is temperature-dependent. Similar to the mpk4 and mekk1 mutants, the mkk1 mkk2 double mutant seedlings accumulate high levels of H2O2, display spontaneous cell death, constitutively express Pathogenesis Related (PR) genes and exhibit pathogen resistance. In addition, activation of MPK4 by flg22 is impaired in the mkk1 mkk2 double mutants, suggesting that MKK1 and MKK2 function together with MPK4 and MEKK1 in a MAP kinase cascade to negatively regulate innate immune responses in plants.     

The bottleneck of JNK signaling: molecular and functional characteristics of MKK4 and MKK7

The functions of mitogen-activated protein kinases (MKKs) 4 and 7 are typically associated with the c-Jun N-terminal kinase (JNK) signaling pathway. Both MKKs synergistically phosphorylate different JNK isoforms and are therefore involved in numerous physiological (e.g. differentiation and proliferation) and pathological (e.g. apoptosis and tumorigenesis) processes. MKK4 and MKK7 share similar molecular characteristics as well as several upstream activators and scaffold proteins. However, their functions are non-redundant and determined by different stimuli, biochemical interactions and differential tissue distribution. The central question is how two MKKs regulate or affect the multiple actions of their JNK substrates. Similar to JNKs, MKK4 and MKK7 can simultaneously exert divergent functions in different cellular compartments and signalosomes. It is also important to realize that the MKK effects are splice variant-specific. The present review not only summarizes the various modes of MKK4 and MKK7 activation and activity, but also their functions. We also extensively describe their impact on JNK signaling, their molecular interactions resulting in the formation of context-specific signalosomes and the functional consequences of JNK deficiency.     

Different properties of SEK1 and MKK7 in dual phosphorylation of stress-induced activated protein kinase SAPK/JNK in embryonic stem cells

Stress-activated protein kinase/c-Jun NH(2)-terminal kinase (SAPK/JNK), belonging to the mitogen-activated protein kinase family, plays an important role in stress signaling. SAPK/JNK activation requires the phosphorylation of both Thr and Tyr residues in its Thr-Pro-Tyr motif, and SEK1 and MKK7 have been identified as the dual specificity kinases. In this study, we generated mkk7(-/-) mouse embryonic stem (ES) cells in addition to sek1(-/-) cells and compared the two kinases in terms of the activation and phosphorylation of JNK. Although SAPK/JNK activation by various stress signals was markedly impaired in both sek1(-/-) and mkk7(-/-) ES cells, there were striking differences in the dual phosphorylation profile. The severe impairment observed in mkk7(-/-) cells was accompanied by a loss of the Thr phosphorylation of JNK without marked reduction in its Tyr-phosphorylated level. On the other hand, Thr phosphorylation of JNK in sek1(-/-) cells was also attenuated in addition to a decreased level of its Tyr phosphorylation. Analysis in human embryonic kidney 293T cells transfected with a kinase-dead SEK1 or a Thr-Pro-Phe mutant of JNK1 revealed that SEK1-induced Tyr phosphorylation of JNK1 was followed by additional Thr phosphorylation by MKK7. Furthermore, SEK1 but not MKK7 was capable of binding to JNK1 in 293T cells. These results indicate that the Tyr and Thr residues of SAPK/JNK are sequentially phosphorylated by SEK1 and MKK7, respectively, in the stress-stimulated ES cells.     

Exploring the function of the JNK (c-Jun N-terminal kinase) signalling pathway in physiological and pathological processes to design novel therapeutic strategies

JNK (c-Jun N-terminal kinase) is a member of the MAPK (mitogen-activated protein kinase) family that regulates a range of biological processes implicated in tumorigenesis and neurodegenerative disorders. For example, genetic studies have demonstrated that the removal of specific Jnk genes can reduce neuronal death associated with cerebral ischaemia. As such, targeting JNK signalling constitutes an obvious opportunity for therapeutic intervention. However, MAPK inhibitors can display toxic effects. Consequently, dual-specificity MKKs (MAPK kinases) may represent more attractive targets. In particular, evidence that blocking JNK activation by removing MKK4 offers an effective therapy to treat pathological conditions has started to emerge. MKK4 was the first JNK activator identified. The remaining level of JNK activity in cells lacking MKK4 expression led to the discovery of a second activator of JNK, named MKK7. Distinct phenotypic abnormalities associated with the targeted deletion of Mkk4 and Mkk7 in mice have revealed that MKK4 and MKK7 have non-redundant function in vivo. Further insights into the specific functions of the JNK activators in cancer cells and in neurons will be of critical importance to validate MKK4 and MKK7 as promising drug targets.     

The MKK7 Gene Encodes a Group of c-Jun NH2-Terminal Kinase Kinases

The c-Jun NH₂-terminal protein kinase (JNK) is a member of the mitogen-activated protein kinase (MAPK) group and is an essential component of a signaling cascade that is activated by exposure of cells to environmental stress. JNK activation is regulated by phosphorylation on both Thr and Tyr residues by a dual-specificity MAPK kinase (MAPKK). Two MAPKKs, MKK4 and MKK7, have been identified as JNK activators. Genetic studies demonstrate that MKK4 and MKK7 serve nonredundant functions as activators of JNK in vivo. We report here the molecular cloning of the gene that encodes MKK7 and demonstrate that six isoforms are created by alternative splicing to generate a group of protein kinases with three different NH₂ termini (α, β, and γ isoforms) and two different COOH termini (1 and 2 isoforms). The MKK7α isoforms lack an NH₂-terminal extension that is present in the other MKK7 isoforms. This NH₂-terminal extension binds directly to the MKK7 substrate JNK. Comparison of the activities of the MKK7 isoforms demonstrates that the MKK7α isoforms exhibit lower activity, but a higher level of inducible fold activation, than the corresponding MKK7β and MKK7γ isoforms. Immunofluorescence analysis demonstrates that these MKK7 isoforms are detected in both cytoplasmic and nuclear compartments of cultured cells. The presence of MKK7 in the nucleus was not, however, required for JNK activation in vivo. These data establish that the MKK4 and MKK7 genes encode a group of protein kinases with different biochemical properties that mediate activation of JNK in response to extracellular stimuli.     

Impaired synergistic activation of stress-activated protein kinase SAPK/JNK in mouse embryonic stem cells lacking SEK1/MKK4: different contribution of SEK2/MKK7 isoforms to the synergistic activation.

Stress-activated protein kinase/c-Jun NH(2)-terminal kinase (SAPK/JNK), which is a member of the mitogen-activated protein kinase (MAPK) family, plays an important role in a stress-induced signaling cascade. SAPK/JNK activation requires the phosphorylation of Thr and Tyr residues in its Thr-Pro-Tyr motif, and SEK1 (MKK4) and MKK7 (SEK2) have been identified as the upstream MAPK kinases. Here we examined the activation and phosphorylation sites of SAPK/JNK and differentiated the contribution of SEK1 and MKK7alpha1, -gamma1, and -gamma2 isoforms to the MAPK activation. In SEK1-deficient mouse embryonic stem cells, stress-induced SAPK/JNK activation was markedly impaired, and this defect was accompanied with a decreased level of the Tyr phosphorylation. Analysis in HeLa cells co-transfected with the two MAPK kinases revealed that the Thr and Tyr of SAPK/JNK were independently phosphorylated in response to heat shock by MKK7gamma1 and SEK1, respectively. However, MKK7alpha1 failed to phosphorylate the Thr of SAPK/JNK unless its Tyr residue was phosphorylated by SEK1. In contrast, MKK7gamma2 had the ability to phosphorylate both Thr and Tyr residues. In all cases, the dual phosphorylation of the Thr and Tyr residues was essentially required for the full activation of SAPK/JNK. These data provide the first evidence that synergistic activation of SAPK/JNK requires both phosphorylation at the Thr and Tyr residues in living cells and that the preference for the Thr and Tyr phosphorylation was different among the members of MAPK kinases.     

MKK3 Was Involved in Larval Settlement of the Barnacle Amphibalanus amphitrite through Activating the Kinase Activity of p38MAPK

The p38 mitogen-activated protein kinase (p38MAPK) plays a key role in larval settlement of the barnacle Amphibalanus amphitrite. To study the signaling pathway associated with p38MAPK during larval settlement, we sought to identify the upstream kinase of p38MAPK. Three MKKs (MKK3, MKK4 and MKK7) and three MAPKs (p38MAPK, ERK and JNK) in A. amphitrite were cloned and recombinantly expressed in E. coli. Through kinase assays, we found that MKK3, but not MKK4 or MKK7, phosphorylated p38MAPK. Furthermore, MKK3 activity was specific to p38MAPK, as it did not phosphorylate ERK or JNK. To further investigate the functional relationship between MKK3 and p38MAPK in vivo, we studied the localization of phospho-MKK3 (pMKK3) and MKK3 by immunostaining. Consistent with the patterns of p38MAPK and phospho-p38MAPK (pp38MAPK), pMKK3 and MKK3 mainly localized to the antennules of the cyprids. Western blot analysis revealed that pMKK3 levels, like pp38MAPK levels, were elevated at cyprid stage, compared to nauplii and juvenile stages. Moreover, pMKK3 levels increased after treatment with adult barnacle crude extracts, suggesting that MKK3 might mediate the stimulatory effects of adult barnacle extracts on the p38MAPK pathway.     

MKK1 and MKK2, which encode Saccharomyces cerevisiae mitogen-activated protein kinase-kinase homologs, function in the pathway mediated by protein kinase C.

The PKC1 gene of Saccharomyces cerevisiae encodes a homolog of mammalian protein kinase C that is required for normal growth and division of yeast cells. We report here the isolation of the yeast MKK1 and MKK2 (for mitogen-activated protein [MAP] kinase-kinase) genes which, when overexpressed, suppress the cell lysis defect of a temperature-sensitive pkc1 mutant. The MKK genes encode protein kinases most similar to the STE7 product of S. cerevisiae, the byr1 product of Schizosaccharomyces pombe, and vertebrate MAP kinase-kinases. Deletion of either MKK gene alone did not cause any apparent phenotypic defects, but deletion of both MKK1 and MKK2 resulted in a temperature-sensitive cell lysis defect that was suppressed by osmotic stabilizers. This phenotypic defect is similar to that associated with deletion of the BCK1 gene, which is thought to function in the pathway mediated by PCK1. The BCK1 gene also encodes a predicted protein kinase. Overexpression of MKK1 suppressed the growth defect caused by deletion of BCK1, whereas an activated allele of BCK1 (BCK1-20) did not suppress the defect of the mkk1 mkk2 double disruption. Furthermore, overexpression of MPK1, which encodes a protein kinase closely related to vertebrate MAP kinases, suppressed the defect of the mkk1 mkk2 double mutant. These results suggest that MKK1 and MKK2 function in a signal transduction pathway involving the protein kinases encoded by PKC1, BCK1, and MPK1. Genetic epistasis experiments indicated that the site of action for MKK1 and MKK2 is between BCK1 and MPK1.     

Activation of JNK3 alpha 1 requires both MKK4 and MKK7: kinetic characterization of in vitro phosphorylated JNK3 alpha 1

JNK3 alpha 1 is predominantly a neuronal specific MAP kinase that is believed to require, like all MAP kinases, both threonine and tyrosine phosphorylation for maximal enzyme activity. In this study we investigated the in vitro activation of JNK3 alpha 1 by MAP kinase kinase 4 (MKK4), MAP kinase kinase 7 (MKK7), and the combination of MKK4 + MKK7. Mass spectral analysis showed that MKK7 was capable of monophosphorylating JNK3 alpha 1 in vitro, whereas both MKK4 and MKK7 were required for bisphosphorylation and maximal enzyme activity. Measuring catalysis under Vmax conditions showed MKK4 + MKK7-activated JNK3 alpha 1 had Vmax 715-fold greater than nonactivated JNK3 alpha 1 and MKK7-activated JNK3 alpha 1 had Vmax 250-fold greater than nonactivated JNK3 alpha 1. In contrast, MKK4-activated JNK3 alpha 1 had no increase in Vmax compared to nonactivated levels and had no phosphorylation on the basis of mass spectrometry. These data suggest that MKK7 was largely responsible for JNK3 alpha 1 activation and that a single threonine phosphorylation may be all that is needed for JNK3 alpha 1 to be active. The steady-state rate constants kcat, Km(GST-ATF2++), and Km(ATP) for both monophosphorylated and bisphosphorylated JNK3 alpha 1 were within 2-fold between the two enzyme forms, suggesting the addition of tyrosine phosphorylation does not affect the binding of ATF2, ATP, or maximal turnover. Finally, the MAP kinase inhibitor, SB203580, had an IC50 value approximately 4-fold more potent on the monophosphorylated JNK3 alpha 1 compared to the bisphosphorylated JNK3 alpha 1, suggesting only a modest effect of tyrosine phosphorylation on inhibitor binding.     

The Arabidopsis MAP kinase kinase MKK1 participates in defence responses to the bacterial elicitor flagellin

Plants sense pathogens through both pathogen-associated molecular patterns and recognition of race-specific virulence factors, which induce basal defence or an accelerated defence (often manifest in the form of local cell death), respectively. A mitogen-activated protein kinase (MAPK) module in Arabidopsis was previously proposed to signal from perception of the bacterial elicitor flagellin to the activation of basal defence-related genes. Here, we present evidence for a parallel MAPK-signalling pathway involved in the response to flg22, a peptide corresponding to the most conserved domain of flagellin. The endogenous Arabidopsis MAP kinase kinase MKK1 is activated in cells treated with flg22, phosphorylates the MAPK MPK4 in vitro, and activates it in vivo in protoplasts. In mkk1 mutant plants, the activation by flg22 of MPK4 and two other flg22-induced MAPKs (MPK3 and MPK6) is impaired. In the mkk1 mutant, a battery of both flg22-induced and flg22-repressed genes show altered expression, indicating that MKK1 negatively regulates the activity of flagellin-responsive genes. Intriguingly, in contrast to the mpk4 mutant, mkk1 shows no morphological anomalies and is compromised in resistance to both virulent and avirulent Pseudomonas syringae strains. Thus, the MKK1 signalling pathway modulates the expression of genes responding to elicitors and plays an important role in pathogen defence.