Characterization of mitogen-activated protein kinase (MAPK) dimers

Phosphorylated ERK2 has an increased capacity to form homodimers relative to unphosphorylated ERK2. We have characterized the nature of the ERK2 dimer and have mutated residues in the crystal dimer interface to examine the impact of dimerization on ERK2 activity. Analysis of the mutants by gel filtration indicates that at least five residues must be mutated simultaneously to produce an ERK2 mutant that is predominantly monomeric. Mutants, whether monomers or dimers, have specific protein kinase activities under fixed assay conditions that are roughly equivalent to wild-type ERK2. The ratio of dimers to monomers is increased as the salt concentration increases, consistent with a strong hydrophobic contribution to the energy of dimer formation. ERK2 dimerization also requires divalent cations. Sedimentation analysis indicates that the related c-Jun N-terminal kinase SAPKalphaI/JNK2 also forms dimers, but dimerization displays no dependence on phosphorylation; the unphosphorylated and phosphorylated forms of the kinase behave similarly, with low micromolar dimer dissociation constants.     

Genetic variation and the mitogen-activated protein kinase (MAPK) signaling pathway

Non-synonymous single nucleotide polymorphisms (nsSNPs) are known to alter protein function, contributing to disease susceptibility. This report explores the nature of nsSNPs in the gene products of the highly conserved mitogen-activated protein kinase (MAPK) signaling pathways already implicated in cancer development. MAPK signaling pathways regulate cellular processes such as proliferation, differentiation, apoptosis, and survival mediated through interconnected signaling cascades. Using the dbSNP database, we have identified 25 nsSNPs in 17 out of 98 MAPK genes studied. Computational algorithms were used to predict whether the amino acid substitutions were evolutionarily tolerated, or affected putative functional units such as phosphorylation sites, protein motifs and domains. This study predicts that 36% of nsSNPs are likely to have functional consequences, based on evolutionary conservation analysis, and 36% based on phosphorylation prediction analysis. All such nsSNPs represent potentially functional and disease-causing/modifying alleles. More interestingly, the epistatic relationships discussed in this report represent potential synergistic/ antagonistic/additive effects of nsSNP combinations found within the same protein, or within members of the same protein complex and cascades. This strategy can effectively determine which nsSNPs potentially alter protein function, and can be utilized to study the genetic architecture and disease association of other biological protein complexes and networks.     

Angiotensin II suppresses growth arrest specific homeobox (Gax) expression via redox-sensitive mitogen-activated protein kinase (MAPK)

Oxidative stress is known to be involved in growth control of vascular smooth muscle cells (VSMCs). We and others have demonstrated that angiotensin II (Ang II) has an important role in vascular remodeling. Several reports suggested that VSMC growth induced by Ang II was elicited by oxidative stress. Gax, growth arrest-specific homeobox is a homeobox gene expressed in the cardiovascular system. Over expression of Gax is demonstrated to inhibit VSMC growth. We previously reported that Ang II down-regulated Gax expression. To address the regulatory mechanism of Gax, we investigated the significance of oxidative stress in Ang II-induced suppression of Gax expression. We further examined the involvement of mitogen-activated protein kinases (MAPKs), which is crucial for cell growth and has shown to be activated by oxidative stress, on the regulation of Gax expression by Ang II. Ang II markedly augmented intracellular H2O2 production which was decreased by pretreatment with N-acetylcystein (NAC), an anti-oxidant. Ang II and H2O2 decreased Gax expression dose-dependently and these effects were blocked by administration of both NAC and pyrrolidine dithiocarbamate (PDTC), another anti-oxidant. Ang II and H2O2 induced marked activation of extracellular signal-responsive kinase1/2 (ERK1/2), which was blocked by NAC. Ang II and H2O2 also activated p38MAPK, and they were blocked by pre-treatment with NAC. However, the level of activated p38MAPK was quite low in comparison with ERK1/2. Ang II- or H2O2 -induced Gax down-regulation was significantly inhibited by PD98059, an ERK1/2 inhibitor but not SB203580, a p38MAPK inhibitor. The present results demonstrated the significance of regulation of Gax expression by redox-sensitive ERK1/2 activation.     

The mitogen-activated protein kinase activator.

The mitogen-activated protein kinase appears to be regulated by another growth factor regulated kinase, the mitogen-activated protein kinase activator. In the past year, much progress has been made in purifying and characterizing the mitogen-activated protein kinase activator, in determining its primary structure, and in identifying another protein kinase that may function upstream to regulate its activity.     

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.     

Involvement of the mitogen-activated protein kinase SIMK in regulation of root hair tip growth

Mitogen-activated protein kinases (MAPKs) are involved in stress signaling to the actin cytoskeleton in yeast and animals. We have analyzed the function of the stress-activated alfalfa MAP kinase SIMK in root hairs. In epidermal cells, SIMK is predominantly nuclear. During root hair formation, SIMK was activated and redistributed from the nucleus into growing tips of root hairs possessing dense F-actin meshworks. Actin depolymerization by latrunculin B resulted in SIMK relocation to the nucleus. Conversely, upon actin stabilization with jasplakinolide, SIMK co-localized with thick actin cables in the cytoplasm. Importantly, latrunculin B and jasplakinolide were both found to activate SIMK in a root-derived cell culture. Loss of tip-focused SIMK and actin was induced by the MAPK kinase inhibitor UO 126 and resulted in aberrant root hairs. UO 126 inhibited targeted vesicle trafficking and polarized growth of root hairs. In contrast, overexpression of gain-of-function SIMK induced rapid tip growth of root hairs and could bypass growth inhibition by UO 126. These data indicate that SIMK plays a crucial role in root hair tip growth.     

Peroxynitrite activates mitogen-activated protein kinase (MAPK) via a MEK-independent pathway: a role for protein kinase C.

In this study we show that phosphorylation of extracellular signal-regulated kinase (ERK1/2; also known as p44/42MAPK) following peroxynitrite (ONOO(-)) exposure occurs via a MAPK kinase (MEK)-independent but PKC-dependent pathway in rat-1 fibroblasts. ONOO(-)-mediated ERK1/2 phosphorylation was not blocked by MEK inhibitors PD98059 and U0126. Furthermore, no increase in MEK phosphorylation was detected upon ONOO(-) treatment. Staurosporine was used to investigate whether protein kinase C (PKC) is involved. This was confirmed by down-regulation of PKC by phorbol-12,13-dibutyrate, which resulted in significant reduction of ERK1/2 phosphorylation by ONOO(-), implying that activation of ERK by ONOO(-) depends on activation of PKC. Indeed, PKCalpha and epsilon were activated upon ONOO(-) exposure. When cells were treated with ONOO(-) in a calcium-free buffer, no activation of PKCalpha was detected. Concomitantly, a reduction of ERK1/2 phosphorylation was observed suggesting that calcium was required for translocation of PKCalpha and ERK phosphorylation by ONOO(-). Indeed, ONOO(-) exposure resulted in increased cytosolic calcium, which depended on the presence of extracellular calcium. Finally, data using G?6976, an inhibitor of calcium-dependent PKC activation, implied that ONOO(-)-mediated ERK1/2 phosphorylation depends on activation of a calcium-dependent PKC.     

Evolutionary history of mitogen-activated protein kinase (MAPK) genes in Lotus,Medicago, and Phaseolus

Mitogen-Activated Protein Kinase (MAPK) genes encode proteins that mediate various signaling pathways associated with biotic and abiotic stress responses in eukaryotes. The MAPK genes form a 3-tier signal transduction cascade between cellular stimuli and physiological responses. Recent identification of soybean MAPKs and availability of genome sequences from other legume species allowed us to identify their MAPK genes. The main objectives of this study were to identify MAPKs in 3 legume species, Lotus japonicus, Medicago truncatula, and Phaseolus vulgaris, and to assess their phylogenetic relationships. We used approaches in comparative genomics for MAPK gene identification and named the newly identified genes following Arabidopsis MAPK nomenclature model. We identified 19, 18, and 15 MAPKs and 7, 4, and 9 MAPKKs in the genome of Lotus japonicus, Medicago truncatula, and Phaseolus vulgaris, respectively. Within clade placement of MAPKs and MAPKKs in the 3 legume species were consistent with those in soybean and Arabidopsis. Among 5 clades of MAPKs, 4 founder clades were consistent to MAPKs of other plant species and orthologs of MAPK genes in the fifth clade-"Clade E" were consistent with those in soybean. Our results also indicated that some gene duplication events might have occurred prior to eudicot-monocot divergence. Highly diversified MAPKs in soybean relative to those in 3 other legume species are attributable to the polyploidization events in soybean. The identification of the MAPK genes in the legume species is important for the legume crop improvement; and evolutionary relationships and functional divergence of these gene members provide insights into plant genome evolution.     

A specific activation of the mitogen-activated protein kinase kinase 1 (MEK1) is required for Golgi fragmentation during mitosis

Incubation of permeabilized cells with mitotic extracts results in extensive fragmentation of the pericentriolarly organized stacks of cisternae. The fragmented Golgi membranes are subsequently dispersed from the pericentriolar region. We have shown previously that this process requires the cytosolic protein mitogen-activated protein kinase kinase 1 (MEK1). Extracellular signal-regulated kinase (ERK) 1 and ERK2, the known downstream targets of MEK1, are not required for this fragmentation (Acharya et al. 1998). We now provide evidence that MEK1 is specifically phosphorylated during mitosis. The mitotically phosphorylated MEK1, upon partial proteolysis with trypsin, generates a different peptide population compared with interphase MEK1. MEK1 cleaved with the lethal factor of the anthrax toxin can still be activated by its upstream mitotic kinases, and this form is fully active in the Golgi fragmentation process. We believe that the mitotic phosphorylation induces a change in the conformation of MEK1 and that this form of MEK1 recognizes Golgi membranes as a target compartment. Immunoelectron microscopy analysis reveals that treatment of permeabilized normal rat kidney (NRK) cells with mitotic extracts, treated with or without lethal factor, converts stacks of pericentriolar Golgi membranes into smaller fragments composed predominantly of tubuloreticular elements. These fragments are similar in distribution, morphology, and size to the fragments observed in the prometaphase/metaphase stage of the cell cycle in vivo.     

Salt stress induces changes in amounts and localization of the mitogen-activated protein kinase SIMK in alfalfa roots

Summary SIMK is an alfalfa mitogen-activated protein kinase (MAPK) that is activated by salt stress and shows a nuclear localization in suspension-cultured cells. We investigated the localization of SIMK in alfalfa (Medicago sati a) roots. Although SIMK was expressed in most tissues of the root apex, cells of the quiescent center and statocytes showed much lower SIMK protein amounts. In cells of the elongation zone, SIMK was present in much higher amounts in epidermal than in cortex cells. In dividing cells of the root tip, SIMK revealed a cell cycle phase-dependent localization, being predominantly nuclear in interphase but associating with the cell plate and the newly formed cell wall in telophase and early G₁ phase. In dividing cells, salt stress resulted in an association of part of the SIMK with the preprophase band. Generally, salt stress resulted in much higher amounts of SIMK in dividing cells of the root apex and epidermal cells of the elongation zone. These data demonstrate that amounts and subcellular localization of SIMK in roots is highly regulated and sensitive to environmental stress.Dedicated to Professor Walter Gustav Url on the occasion of his 70th birthday     

3pK, a novel mitogen-activated protein (MAP) kinase-activated protein kinase, is targeted by three MAP kinase pathways.

Recently we have identified a mitogen-activated protein kinase (MAPK)-activated protein kinase, named 3pK (G. Sithanandam, F. Latif, U. Smola, R. A. Bernal, F.-M. Duh, H. Li, I. Kuzmin, V. Wixler, L. Geil, S. Shresta, P. A. Lloyd, S. Bader, Y. Sekido, K. D. Tartof, V. I. Kashuba, E. R. Zabarovsky, M. Dean, G. Klein, B. Zbar, M. I. Lerman, J. D. Minna, U. R. Rapp, and A. Allikmets, Mol. Cell. Biol. 16:868-876, 1996). In vitro characterization of the kinase revealed that 3pK is activated by ERK. It was further shown that 3pK is phosphorylated in vivo after stimulation of cells with serum. However, the in vivo relevance of this observation in terms of involvement of the Raf/MEK/ERK cascade has not been established. Here we show that 3pK is activated in vivo by the growth inducers serum and tetradecanoyl phorbol acetate in promyelocytic HL60 cells and transiently transfected embryonic kidney 293 cells. Activation of 3pK was Raf dependent and was mediated by the Raf/MEK/ERK kinase cascade. 3pK was also shown to be activated after stress stimulation of cells. In vitro studies with recombinant proteins demonstrate that in addition to ERK, members of other subgroups of the MAPK family, namely, p38RK and Jun-N-terminal kinases/stress-activated protein kinases, were also able to phosphorylate and activate 3pK. Cotransfection experiments as well as the use of a specific inhibitor of p38RK showed that these in vitro upstream activators also function in vivo, identifying 3pK as the first kinase to be activated through all three MAPK cascades. Thus, 3pK is a novel convergence point of different MAPK pathways and could function as an integrative element of signaling in both mitogen and stress responses.     

Convergence and divergence of stress-induced mitogen-activated protein kinase signaling pathways at the level of two distinct mitogen-activated protein kinase kinases

Plants respond to biotic and abiotic stresses by inducing overlapping sets of mitogen-activated protein kinases (MAPKs) and response genes. To define the mechanisms of how different signals can activate a common signaling pathway, upstream activators of SIMK, a salt stress- and pathogen-induced alfalfa MAPK, were identified. Here, we compare the properties of SIMKK, a MAPK kinase (MAPKK) that mediates the activation of SIMK by salt stress, with those of PRKK, a distantly related novel MAPKK. Although both SIMKK and PRKK show strongest interaction with SIMK, SIMKK can activate SIMK without stimulation by upstream factors. In contrast, PRKK requires activation by an upstream activated MAPKK kinase. SIMKK mediates pathogen elicitor signaling and salt stress, but PRKK transmits only elicitor-induced MAPK activation. Of four tested MAPKs, PRKK activates three of them (SIMK, MMK3, and SAMK) upon elicitor treatment of cells. However, PRKK is unable to activate any MAPK upon salt stress. In contrast, SIMKK activates SIMK and MMK3 in response to elicitor, but it activates only SIMK upon salt stress. These data show that (1) MAPKKs function as convergence points for stress signals, (2) MAPKKs activate multiple MAPKs, and (3) signaling specificity is obtained not only through the inherent affinities of MAPKK-MAPK combinations but also through stress signal-dependent intracellular mechanisms.     

Dynamin is required for the activation of mitogen-activated protein (MAP) kinase by MAP kinase kinase

Internalization of activated receptors from the plasma membrane has been implicated in the activation of mitogen-activated protein (MAP) kinase. However, the mechanism whereby membrane trafficking may regulate mitogenic signaling remains unclear. Here we report that dominant-negative dynamin (K44A), an inhibitor of endocytic vesicle formation, abrogates MAP kinase activation in response to epidermal growth factor, lysophosphatidic acid, and protein kinase C-activating phorbol ester. In contrast, dynamin-K44A does not affect the activation of Ras, Raf, and MAP kinase kinase (MEK) by either agonist. Through immunofluorescence and subcellular fractionation studies, we find that activated MEK is present both at the plasma membrane and in intracellular vesicles but not in the cytosol. Our findings suggest that dynamin-regulated endocytosis of activated MEK, rather than activated receptors, is a critical event in the MAP kinase activation cascade.     

Inhibition of mitogen-activated protein kinase (MAPK) and cyclin-dependent kinase 2 (Cdk2) by platinum(II) phenanthroline complexes

Inhibition of protein kinases in the fight against disease remains a constant challenge for medicinal chemists, who have screened multitudes of predominantly planar organic scaffolds, natural and synthetic, to identify potent—albeit not always selective—kinase inhibitors. Herein, in an effort to investigate the potential biological utility of metal-based compounds as inhibitors against the cancer-relevant targets mitogen-activated protein kinase and cyclin-dependent kinase 2, we explore various parameters in planar platinum(II) complexes with substituted phenanthroline ligands and aliphatic diamine chelate co-ligands, to identify combinations that yield promising inhibitory activity. The individual ligands’ steric requirements as well as their pattern of hydrogen bond donors/acceptors appear to alter inhibitory potency when modulated.     

Dexamethasone causes sustained expression of mitogen-activated protein kinase (MAPK) phosphatase 1 and phosphatase-mediated inhibition of MAPK p38

The stress-activated protein kinase p38 stabilizes a number of mRNAs encoding inflammatory mediators, such as cyclooxygenase 2 (Cox-2). In HeLa cells the anti-inflammatory glucocorticoid dexamethasone destabilizes Cox-2 mRNA by inhibiting p38 function. Here we demonstrate that this effect is phosphatase dependent. Furthermore, in HeLa cells dexamethasone induced the sustained expression of mitogen-activated protein kinase phosphatase 1 (MKP-1), a potent inhibitor of p38 function. The inhibition of p38 and the induction of MKP-1 by dexamethasone occurred with similar dose dependence and kinetics. No other known p38 phosphatases were induced by dexamethasone, and other cell types which failed to express MKP-1 also failed to inhibit p38 in response to dexamethasone. The proinflammatory cytokine interleukin 1 (IL-1) induced MKP-1 expression in a p38-dependent manner and acted synergistically with dexamethasone to induce MKP-1 expression. In HeLa cells treated with IL-1 or IL-1 and dexamethasone, the dynamics of p38 activation mirrored the expression of MKP-1. These observations suggest that MKP-1 participates in a negative-feedback loop which regulates p38 function and that dexamethasone may inhibit proinflammatory gene expression in part by inducing MKP-1 expression.     

Uniaxial cyclic stretch induces focal adhesion kinase (FAK) tyrosine phosphorylation followed by mitogen-activated protein kinase (MAPK) activation

We investigated the role of tyrosine phosphorylation of FAK in the stretch-induced MAPKs (extracellular signal-regulated kinase (ERK), p38MAPK) activation in mutant FAK-transfected fibroblasts. In response to uniaxial cyclic stretch (1 Hz, 120% in length), the levels of tyrosine phosphorylation of the Tyr-397 and Tyr-925 of FAK in control cells increased and peaked at 5 min (2.75 +/- 0.51, n = 3), and 20 min (2.98 +/- 0.58, n = 3), respectively, and the activities of MAPKs increased and peaked at approximately 10 min. On the other hand, in the mutant FAK-transfected cells, the stretch-induced MAPKs activation was significantly inhibited. The stretch-induced activation of MAPKs was also significantly abolished by either treatment with Gd(3+) or extracellular Ca(2+) removal which may inhibit intracellular Ca(2+) increase caused by the activation of cation selective (Ca(2+)-permeable) stretch activated (SACatC) channels. These results suggest that the stretch-induced tyrosine-phosphorylation of FAK via SACatC activation is critical for the stretch-induced MAPKs activation.