Table of contents
MEK, the MAP kinase kinase activator of MAP kinase
Ascidians are invertebrate chordates with a larval body plan similar to that of vertebrates. The ascidian larval CNS is divided along the anteroposterior axis into sensory vesicle, neck, visceral ganglion and tail nerve cord. The anterior part of the sensory vesicle comes from the a-line animal blastomeres, whereas the remaining CNS is largely derived from the A-line vegetal blastomeres. The role of the Ras/MEK/ERK signalling pathway in the formation of the larval CNS has been analyzed in Ciona intestinalis. This pathway is required, during the cleavage stages, for the acquisition of (1) neural fates in otherwise epidermal cells (in a-line cells) and (2) the posterior identity of tail nerve cord precursors that otherwise adopt a more anterior neural character (in A-line cells). Altogether, the MEK signalling pathway appears to play evolutionary conserved roles in these processes in ascidians and vertebrates, suggesting that this may represent an ancestral chordate strategy (Hudson, 2003).
In vertebrates, dorsal mesoderm (organizer) has been implicated in the induction of neural tissue. Furthermore, it has been shown in ascidians that recombination of notochord blastomeres with a4.2 explants results in the induction of the a-line derived pigment cells. Therefore, one possibility, which could account for the observed loss of the a-line neural tissue upon MEK inhibition, is that this is a direct result of the loss of notochord in these embryos. However, there is a wealth of evidence that the notochord is not fully specified until the 64-cell stage. This is after the MEK-dependent activation of ERK in a-line neural precursors and the MEK-dependent expression of ascidian otx in these cells. Hence, it is possible that there is no requirement for a fully specified notochord for the onset of a-line neural induction. Consistently, in one experiment where embryos were placed in MEK inhibitor PD184352 at the late 32-cell stage, early neural marker Ci-ETR-1 was expressed in prospective notochord precursors (100%) and Ci-otx was expressed in a-line neural precursors (58%). However, this does not rule out that the notochord precursors secrete neural inducing signals (Hudson, 2003).
FGF constitutes a good candidate for the neural inducing signal. It can induce expression of Ci-otx in animal pole explants at the 44-cell stage. Furthermore, it has recently been shown in Ciona savigni that Cs-FGF4/6/9 is expressed in the vegetal cells of 16-32-cell stage embryos, thus at a time and in a place consistent with a role in a-line neural induction. There is an ongoing requirement for MEK signalling in order for a-line neural precursors to fully adopt neural fate, express Ci-ETR-1 and generate differentiated neurons and pigment cells, suggesting MEK may also be required for maintenance. The observation that FGF treatment of animal explants can induce neural and neuronal markers in the absence of the induction of non-neural tissues, suggests that a MEK-dependent signal may be acting directly to instruct these fates, though this requires further investigation. In summary, the results support the idea that the acquisition of neural fate and the differentiation of specific neural cell types in a-line neural cells may involve multiple MEK-dependent steps (Hudson, 2003).
Upon stimulation, MAPK is translocated to the nucleus, whereas MAPKK stays in the cytoplasm. It has been shown recently that the cytoplasmic localization of MAPKK is determined by its nuclear export signal (NES) in the near N-terminal region (residues 33-44). However, the mechanism determining the subcellular distribution of MAPK has been poorly understood. Introduction of v-Ras, active STE11 or constitutively active MAPKK can induce nuclear translocation of MAPK in mammalian cultured cells. MAPK is localized to the cytoplasm through its specific association with MAPKK; nuclear accumulation of MAPK is accompanied by dissociation of a complex between MAPK and MAPKK following activation of the MAPK pathway. The MAPK-binding site of MAPKK has been identified as its N-terminal residues 1-32. A peptide encompassing the MAPK-binding site and the NES sequence of MAPKK has been shown to be sufficient to retain MAPK in the cytoplasm. These findings reveal the molecular basis regulating subcellular distribution of MAPK, and identify a novel function of MAPKK as a cytoplasmic anchoring protein for MAPK (Fukuda, 1997).
To discern the MEK1 and MEK2 specificity for their substrate, extracellular signal-regulated kinase (ERK), site-directed mutagenesis was performed on the amino acid residues flanking the regulatory phosphorylation sites of ERK1. These ERK1 mutants were analyzed for the ability to act as a substrate for MEK1 and MEK2. Based on both phosphorylation and activation analyses, the mutants can be divided into four classes: 1) dramatically decreased phosphorylation and activation, 2) enhanced basal kinase activity, 3) preferentially enhanced phosphorylation of tyrosine and decreased phosphorylation of threonine, and 4) increased threonine phosphorylation with an increase in activation. In general, the residues proximal to the regulatory phosphorylation sites of ERK1 have greater influence on both phosphorylation and activation. This is consistent with the highly specific recognition of the ERK1 regulatory sites by MEK. Mutation of Arg-208 or Thr-207 to an alanine residue significantly alters the relative phosphorylation on Thr-202 and Tyr-204. The Arg-208 to alanine mutant increases the phosphorylation of Tyr-204 approximately 4-fold, yet almost completely eliminates the phosphorylation on Thr-202. In contrast, mutation of Gly-199 to alanine results in an increased phosphorylation of Thr-202 relative to Tyr-204. This suggests that both Gly-199 and Arg-208 play important roles in determining the relative phosphorylation of Thr-202 and Tyr-204. These results demonstrate that residues in the phosphorylation lip of ERK play an important role in the recognition and phosphorylation by MEK (Butch, 1996).
The enzyme activity of mitogen-activated protein kinase (MAP kinase) increases in response to agents acting on a variety of cell surface receptors, including receptors linked to heterotrimeric G proteins. In this report, it is demonstrated that Raf-1 protein kinase activity in the mouse parotid glands is induced by chronic isoproterenol administration in whole animals. To investigate the molecular nature underlying cellular responses to Raf-1 activation, rat salivary epithelial Pa-4 cells were stably transfected with human Raf-1-estrogen receptor fusion gene (DeltaRaf-1:ER) and mRNA differential display was used in search of messages induced by DeltaRaf-1:ER activation. Through this approach, the gene encoding non-histone chromosomal protein HMGI-C was identified as one of the target genes activated by oncogenic Raf-1 kinase. Activation of Raf-1 kinase results in a delayed and sustained increase of HMGI-C expression in the Pa-4 cells. The induction of HMGI-C mRNA level is sensitive to both the protein synthesis inhibitor cycloheximide and transcription inhibitor actinomycin D. The role of the extracellular signal-related kinase (ERK) signaling pathway in the HMGI-C induction was highlighted by the result that a MAP kinase kinase (MEK) inhibitor blocks DeltaRaf-1:ER- and phorbol ester-stimulated HMGI-C induction. Altogether, these findings support the notion that the Raf/MEK/ERK signaling module, at least in part, regulates transcriptional activation of the chromosomal architectural protein HMGI-C (Li, 1997).
MEK kinases (MEKKs) 1, 2, 3 and 4 are members of sequential kinase pathways that regulate MAP kinases including c-Jun NH2-terminal kinases (JNKs) and extracellular regulated kinases (ERKs). Confocal immunofluorescence microscopy of COS cells demonstrates differential MEKK subcellular localization: MEKK1 is nuclear and found in post-Golgi vesicular-like structures; MEKK2 and 4 are localized to distinct Golgi-associated vesicles that are dispersed by brefeldin A. MEKK1 and 2 are activated by EGF; kinase-inactive mutants of each MEKK partially inhibit EGF-stimulated JNK activity. Kinase-inactive MEKK1, but not MEKK2, 3 or 4, strongly inhibits EGF-stimulated ERK activity. In contrast to MEKK2 and 3, MEKK1 and 4 specifically associates with Rac (see Drosophila Rac) and Cdc42; kinase-inactive mutants block Rac/Cdc42 stimulation of JNK activity. Inhibitory mutants of MEKK1-4 do not affect p21-activated kinase (PAK) activation of JNK, indicating that the PAK-regulated JNK pathway is independent of MEKKs. Thus, in different cellular locations, specific MEKKs are required for the regulation of MAPK family members, and MEKK1 and 4 are involved in the regulation of JNK activation by Rac/Cdc42, independent of PAK. Differential MEKK subcellular distribution and interaction with small GTP-binding proteins provides a mechanism to regulate MAP kinase responses in localized regions of the cell and to different upstream stimuli (Fanger, 1997).
Mek is a dual-specificity kinase that activates the extracellular-signal-regulated (Erk) mitogen-activated protein (MAP) kinases upon agonist binding to receptors. The Erk MAP kinase cascade is involved in cell-fate determination in many organisms. In mammals, this pathway is proposed to regulate cell growth and differentiation. Genetic studies have shown that although a single mek gene is present in Caenorhabditis elegans, Drosophila and Xenopus, two mek homologs, Mek1 and Mek2, are present in the mammalian cascade. A mutant mouse line is described in which the mek1 gene has been disrupted by insertional mutagenesis. The null mutation is recessive lethal, since the homozygous mutant embryos die at 10.5 days of gestation. Histopathological analyses reveals a reduction in vascularization of the placenta that is due to a marked decrease of vascular endothelial cells in the labyrinthine region. The failure to establish a functional placenta probably explains the death of the mek1-/- embryos. Cell-migration assays indicate that mek1-/- fibroblasts can not be induced to migrate by fibronectin, although the levels of Mek2 protein and Erk activation are normal. Re-expression of Mek1 in the mutant mouse embryonic fibroblasts (MEFs) restores their ability to migrate. These findings provide genetic evidence that establishes the unique role played by Mek1 in signal transduction. They also suggest that mek1 function is required for normal response to angiogenic signals that might promote vascularization of the labyrinthine region of the placenta. It is argued that Mek1 and Mek2 play non-redundant roles in signal transduction (Giroux, 1999).
The c-jun proto-oncogene encodes a transcription factor that is activated by mitogens both transcriptionally and as a result of phosphorylation by Jun N-terminal kinase (JNK). The cellular signaling pathways involved in epidermal growth factor (EGF) induction of the c-jun promoter have been investigated. Two sequence elements that bind ATF1 (a leucine zipper DNA binding protein) and MEF2D transcription factors are required in HeLa cells, although these elements are not sufficient for maximal induction. Activated forms of Ras, RacI, Cdc42Hs, and MEKK increase expression of the c-jun promoter, while dominant negative forms of Ras, RacI, and MEK kinase (MEKK) inhibit EGF induction. These results suggest that EGF activates the c-jun promoter by a Ras-to-Rac-to-MEKK pathway. No change is found in protein binding to the jun ATF1 site in EGF-treated cells. A potential mechanism for regulation of ATF1 and CREB is phosphorylation (Clarke, 1997).
Mitogens promote cell growth through integrated signal transduction networks that alter cellular metabolism, gene expression and cytoskeletal organization. Many such signals are propagated through activation of MAP kinase cascades that are partly regulated by upstream small GTP-binding proteins. Interactions among cascades are suspected but not defined. Rho family small G proteins such as Rac1 and Cdc42hs, which activate the JNK/SAPK pathway, cooperate with Raf-1 to activate the ERK pathway. This causes activation of ternary complex factors (TCFs), which regulate c-fos (see Drosophila Fos related antigen) gene expression through the serum response element. Examination of ERK pathway kinases shows that neither MEK1 nor Ras will synergize with Rho-type proteins, and that only MEK1 is fully activated, indicating that MEKs are a focal point for cross-cascade regulation. Rho family proteins utilize PAKs for this effect (information about PAK is provided in the Myospheroid site), as expression of an active PAK1 mutant can substitute for Rho family small G proteins, and expression of an interfering PAK1 mutant blocks the Rho-type protein stimulation of ERKs. PAK1 phosphorylates MEK1 on Ser298, a site important for the binding of Raf-1 to MEK1 in vivo. Expression of interfering PAK1 also reduces stimulation of TCF function by serum growth factors, while expression of active PAK1 enhances EGF-stimulated MEK1 activity. This demonstrates interaction among MAP kinase pathway elements not previously recognized and suggests an explanation for the cooperative effect of Raf-1 and Rho family proteins on cellular transformation (Frost, 1997).
Growth factor stimulated receptor tyrosine kinases activate a protein kinase cascade via the serine/threonine protein kinase Raf-1. Direct upstream activators of Raf-1 are Ras and Src. This study shows that MEK1, the direct downstream effector of Raf-1, can also stimulate Raf-1 kinase activity by a positive feedback loop. Activated MEK1 mediates hyperphosphorylation of the amino terminal regulatory as well as of the carboxy terminal catalytic domain of Raf-1. The hyperphosphorylation of Raf-1 correlates with a change in the tryptic phosphopeptide pattern only at the carboxy terminus of Raf-1 and an increase in Raf-1 kinase activity. MEK1-mediated Raf-1 activation is inhibited by co-expression of the MAPK specific phosphatase MKP-1, indicating that the MEK1 effect is exerted through a MAPK dependent pathway. Stimulation of Raf-1 activity by MEK1 is independent of Ras, Src and tyrosine phosphorylation of Raf-1. However, MEK1 can synergize with Ras and leads to further increase of the Raf-1 kinase activity. Thus, MEK1 can mediate activation of Raf-1 by a novel positive feedback mechanism that allows fast signal amplification and could prolong activation of Raf-1 (Zimmermann, 1997).
Genetic screens in Drosophila melanogaster and Caenorhabditis elegans have identified the Kinase suppressor of ras, Ksr, as a new component in the Ras intracellular signaling pathway. In these organisms, mutations in Ksr results in attenuation of Ras-mediated signaling. Homologs of Ksr have also been isolated from mice and humans; their precise role in Ras signaling is not yet well defined. Interactions between the murine form of Ksr (mKsr-1) and other components of the Ras pathway have now been studied. To gain insight into the biological function of Ksr, a yeast two-hybrid screen was used and an interaction between the carboxy-terminal region of mKsr-1 and mitogen-activated protein (MAP) kinase kinase 1 (MAPKK-1 or MEK-1) was found. An interaction is also detected between MAP kinase (also called extracellular signal-regulated kinase, or ERK), and the amino-terminal region of mKsr-1. These interactions are recapitulated in COS-7 cells. When COS-7 cells are transfected with either full-length mKsr-1 or just its carboxy-terminal region, an inhibition of serum-stimulated MAP kinase activation is observed. Microinjection of full-length mKsr-1 or its carboxy-terminal, but not its amino-terminal region, blocks serum-induced DNA synthesis in rat embryo fibroblasts. Co-injection of mKsr-1 with MEK-1 reverses the blockage. Together with the data from genetic analyses, these findings lead to the proposal that mKsr-1 may control MAP kinase signaling by serving as a scaffold protein that links MEK and its substrate ERK (Yu, 1998).
Ksr (kinase supressor of Ras) was identified as a regulator of the Ras-MAP kinase (mitogen-activated protein kinase) pathway using genetic screens in Drosophila and Caenorhabditis elegans. Ksr is a kinase with similarities to the three conserved regions of Raf kinases, especially within the kinase domain. To investigate whether these structural similarities correlate with common functional properties, the ability of mKsr-1 (the murine homolog of Ksr) to interact with components of the vertebrate MAP kinase pathway was examined. In the yeast two-hybrid interaction assay, mKsr-1 does not bind to either Ras, B-Raf or Raf-1, but interacts strongly with both MEK-1 and MEK-2, activators of MAP kinase. The Ksr-MEK interaction was confirmed by co-immunoprecipitation experiments. Ectopically expressed mKsr-1 co-precipitates with endogenous MEK-1 in COS-1 cells, and endogenous Ksr and MEK co-precipitate from PC12 cells. Phosphorylation of MEK by mKsr-1 was not detected, however. In contrast, neither the MEK subpopulation complexed with mKsr-1 in COS-1 cells nor the PC12 cells display kinase activity. This ability of Ksr to block MEK in an inactive form correlates with a biological response: mKsr-1 does not transform NIH3T3 cells, and, furthermore, mKsr-1 reduces Ras-induced transformation. Similarly, mKsr-1 inhibits the proliferation of embryonic neuroretina cells induced by Ras and B-Raf but not that induced by MEK. These results suggest a novel mechanism for Ksr in regulating the MAP kinase pathway, at least in part through an ability to interact with MEK (Denouel-Galy, 1998).
Kinase suppressor of Ras (KSR) is a loss-of-function allele that suppresses the rough eye phenotype of activated Ras in Drosophila and the multivulval phenotype of activated Ras in Caenorhabditis elegans. Genetic and biochemical studies suggest that KSR is a positive regulator of Ras signaling that functions between Ras and Raf or in a pathway parallel to Raf. The effect of mammalian KSR expression was examined on the activation of extracellular ligand-regulated (ERK) mitogen-activated protein (MAP) kinase in fibroblasts. Ectopic expression of KSR inhibits the activation of ERK MAP kinase by insulin, phorbol ester, or activated alleles of Ras, Raf, and mitogen and extracellular-regulated kinase. Expression of deletion mutants of KSR demonstrates that the KSR kinase domain is necessary and sufficient for the inhibitory effect of KSR on ERK MAP kinase activity. KSR inhibits cell transformation by activated RasVal-12 but has no effect on the ability of RasVal-12 to induce membrane ruffling. These data indicate that KSR is a potent modulator of a signaling pathway essential to normal and oncogenic cell growth and development (Joneson, 1998).
Signal transduction is controlled both by regulation of enzyme activation and by organization of enzymatic complexes with nonenzymatic adapters, scaffolds, and anchor proteins. The extracellular signal-regulated kinase (ERK) cascade is one of several evolutionarily conserved mitogen-activated protein (MAP) kinase cascades important in the regulation of growth, apoptosis, and differentiation. A two-hybrid screen was conducted to identify nonenzymatic components of this signaling cascade that might be important in regulating its activity. A protein called MP1 (MEK Partner 1) has been identified that binds specifically to MEK1 and ERK1 and facilitates their activation. When overexpressed in cultured cells, MP1 enhances activation of ERK1 and activation of a reporter driven by the transcription factor Elk-1. Expression of MP1 in cells increases binding of ERK1 to MEK1. MP1 apparently functions as an adapter to enhance the efficiency of the MAP kinase cascade (Schaeffer, 1998).
A constitutively active form of mitogen-activated protein kinase kinase (MEK1) was synthesized under control of a zinc-inducible promoter in NIH 3T3 fibroblasts. Zinc treatment of serum-starved cells activates extracellular signal-regulated protein kinases (ERKs) and induces expression of cyclin D1. Newly synthesized cyclin D1 assembles with cyclin-dependent kinase-4 (CDK4) to form holoenzyme complexes that inefficiently phosphorylate the retinoblastoma protein. Activation of the MEK1/ERK pathway triggers neither degradation of the CDK inhibitor kinase inhibitory protein-1 (p27Kip1) nor leads to activation of cyclin E- and A-dependent CDK2, and such cells do not enter the DNA synthetic (S) phase of the cell division cycle. In contrast, zinc induction of active MEK1 in cells also engineered to ectopically overexpress cyclin D1 and CDK4 subunits, generates levels of cyclin D-dependent retinoblastoma protein kinase activity approximating those achieved in cells stimulated by serum. In this setting, p27Kip1 is mobilized into complexes containing cyclin D1; cyclin E- and A-dependent CDK2 complexes are activated, and serum-starved cells enter S phase. Thus, although the activity of p27Kip1 normally is canceled through a serum-dependent degradative process, overexpressed cyclin D1-CDK complexes sequester p27Kip1 and reduce the effective inhibitory threshold through a stoichiometric mechanism. A fraction of these cells complete S phase and divide, but they are unable to continuously proliferate, indicating that other serum-responsive factors ultimately become rate limiting for cell cycle progression. Therefore, the MEK/ERK pathway not only acts transcriptionally to induce the cyclin D1 gene but functions posttranslationally to regulate cyclin D1 assembly with CDK4 and to thereby help cancel p27Kip1-mediated inhibition (Cheng, 1998).
The synthesis of cyclin D1 and its assembly with cyclin-dependent kinase 4 (CDK4) to form an active complex is a rate-limiting step in progression through the G1 phase of the cell cycle. Using an activated allele of mitogen-activated protein kinase kinase 1 (MEK1), it has been shown that this kinase plays a significant role in positively regulating the expression of cyclin D1. This was found both in quiescent serum-starved cells and in cells expressing dominant-negative Ras. Despite the observation that cyclin D1 is a target of MEK1, in cycling cells it has been found that activated MEK1, but not cyclin D1, is capable of overcoming a G1 arrest induced by Ras inactivation. Either wild-type or catalytically inactive CDK4 cooperates with cyclin D1 in reversing the G1 arrest induced by inhibition of Ras activity. In quiescent NIH 3T3 cells expressing either ectopic cyclin D1 or activated MEK1, cyclin D1 is able to efficiently associate with CDK4; however, the complex is inactive. A significant percentage of the cyclin D1-CDK4 complexes are associated with p27 in serum-starved activated MEK1 or cyclin D1 cell lines. Reduction of p27 levels by expression of antisense p27 allows for S-phase entry from quiescence in NIH 3T3 cells expressing ectopic cyclin D1, but not in parental cells (Ladha, 1998).
An assay using permeabilized cells was developed to monitor fragmentation of the Golgi complex that occurs during mitosis. Golgi stacks, in permeabilized interphase normal rat kidney (NRK) cells, upon incubation with mitotic extracts, undergo extensive fragmentation; the fragmented Golgi membranes are then dispersed throughout the cytoplasm. Continued presence of p34cdc2, the mitosis initiation kinase, is not necessary for Golgi fragmentation. Instead, fragmentation depends on cytosolic mitogen-activated protein kinase kinase 1 (MEK1 or MAPKK1). However, the known cytoplasmic substrates for MEK1, ERK1, and ERK2 are not required for this process. Interestingly, a Golgi-associated ERK has been found that is the likely target for MEK1 in Golgi fragmentation (Acharya, 1998).
Genetic screens for modifiers of activated Ras phenotypes have identified a novel protein, kinase suppressor of Ras (KSR), which shares significant sequence homology with Raf family protein kinases. Studies using Drosophila and C. elegans predict that KSR positively regulates Ras signaling; however, the function of mammalian KSR is not well understood. Two predicted kinase-dead mutants of KSR retain the ability to complement ksr-1 loss-of-function alleles in C. elegans, suggesting that KSR may have physiological, kinase-independent functions. Furthermore, murine KSR forms a multimolecular signaling complex in human embryonic kidney 293T cells composed of HSP90, HSP70, HSP68, p50(CDC37), MEK1, MEK2, 14-3-3, and several other, unidentified proteins. Treatment of cells with geldanamycin, an inhibitor of HSP90, decreases the half-life of KSR, suggesting that HSPs may serve to stabilize KSR. Both nematode and mammalian KSRs are capable of binding to MEKs, and three-point mutants of KSR, corresponding to C. elegans loss-of-function alleles, are specifically compromised in MEK binding. KSR does not alter MEK activity or activation. However, KSR-MEK binding shifts the apparent molecular mass of MEK from 44 to greater than 700 kDa, and this results in the appearance of MEK in membrane-associated fractions. Together, these results suggest that KSR may act as a scaffolding protein for the Ras-mitogen-activated protein kinase pathway (Stewart, 1999).
During vertebrate eye development, the optic vesicle originating from the neuroectoderm is partitioned into a domain that will give rise to the neural retina (NR) and another that will give rise to the retinal pigmented epithelium (RPE). Previous studies have shown that ectopic expression of FGFs in the RPE induces RPE-to-NR transdifferentiation. Similarly, a naturally occurring mutation of the transcription factor Mitf in mouse results in the formation of a second neural retina in place of the dorsal RPE, but the putative signaling pathway linking FGF to Mitf regulation is presently unknown. In cultures of neural crest-derived melanocytes, the MAPK pathway has been shown to target the Mitf transcription factor for ubiquitin-dependent proteolysis, resulting in a rapid degradation and downregulation. Ectopic expression of a constitutively activated allele of MEK-1, the immediate upstream activator of the MAPK ERK, in chicken embryonic retina in ovo, induces transdifferentiation of the RPE into a neural-like epithelium that is correlated with a downregulation of Mitf expression in the presumptive RPE (Galy, 2002).
Mammalian neurogenesis is determined by an interplay between intrinsic genetic mechanisms and extrinsic cues such as growth factors. A signaling cascade, a MEK-C/EBP pathway, has been defined that is essential for cortical progenitor cells to become postmitotic neurons. Inhibition of MEK or of the C/EBP family of transcription factors inhibits neurogenesis while expression of a C/EBPß mutant that is a phosphorylation-mimic at a MEK-Rsk site enhances neurogenesis. C/EBP mediates this positive effect by direct transcriptional activation of neuron-specific genes such as Talpha1 alpha-tubulin. Conversely, inhibition of C/EBP-dependent transcription enhances CNTF-mediated generation of astrocytes from the same progenitor cells. Thus, activation of a MEK-C/EBP pathway enhances neurogenesis and inhibits gliogenesis, thereby providing a mechanism whereby growth factors can selectively bias progenitors to become neurons during development (Ménard, 2002).
Spatiotemporal control of the Ras/ERK MAP kinase signaling pathway is among the key mechanisms for regulating a wide variety of cellular processes. Human Sef (hSef), a recently identified inhibitor whose action mechanism has not been fully defined, acts as a molecular switch for ERK signaling by specifically blocking ERK nuclear translocation without inhibiting its activity in the cytoplasm. Thus, hSef binds to activated forms of MEK, inhibits the dissociation of the MEK-ERK complex, and blocks nuclear translocation of activated ERK. Consequently, hSef inhibits phosphorylation and activation of the nuclear ERK substrate Elk-1, while it does not affect phosphorylation of the cytoplasmic ERK substrate RSK2. Downregulation of endogenous hSef by hSef siRNA enhances the stimulus-induced ERK nuclear translocation and the activity of Elk-1. These results thus demonstrate that hSef acts as a spatial regulator for ERK signaling by targeting ERK to the cytoplasm (Torii, 2004).
Functions of MEKK
NF-kappaB is activated by various stimuli, including inflammatory cytokines and stresses. A key step in the activation of NF-kappaB is the phosphorylation of its inhibitors, IkappaBs (Drosophila homolog: Cactus), by an IkappaB kinase (IKK) complex. Recently, two closely related kinases, designated IKKalpha and IKKbeta, have been identified as the components of the IKK complex that phosphorylate critical serine residues of IkappaBs for degradation. A previously identified NF-kappaB-inducing kinase (NIK), which mediates NF-kappaB activation by TNFalpha and IL-1, has been demonstrated to activate IKK. Previous studies have shown that mitogen-activated protein kinase/ERK kinase kinase-1 (MEKK1), which constitutes the c-Jun N-terminal kinase/stress-activated protein kinase pathway, also activates NF-kappaB by an undefined mechanism. Overexpression of MEKK1 preferentially stimulates the kinase activity of IKKbeta, which results in phosphorylation of IkappaBs. Moreover, a catalytically inactive mutant of IKKbeta blocks the MEKK1-induced NF-kappaB activation. By contrast, overexpression of NIK stimulates kinase activities of both IKKalpha and IKKbeta comparably, suggesting a qualitative difference between NIK- and MEKK1-mediated NF-kappaB activation pathways. Collectively, these results indicate that NIK and MEKK1 independently activate the IKK complex and that the kinase activities of IKKalpha and IKKbeta are differentially regulated by two upstream kinases, NIK and MEKK1, which are responsive to distinct stimuli (Nakano, 1998).
A critical step in the signal-induced activation of the transcription factor NF-kappaB is the site-specific phosphorylation of its inhibitor, IkappaB, that targets the latter for degradation by the ubiquitin-proteasome pathway. Mitogen-activated protein kinase/ERK kinase kinase 1 (MEKK1) can induce both this site-specific phosphorylation of IkappaBalpha at Ser-32 and Ser-36 in vivo and the activity of a high molecular weight IkappaB kinase complex in vitro. Subsequently, others have identified two proteins, IkappaB kinase alpha (IKK-alpha) and IkappaB kinase beta (IKK-beta), that are present in a tumor necrosis factor alpha-inducible, high molecular weight IkappaB kinase complex. These kinases are believed to directly phosphorylate IkappaB based on the examination of the kinase activities of IKK immunoprecipitates, but more rigorous proof of this has yet to be demonstrated. Recombinant IKK-alpha and IKK-beta can, in fact, directly phosphorylate IkappaBalpha at Ser-32 and Ser-36, as well as homologous residues in IkappaBbeta in vitro, and thus are bona fide IkappaB kinases. MEKK1 can induce the activation of both IKK-alpha and IKK-beta in vivo. IKK-alpha is present in the MEKK1-inducible, high molecular weight IkappaB kinase complex and treatment of this complex with MEKK1 induces phosphorylation of IKK-alpha in vitro. It is concluded that IKK-alpha and IKK-beta can mediate the NF-kappaB-inducing activity of MEKK1 (Lee, 1998).
Interleukin-1 (IL-1) and tumor necrosis factor (TNF-alpha) stimulate transcription factors AP-1 and NF-kappaB through activation of the MAP kinases (JNK and p38) and the IkappaB kinase (IKK), respectively. The TNF-alpha and IL-1 signals are transduced through TRAF2 and TRAF6, respectively. Overexpressed TRAF2 or TRAF6 activate JNK, p38, or IKK in the absence of extracellular stimulation. By replacing the carboxy-terminal TRAF domain of TRAF2 and TRAF6 with repeats of the immunophilin FKBP12, it has been demonstrated that their effector domains are composed of their amino-terminal Zn and RING fingers. Oligomerization of the TRAF2 effector domain results in specific binding to MEKK1, a protein kinase capable of JNK, p38, and IKK activation, and induction of TNF-alpha and IL-1 responsive genes. TNF-alpha also enhances the binding of native TRAF2 to MEKK1 and stimulates the kinase activity of the latter. Thus, TNF-alpha and IL-1 signaling is based on oligomerization of TRAF2 and TRAF6 leading to activation of effector kinases (Baud, 1999).
A developmentally regulated, putative MEK kinase (MEKKalpha) has been identified that contains an F-box and WD40 repeats and plays a complex role in regulating cell-type differentiation and spatial patterning. Although components of three MAP kinase pathways required for chemotaxis, activation of adenylyl cyclase, and prespore cell differentiation have been identified previously in Dictyostelium, it is expected that these are independent pathways and unrelated to the pathway containing MEKK. Cells deficient in MEKKalpha develop precociously and exhibit abnormal cell-type patterning with an increase in one of the prestalk compartments (pstO), a concomitant reduction in the prespore domain, and a loss of the sharp compartment boundaries, resulting in overlapping prestalk and prespore domains. Overexpression of MEKKalpha or MEKKalpha lacking the WD40 repeats results in very delayed development and a severe loss of compartment boundaries. Prespore and prestalk cells are interspersed throughout the slug. Analysis of chimeric organisms suggests that MEKKalpha function is required for the proper induction and maintenance of prespore cell differentiation. The WD40 repeats target MEKKalpha to the cortical region of the cell, whereas the F-box/WD40 repeats direct ubiquitin-mediated MEKKalpha degradation. A UBC and a UBP (ubiquitin hydrolase) have been identified that interact with the F-box/WD40 repeats. These findings indicate that cells lacking the ubiquitin hydrolase have phenotypes similar to those of MEKKalpha null (mekkalpha-) cells, further supporting a direct genetic and biochemical interaction between MEKKalpha, the UBC, and the UBP. UBC and UBP differentially control MEKKalpha ubiquitination/deubiquitination and degradation through the F-box/WD40 repeats in a cell-type-specific and temporally regulated manner. These results represent a novel mechanism that includes targeted protein degradation by which MAP kinase cascade components can be controlled. More importantly, these findings suggest a new paradigm of spatial and temporal control of the kinase activity controlling spatial patterning during multicellular development, which parallels the temporally regulated degradation of proteins required for cell-cycle progression (Chung, 1998).
ERK1/2 MAP kinases are important regulators in cellular signaling, whose activity is normally reversibly regulated by threonine-tyrosine phosphorylation. In contrast, stress-induced ERK1/2 activity is downregulated by ubiquitin/proteasome-mediated degradation of ERK1/2. The PHD domain of MEKK1, a RING finger-like structure, exhibits E3 ubiquitin ligase activity toward ERK2 in vitro and in vivo. Moreover, both MEKK1 kinase activity and the docking motif on ERK1/2 were involved in ERK1/2 ubiquitination. Significantly, cells expressing ERK2 with the docking motif mutation are resistant to sorbitol-induced apoptosis. Therefore, MEKK1 functions not only as an upstream activator of the ERK and JNK through its kinase domain, but also as an E3 ligase through its PHD domain, providing a negative regulatory mechanism for decreasing ERK1/2 activity (Lu, 2002).
The activation of ERK1/2 provides an antiapoptotic effect, while JNK activation promotes proapoptotic signaling. Both ERK1/2 and JNK are downstream targets of MEKK1. Therefore, MEKK1 signaling, during stress stimuli like sorbitol treatment, results in the simultaneous activation of both the MEK1/2-ERK1/2 antiapoptotic and MKK4-JNK proapoptotic pathways. The results of this study demonstrate that constitutive MEKK1 activation, by itself, is sufficient to cause apoptosis and downregulation of MEK1-ERK1/2 survival signals through Ub/proteasome degradation pathways. These data suggest that MEKK1, possibly in conjunction with other unknown E3 ligases mediates ubiquitination and degradation of ERK1/2 that functions as a negative feedback mechanism for regulation of survival factors during persistent stress stimuli. This hypothesis provides a model for a dynamic balance between survival signals and pro-apoptotic signals during stress-response in living cells. Simultaneous activation of survival and proapoptotic signaling provides a choice between conflicting fates during initial response to stress. This would allow cells to recover from transient stress stimuli, but if the stress stimulus persists for prolonged periods, the survival signal pathway is downregulated, allowing the cell to commit to an apoptotic response. This model may represent a general mechanism for stress-induced apoptosis (Lu, 2002).
The SMRT (silencing mediator of retinoic acid and thyroid hormone receptor) corepressor participates in the repression of target gene expression by a variety of transcription factors, including the nuclear hormone receptors, promyelocytic leukemia zinc finger protein, and B-cell leukemia protein 6. The ability of SMRT to associate with these transcription factors and thereby to mediate repression is strongly inhibited by activation of tyrosine kinase signaling pathways, such as that represented by the epidermal growth factor receptor. SMRT function is potently inhibited by a mitogen-activated protein kinase (MAPK) kinase kinase (MAPKKK) cascade that operates downstream of this growth factor receptor. Intriguingly, the SMRT protein is a substrate for phosphorylation by protein kinases operating at multiple levels in this MAPKKK pathway, including the MAPKs, MAPK-extracellular signal-regulated kinase 1 (MEK-1), and MEK-1 kinase (MEKK-1). Phosphorylation of SMRT by MEKK-1 and, to a lesser extent, MEK-1 inhibits the ability of SMRT to physically tether to its transcription factor partners. Notably, activation of MEKK-1 or MEK-1 signaling in transfected cells also leads to a redistribution of the SMRT protein from a nuclear compartment to a more perinuclear or cytoplasmic compartment. It is suggested that SMRT-mediated repression is regulated by the MAPKKK cascade and that changes both in the affinity of SMRT for its transcription factors and in the subcellular distribution of SMRT contribute to the loss of SMRT function that is observed in response to kinase signal transduction (Hong, 2000).
The p42/p44 mitogen-activated protein kinase (MAPK) cascade includes Ras, Raf, Mek, and Erk MAPK. To determine the effect of a full knockout at a single level of this signaling pathway in mammals, and to investigate functional redundancy between Mek1 and Mek2, these genes were disrupted in murine and human epidermis. Loss of either protein alone produced no phenotype, whereas combined Mek1/2 deletion in development or adulthood abolished Erk1/2 phosphorylation and led to hypoproliferation, apoptosis, skin barrier defects, and death. Conversely, a single copy of either allele was sufficient for normal development. Combined Mek1/2 loss also abolished Raf-induced hyperproliferation. Human tissue deficient in either Mek isoform was normal, whereas loss of both proteins led to hypoplasia, which was rescued by active Erk2 expression. These data indicate that Mek1/2 are functionally redundant in the epidermis, where they act as a linear relay in the MAPK pathway to mediate development and homeostasis (Scholl, 2007).
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