MAPK/ERK kinase (MEK) signalling is required for resumption of meiosis in cultured cumulus-enclosed pig oocytes

Resumption of meiosis of mammalian oocytes is facilitated by the maturation promoting factor (MPF) and accompanied by activation of mitogen activated protein kinases (MAPK) which are phosphorylated by the MAPK kinase (MEK). In this study we examined the effects of PD 98059, which inhibits the activity of MEK, on in vitro maturation of pig oocytes. Cumulus-oocyte complexes (COCs) were cultured in the presence or absence of the drug (50 microM) for various time periods. To elucidate the influence of cumulus cells, COCs were first cultured in inhibitor-free medium, subsequently denuded, and incubated further in PD 98059 supplemented medium. Reversibility of drug action as tested following PD 98059 treatment of COCs by transferring them to drug-free medium. Culture of COCs in medium supplemented with PD 98059 prevents resumption of nuclear maturation in the majority of COCs. This inhibition was reversible and accompanied by a non-activation of both MAP and MPF. Addition of the MEK inhibitor to extracts of in vitro matured oocytes revealed that the kinase activities were not directly influenced by the inhibitor, suggesting a link between MAP and MPF kinases. Preincubation of COCs in inhibitor-free medium for 6 h followed by further culture of COCs or denuded oocytes in the presence of PD 98059 for various periods resulted in elevated MAP and MPF kinase activities, indicating an early and transient MEK signalling in the oocyte itself. These results support the idea that MAP and MPF are involved in the induction of germinal vesicle breakdown in porcine oocytes.     

MAPK/ERK signalling is required for zebrafish cardiac regeneration

Objectives

To better understand the molecular mechanisms of regeneration and explore the potential signalling pathways as therapeutic targets for heart attacks.

Results

After treatment with the MEK inhibitor AZD6244 upon cardiac injury, the core members in MAPK/ERK signalling—mek and erk—demonstrate elevated expression, and these proteins are deposited at the injury site in zebrafish. pERK is also induced in non-cardiomyocytes near the injury site. Furthermore, the induced expression of a dominant-negative form of MEK1 inhibits zebrafish cardiac regeneration, characterized by increased cardiac fibrosis (a hallmark of regenerative failure), reduced or delayed production of regenerative myocardium, and migration of FLI1⁺ endothelial cells, without direct inhibition of cardiomyocyte proliferation.

Conclusion

Appropriate activation of MAPK/ERK signalling is essential for zebrafish cardiac regeneration.

     

Integrin and FAK-mediated MAPK activation is required for cyclic strain mitogenic effects in Caco-2 cells

Rhythmic strain stimulates Caco-2 proliferation. We asked whether mitogen-activated protein kinase (MAPK) activation mediates strain mitogenicity and characterized upstream signals regulating MAPK. Caco-2 cells were subjected to strain on collagen I-precoated membranes or antibodies to integrin subunits. Twenty-four hours of cyclic strain increased cell numbers compared with static conditions. MAPK-extracellular signal-regulated kinase (ERK) kinase inhibition (20 microM PD-98059) blocked strain mitogenicity. p38 Inhibition (10 microM SB-202190) did not. Strain rapidly and time-dependently activated focal adhesion kinase (FAK), paxillin, ERK1 and 2, and p38 on collagen. c-Jun NH(2)-terminal kinase (JNK)1 and 2 exhibited delayed activation. Similar activation occurred when Caco-2 cells were subjected to strain on a substrate of functional antibody to the alpha2-, alpha3-, alpha6-, or beta1-integrin subunits but not on a substrate of functional antibody to the alpha5-subunit. FAK inhibition by FAK397 transfection blocked ERK2 and JNK1 activation by in vitro kinase assays, but pharmacological protein kinase C inhibition did not block ERK1 or 2 activation by strain. Strain-induced ERK signals mediate strain's mitogenic effects and may require integrins and FAK activation.     

PAK1 phosphorylation of MEK1 regulates fibronectin-stimulated MAPK activation

Activation of the Ras-MAPK signal transduction pathway is necessary for biological responses both to growth factors and ECM. Here, we provide evidence that phosphorylation of S298 of MAPK kinase 1 (MEK1) by p21-activated kinase (PAK) is a site of convergence for integrin and growth factor signaling. We find that adhesion to fibronectin induces PAK1-dependent phosphorylation of MEK1 on S298 and that this phosphorylation is necessary for efficient activation of MEK1 and subsequent MAPK activation. The rapid and efficient activation of MEK and phosphorylation on S298 induced by cell adhesion to fibronectin is influenced by FAK and Src signaling and is paralleled by localization of phospho-S298 MEK1 and phospho-MAPK staining in peripheral membrane-proximal adhesion structures. We propose that FAK/Src-dependent, PAK1-mediated phosphorylation of MEK1 on S298 is central to the organization and localization of active Raf-MEK1-MAPK signaling complexes, and that formation of such complexes contributes to the adhesion dependence of growth factor signaling to MAPK.     

Concomitant recruitment of ERK1/2 and p38 MAPK signalling pathway is required for activation of cytoplasmic phospholipase A2 via ATP in articular chondrocytes

Extracellular ATP is a pro-inflammatory mediator involved in the release of prostaglandin from articular chondrocytes, but little is known about its effects on intracellular signaling. ATP triggered the rapid release of prostaglandin E(2) (PGE(2)) by acting on P2Y(2) receptors in rabbit articular chondrocytes. We have explored the signaling events involved in this synthesis. ATP significantly increased arachidonic acid production, which involved the activation of the 85-kDa cytosolic phospholipase A(2) (cPLA(2)) but not a secreted form of PLA(2), as demonstrated by various PLA(2) inhibitors and translocation experiments. We also showed that ATP induced the phosphorylation of p38 and ERK1/2 mitogen-activated-protein kinases (MAPKs). Both PD98059, an inhibitor of the ERK pathway, and SB203580, an inhibitor of p38 MAPK, completely inhibited the ATP-induced release of PGE(2). Finally, dominant-negative plasmids encoding p38 and ERK transfected alone into the cells impaired the ATP-induced release of PGE(2) to about the same extent as both plasmids transfected together. These results suggest that PGE(2) production induced by ATP requires the activation of both ERK1/2 and p38 MAPKs. Thus, ATP acts via P2Y(2)-purine receptors to recruit cPLA(2) by activating both ERK1/2 and p38 MAPKs and stimulates the release of PGE(2) from articular chondrocytes.     

Phosphoinositide 3-kinase activation in late G1 is required for c-Myc stabilization and S phase entry

Phosphoinositide 3-kinase (PI3K) is one of the early-signaling molecules induced by growth factor (GF) receptor stimulation that are necessary for cell growth and cell cycle entry. PI3K activation occurs at two distinct time points during G(1) phase. The first peak is observed immediately following GF addition and the second in late G(1), before S phase entry. This second activity peak is essential for transition from G(1) to S phase; nonetheless, the mechanism by which this peak is induced and regulates S phase entry is poorly understood. Here, we show that activation of Ras and Tyr kinases is required for late-G(1) PI3K activation. Inhibition of late-G(1) PI3K activity results in low c-Myc and cyclin A expression, impaired Cdk2 activity, and reduced loading of MCM2 (minichromosome maintenance protein) onto chromatin. The primary consequence of inhibiting late-G(1) PI3K was c-Myc destabilization, as conditional activation of c-Myc in advanced G(1) as well as expression of a stable c-Myc mutant rescued all of these defects, restoring S phase entry. These results show that Tyr kinases and Ras cooperate to induce the second PI3K activity peak in G(1), which mediates initiation of DNA synthesis by inducing c-Myc stabilization.     

Raf-1/MEK/MAPK pathway is necessary for the G2/M transition induced by nocodazole

The dynamic balance between polymerization and depolymerization of microtubules is critical for cells to enter and exit mitosis, and drugs that disrupt this balance, such as taxol, colchicine, and nocodazole, arrest the cell cycle in mitosis. Although the Raf/MEK/MAPK pathway can be activated by these drugs, its role in mitosis has not been addressed. Here, we characterize activation of Raf/MEK/MAPK by nocodazole when mitosis is induced. We find that at early time points (up to 3 h) in nocodazole induction, Raf/MEK/MAPK is activated, and inhibition of MAPK activation by a MEK inhibitor, PD98059 or U0126, reduces the number of cells entering mitosis by creating a block at G(2). At later time points and in mitosis, activation of MEK/MAPK is severely inhibited, even though Raf-1 activity remains high and can be further increased by growth factor. This inhibition is reversed when cells are released from metaphase and enter G(0)/G(1) phase. In addition, we find that binding of Raf-1 to 14-3-3 is progressively induced by nocodazole, reaching a maximum in mitosis, and that this binding is necessary to maintain mitotic Raf-1 activity. Our present study indicates that activation of the Raf/MEK/MAPK pathway is necessary for the G(2)/M progression.     

SDF-1alpha-induced intracellular calcium transient involves Rho GTPase signalling and is required for migration of hematopoietic progenitor cells

Signalling through the chemokine stromal derived factor (SDF)-1alpha and its receptor CXCR4 has been recognized as a key event in the migratory response of hematopoietic stem and progenitor cells (HPC). Small GTPases of the Rho/Rac family might be involved in SDF-1alpha signalling at several different levels. In the present study we report that two toxins from Clostridium species which inhibit the small GTPase Rho suppressed SDF-1alpha-induced generation of intracellular calcium transients in HPC. Chelation of intracellular Ca(2+) with BAPTA or depletion of intracellular Ca(2+) stores with thapsigargin demonstrated that calcium transients are essential for SDF-1alpha-induced chemotactic migration of HPC. Furthermore, transplantation of HPC pretreated with Ca(2+) flux inhibitors into mice revealed a suppression of HPC homing to the bone marrow and increased levels of cells remaining in the bloodstream or circulating to the spleen. Our data indicate that the small GTPase Rho is required for the induction of Ca(2+) transients in HPC, which in turn are necessary for the coordinated migratory response of HPC both in vitro and in vivo.     

A proline-rich sequence unique to MEK1 and MEK2 is required for raf binding and regulates MEK function.

Mammalian MEK1 and MEK2 contain a proline-rich (PR) sequence that is absent both from the yeast homologs Ste7 and Byr1 and from a recently cloned activator of the JNK/stress-activated protein kinases, SEK1/MKK4. Since this PR sequence occurs in MEKs that are regulated by Raf family enzymes but is missing from MEKs and SEKs activated independently of Raf, we sought to investigate the role of this sequence in MEK1 and MEK2 regulation and function. Deletion of the PR sequence from MEK1 blocked the ability of MEK1 to associate with members of the Raf family and markedly attenuated activation of the protein in vivo following growth factor stimulation. In addition, this sequence was necessary for efficient activation of MEK1 in vitro by B-Raf but dispensable for activation by a novel MEK1 activator which we have previously detected in fractionated fibroblast extracts. Furthermore, we found that a phosphorylation site within the PR sequence of MEK1 was required for sustained MEK1 activity in response to serum stimulation of quiescent fibroblasts. Consistent with this observation, we observed that MEK2, which lacks a phosphorylation site at the corresponding position, was activated only transiently following serum stimulation. Finally, we found that deletion of the PR sequence from a constitutively activated MEK1 mutant rendered the protein nontransforming in Rat1 fibroblasts. These observations indicate a critical role for the PR sequence in directing specific protein-protein interactions important for the activation, inactivation, and downstream functioning of the MEKs.     

Polycomb group protein-associated chromatin is reproduced in post-mitotic G1 phase and is required for S phase progression

Polycomb group (PcG) proteins form two distinct complexes, PRC1 and PRC2, to regulate developmental target genes by maintaining the epigenetic state in cells. PRC2 methylates histone H3 at lysine 27 (H3K27), and PRC1 then recognizes methyl-H3K27 to form repressive chromatin. However, it remains unknown how PcG proteins maintain stable and plastic chromatin during cell division. Here we report that PcG-associated chromatin is reproduced in the G(1) phase in post-mitotic cells and is required for subsequent S phase progression. In dividing cells, H3K27 trimethylation (H3K27Me(3)) marked mitotic chromosome arms where PRC2 (Suz12 and Ezh2) co-existed, whereas PRC1 (Bmi1 and Pc2) appeared in distinct foci in the pericentromeric regions. As each PRC complex was increasingly assembled from mitosis to G(1) phase, PRC1 formed H3K27Me(3)-based chromatin intensively during middle and late G(1) phase; this chromatin was highly resistant to in situ nuclease treatment. Thus, the transition from mitosis to G(1) phase is crucial for PcG-mediated chromatin inheritance. Knockdown of Suz12 markedly reduced the amount of H3K27Me(3) on mitotic chromosomes, and as a consequence, PRC1 foci were not fully transmitted to post-mitotic daughter cells. S phase progression was markedly delayed in these Suz12-knockdown cells. The fact that PcG-associated chromatin is reproduced during post-mitotic G(1) phase suggests the possibility that PcG proteins enable their target chromatin to be remodeled in response to stimuli in the G(1) phase.     

Cleavage of the signaling mucin Msb2 by the aspartyl protease Yps1 is required for MAPK activation in yeast

Signaling mucins are cell adhesion molecules that activate RAS/RHO guanosine triphosphatases and their effector mitogen-activated protein kinase (MAPK) pathways. We found that the Saccharomyces cerevisiae mucin Msb2p, which functions at the head of the Cdc42p-dependent MAPK pathway that controls filamentous growth, is processed into secreted and cell-associated forms. Cleavage of the extracellular inhibitory domain of Msb2p by the aspartyl protease Yps1p generated the active form of the protein by a mechanism incorporating cellular nutritional status. Activated Msb2p functioned through the tetraspan protein Sho1p to induce MAPK activation as well as cell polarization, which involved the Cdc42p guanine nucleotide exchange factor Cdc24p. We postulate that cleavage-dependent activation is a general feature of signaling mucins, which brings to light a novel regulatory aspect of this class of signaling adhesion molecule.     

MEK1-independent activation of MAPK and MEK1-dependent activation of p70 S6 kinase by stem cell factor (SCF) in ovarian cancer cells

We discovered a stem cell factor (SCF)-triggered, MEK1-independent, and PI3K-dependent MAPK activation pathway in the Kit-expressing ovarian cancer cell line HEY. When we knocked down MEK1 with RNA interference (RNAi) to study the function of MEK1 on the proliferation and survival of ovarian cancer cells, we found that impaired cell growth still occurred after MEK1 expression had been suppressed, although MAPK activation remained intact. This suggests that there is MEK1-independent activation of MAPK in the SCF-induced ovarian cancer cell growth process, and that MEK1 still plays a crucial role in maintaining the malignant properties of ovarian cancer cells even when it fails to activate MAPK as expected.     

PKC-dependent delayed metabolic preconditioning is independent of transient MAPK activation

In this study we used an in vitro model of delayed preconditioning to investigate activation of mitogen-activated protein kinases (MAPKs) and their potential role in protection. Neonatal rat cardiomyocytes were preconditioned using a buffer containing glycolytic inhibitors and low pH (minimal metabolic preconditioning; MMPC) consisting of modified Krebs buffer, 10 mM 2-deoxyglucose, and 20 mM lactate, pH 6.8, for 2 h followed by 24 h of simulated reperfusion before lethal simulated ischemia (LSI). MAPK activation during the MMPC protocol was determined using phospho-specific antisera and the effect on protection determined following LSI. Rapid, transient phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38 MAPK was observed during each of the MMPC, reperfusion, and LSI phases; an effect blocked by MAPK inhibitors PD-98059 and SB-203580, respectively, but not by the protein kinase C (PKC) inhibitor Ro31-8220. However, although MMPC was blocked by Ro31-8220, treatment with the MAPK inhibitors during the preconditioning protocol did not block delayed protection conferred by MMPC. Thus the data suggest that, in this model of delayed preconditioning, protection appears to be PKC dependent but independent of ERK1/2 or p38 MAPK activation.     

p90Rsk is required for G1 phase arrest in unfertilized starfish eggs

The cell cycle in oocytes generally arrests at a particular meiotic stage to await fertilization. This arrest occurs at metaphase of meiosis II (meta-II) in frog and mouse, and at G1 phase after completion of meiosis II in starfish. Despite this difference in the arrest phase, both arrests depend on the same Mos-MAPK (mitogen-activated protein kinase) pathway, indicating that the difference relies on particular downstream effectors. Immediately downstream of MAPK, Rsk (p90 ribosomal S6 kinase, p90(Rsk)) is required for the frog meta-II arrest. However, the mouse meta-II arrest challenges this requirement, and no downstream effector has been identified in the starfish G1 arrest. To investigate the downstream effector of MAPK in the starfish G1 arrest, we used a neutralizing antibody against Rsk and a constitutively active form of Rsk. Rsk was activated downstream of the Mos-MAPK pathway during meiosis. In G1 eggs, inhibition of Rsk activity released the arrest and initiated DNA replication without fertilization. Conversely, maintenance of Rsk activity prevented DNA replication following fertilization. In early embryos, injection of Mos activated the MAPK-Rsk pathway, resulting in G1 arrest. Moreover, inhibition of Rsk activity during meiosis I led to parthenogenetic activation without meiosis II. We conclude that immediately downstream of MAPK, Rsk is necessary and sufficient for the starfish G1 arrest. Although CSF (cytostatic factor) was originally defined for meta-II arrest in frog eggs, we propose to distinguish ;G1-CSF' for starfish from ;meta-II-CSF' for frog and mouse. The present study thus reveals a novel role of Rsk for G1-CSF.     

The N-terminal ERK-binding site of MEK1 is required for efficient feedback phosphorylation by ERK2 in vitro and ERK activation in vivo

An ERK2-binding site at the N terminus of MEK1 was reported to mediate their stable association. We examined the importance of this binding site in the feedback phosphorylation of MEK1 on Thr(292) and Thr(386) by ERK2, the phosphorylation and activation of ERK2 by MEK1, and the interaction of MEK1 with ERK2 and Raf-1. Deletion of the binding site from MEK1 reduced its phosphorylation by ERK2, but had no effect on its phosphorylation by p21-activated protein kinase-1 (PAK1). A MEK1 N-terminal peptide containing the binding site inhibited MEK1 phosphorylation by ERK2. However, it did not affect MEK1 phosphorylation by p21-activated protein kinase or myelin basic protein phosphorylation by ERK2. Deletion of the N-terminal ERK-binding domain of MEK1 also reduced its ability to phosphorylate ERK2 in vitro, to co-immunoprecipitate with ERK2, and to stimulate ERK2 activation in transfected cells, but it did not alter the association with endogenous Raf-1. Using ERK2-p38 chimeras and an ERK2 deletion mutant, a MEK1-binding site of ERK2 was localized to its N terminus.     

Shoc2 is targeted to late endosomes and required for Erk1/2 activation in EGF-stimulated cells

Shoc2 is the putative scaffold protein that interacts with RAS and RAF, and positively regulates signaling to extracellular signal-regulated protein kinases 1 and 2 (ERK1/2). To elucidate the mechanism by which Shoc2 regulates ERK1/2 activation by the epidermal growth factor (EGF) receptor (EGFR), we studied subcellular localization of Shoc2. Upon EGFR activation, endogenous Shoc2 and red fluorescent protein tagged Shoc2 were translocated from the cytosol to a subset of late endosomes containing Rab7. The endosomal recruitment of Shoc2 was blocked by overexpression of a GDP-bound H-RAS (N17S) mutant and RNAi knockdown of clathrin, suggesting the requirement of RAS activity and clathrin-dependent endocytosis. RNAi depletion of Shoc2 strongly inhibited activation of ERK1/2 by low, physiological EGF concentrations, which was rescued by expression of wild-type recombinant Shoc2. In contrast, the Shoc2 (S2G) mutant, that is myristoylated and found in patients with the Noonan-like syndrome, did not rescue ERK1/2 activation in Shoc2-depleted cells. Shoc2 (S2G) was not located in late endosomes but was present on the plasma membrane and early endosomes. These data suggest that targeting of Shoc2 to late endosomes may facilitate EGFR-induced ERK activation under physiological conditions of cell stimulation by EGF, and therefore, may be involved in the spatiotemporal regulation of signaling through the RAS-RAF module.     

RAS signalling is abnormal in a c-raf1 MEK1 double mutant.

A mutant rat cell clone that suppresses the transformation defects of RAS effector loop substitutions is heterozygous for mutations in c-raf1 and MEK1. The mutant cells can be transformed by many otherwise defective RAS effector mutants, including RAS genes with the effector regions of distantly related GTPases, even though the encoded RAS proteins do not interact with either the mutant or wild-type RAF in Saccharomyces cerevisiae. While the significance of the c-raf1 mutation is unclear, the MEK1 mutation increases MEK1 activity and leads to activation of mitogen-activated protein kinase. The mutant MEK1 is coupled to the epidermal growth factor pathway but exhibits decreased physical interaction with RAF. When overexpressed, the MEK1 mutation is transforming and causes hyperphosphorylation of RAF. Signalling from RAS to MEK1 may be mediated by something other than RAF alone, but signalling through MEK1 is probably sufficient for RAS transformation.     

A cyclin D-Cdk4 activity required for G2 phase cell cycle progression is inhibited in ultraviolet radiation-induced G2 phase delay.

Cyclin D-Cdk4 complexes have a demonstrated role in G1 phase, regulating the function of the retinoblastoma susceptibility gene product (Rb). Previously, we have shown that following treatment with low doses of UV radiation, cell lines that express wild-type p16 and Cdk4 responded with a G2 phase cell cycle delay. The UV-responsive lines contained elevated levels of p16 post-treatment, and the accumulation of p16 correlated with the G2 delay. Here we report that in UV-irradiated HeLa and A2058 cells, p16 bound Cdk4 and Cdk6 complexes with increased avidity and inhibited a cyclin D3-Cdk4 complex normally activated in late S/early G2 phase. Activation of this complex was correlated with the caffeine-induced release from the UV-induced G2 delay and a decrease in the level of p16 bound to Cdk4. Finally, overexpression of a dominant-negative mutant of Cdk4 blocked cells in G2 phase. These data indicate that the cyclin D3-Cdk4 activity is necessary for cell cycle progression through G2 phase into mitosis and that the increased binding of p16 blocks this activity and G2 phase progression after UV exposure.