Regulation of Drosophila p38 activation by specific MAP2 kinase and MAP3 kinase in response to different stimuli

The p38 mitogen-activated protein kinase (MAPK) signaling pathway plays an important role in cellular responses to inflammatory stimuli and environmental stress. Activation of p38 is mediated through phosphorylation by upstream MAPKK, which in turn is activated by MAPKKK. However, the mechanism of how different upstream MAP2Ks and MAP3Ks specifically contribute to p38 activation in response to different stimuli is still not clearly understood. By using double-stranded RNA-mediated interference (RNAi) in Drosophila cells, we demonstrate that D-MKK3 is a major MAP2K responsible for D-p38 activation by UV, heat shock, NaCl or peptiodglycan (PGN). Stimulation of UV and PGN activates D-p38 through D-MEKK1, heat shock-induced activation of D-p38 signals through both D-MEKK1 and D-ASK1. On the other hand, maximal activation of D-p38 by NaCl requires the expression of four MAP3Ks.     

Mip1, an MEKK2-interacting protein, controls MEKK2 dimerization and activation

Mitogen-activated protein kinase (MAPK) cascades are central components of the intracellular signaling networks used by eukaryotic cells to respond to a wide spectrum of extracellular stimuli. An MAPK is activated by an MAPK kinase, which in turn is activated by an MAPK kinase kinase (MAP3K). However, little is known about the molecular aspects of the regulation and activation of large numbers of MAP3Ks that are crucial in relaying upstream receptor-mediated signals through the MAPK cascades to induce various physiological responses. In this study, we identified a novel MEKK2-interacting protein, Mip1, that regulates MEKK2 dimerization and activation by forming a complex with inactive and nonphosphorylated MEKK2. In particular, Mip1 prevented MEKK2 activation by blocking MEKK2 dimer formation, which in turn blocked JNKK2, c-Jun N-terminal kinase 1 (JNK1), extracellular signal-regulated kinase 5, and AP-1 reporter gene activation by MEKK2. Furthermore, we found that the endogenous Mip1-MEKK2 complex was dissociated transiently following epidermal growth factor stimulation. In contrast, the knockdown of Mip1 expression by siRNA augmented the MEKK2-mediated JNK and AP-1 reporter activation. Together, our data suggest a novel model for MEKK2 regulation and activation.     

MAPK signaling by the D quadrant embryonic organizer of the mollusc Ilyanassa obsoleta

Classical experiments performed on the embryo of the mollusc Ilyanassa obsoleta demonstrate that the 3D macromere acts as an embryonic organizer, by signaling to other cells and inducing them to assume the correct pattern of cell fates. We have discovered that MAP kinase signaling is activated in the cells that require the signal from 3D for normal differentiation. Preventing specification of the D quadrant lineage by removing the polar lobe disrupts the pattern of MAPK activation, as does ablation of the 3D macromere itself. Blocking MAPK activation with the MAP Kinase inhibitor U0126 produces larvae that differentiate the same limited complement of tissues as D quadrant deletions. Our results suggest that the MAP Kinase signaling cascade transduces the inductive signal from 3D and specifies cell fate among the cells that receive the signal.     

p38 MAPK: A dual role in hepatocyte proliferation through reactive oxygen species

Abstract

p38 MAPKs are important mediators of signal transduction that respond to a wide range of extracellular stressors such as UV radiation, osmotic shock, hypoxia, pro-inflammatory cytokines, and oxidative stress. The most abundant family member is p38α, which helps to couple cell proliferation and growth in response to certain damaging stimuli. In fact, increased proliferation and impaired differentiation are hallmarks of p38α-deficient cells. It has been reported that reactive oxygen species (ROS) play a critical role in cytokine-induced p38α activation. Under physiological conditions, p38α can function as a mediator of ROS signaling and either activate or suppress cell cycle progression depending on the activation stimulus. The interplay between cell proliferation, p38 MAPK activation, and ROS production plays an important role in hepatocytes. In fact, low levels of ROS seem to be needed to activate several signaling pathways in response to hepatectomy and to orchestrate liver regeneration. p38 MAPK works as a sensor of oxidative stress and cells that have developed mechanisms to uncouple p38 MAPK activation from oxidative stress are more likely to become tumorigenic. So far, p38α influences the redox balance, determining cell survival, terminal differentiation, proliferation, and senescence. Further studies would be necessary in order to clarify the precise role of p38 MAPK signaling as a redox therapeutical target.     

Yersinia effector YopJ inhibits yeast MAPK signaling pathways by an evolutionarily conserved mechanism

Yersinia effector, YopJ, inhibits the innate immune response by blocking MAP kinase and NFkappaB signaling pathways in mammalian cells. Herein, YopJ is shown to disrupt the MAP kinase signaling pathways in Saccharomyces cerevisiae. Expression of YopJ in yeast blocks the ability of yeast to respond to alpha factor by disrupting activation of the pheromone signaling pathway upstream of the activation of the MAPK Fus3p. YopJ also blocks the high osmolarity growth (HOG) MAP kinase pathway in yeast upstream of the activation of the MAPK Hog1p. YopJ is proposed to block the MAP kinase pathways in yeast in a similar manner to the way it blocks mammalian signaling pathways, implicating that a novel, evolutionarily conserved mechanism of regulation is utilized for signal transduction by these pathways.     

Caenorhabditis elegans mom-4 is required for the activation of the p38 MAPK signaling pathway in the response to Pseudomonas aeruginosa infection

The p38 mitogen-activated protein kinase (MAPK) plays an evolutionarily conserved role in the cellular response to microbial infection and environmental stress. Activation of p38 is mediated through phosphorylation by upstream MAPKK, which in turn is activated by MAPKKK. In the Caenorhabditis elegans, the p38 MAPK (also called PMK-1) signaling pathway has been shown to be required in its resistance to bacterial infection. However, how different upstream MAP2Ks and MAP3Ks specifically contribute to the activation of PMK-1 in response to bacterial infection still is not clearly understood. By using double-stranded RNA-mediated interference (RNAi) and genetic mutants of C. elegans, we demonstrate that C. elegans MOM-4, a mammalian TAK1 homolog, is required for the resistance of C. elegans to a P. aeruginosa infection. We have also found that the MKK-4 of C. elegans is required for P. aeruginosa resistance, but not through the regulation of DLK-1. In summary, our results indicate that different upstream MAPKKKs or MAPKKs regulate the activation of PMK-1 in response to P. aeruginosa.     

Redox control of cell fate by MAP kinase: physiological roles of ASK1-MAP kinase pathway in stress signaling

The intracellular redox state is a key determinant of cell fate, such as cell survival, proliferation, differentiation, and apoptosis. Redox imbalance is closely linked to a variety of human diseases, so that the intracellular redox condition should be tightly regulated. The redox state of the cell is a consequence of the precise balance between the levels of oxidizing and reducing equivalents, such as reactive oxygen species (ROS) and endogenous antioxidants. ROS are not only toxicants to the cell, but also second messengers in intracellular signal transduction, and control the action of several signaling pathways, including mitogen-activated protein (MAP) kinases. Apoptosis signal-regulating kinase 1 (ASK1) is a MAP kinase kinase kinase of the c-Jun N-terminal kinase (JNK) and p38 MAP kinase pathways, which is preferentially activated in response to various types of stress such as oxidative stress and plays pivotal roles in a wide variety of cellular responses. Recent studies have revealed that ASK1 is also required for innate immune response through ROS production. In this review, we focus on redox control of cell function by MAP kinase signaling, and provide the advanced mechanism of redox-regulated ASK1 activation and physiological roles of the ASK1-MAP kinase pathway in stress signaling.     

Pathological roles of MAPK signaling pathways in human diseases

The mammalian family of mitogen-activated protein kinases (MAPKs) includes extracellular signal-regulated kinase (ERK), p38, and c-Jun NH₂-terminal kinase (JNK), with each MAPK signaling pathway consisting of at least three components, a MAPK kinase kinase (MAP3K), a MAPK kinase (MAP2K), and a MAPK. The MAPK pathways are activated by diverse extracellular and intracellular stimuli including peptide growth factors, cytokines, hormones, and various cellular stressors such as oxidative stress and endoplasmic reticulum stress. These signaling pathways regulate a variety of cellular activities including proliferation, differentiation, survival, and death. Deviation from the strict control of MAPK signaling pathways has been implicated in the development of many human diseases including Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS) and various types of cancers. Persistent activation of the JNK or p38 signaling pathways has been suggested to mediate neuronal apoptosis in AD, PD, and ALS, whereas the ERK signaling pathway plays a key role in several steps of tumorigenesis including cancer cell proliferation, migration, and invasion. In this review, we summarize recent findings on the roles of MAPK signaling pathways in human disorders, focusing on cancer and neurodegenerative diseases including AD, PD, and ALS.     

Stimulus-specific requirements for MAP3 kinases in activating the JNK pathway

Mitogen-activated protein kinases (MAPKs) are activated by numerous ligands typically through a protein kinase cascade minimally composed of the MAPK in series with a MAP2 kinase (MAP2K) and a MAP3K. This arrangement is thought to confer specificity and appropriate kinetic properties on the activation of MAPKs in response to physiological stimuli. Surprisingly, more than a dozen MAP3Ks have been identified that activate the c-Jun N-terminal kinases (JNKs) when overexpressed, but there is no clear understanding of which kinases actually mediate JNK activation by ligands. Here, we use double-stranded RNA-mediated interference of gene expression to reveal the explicit participation of discrete MAP3Ks in controlling JNK activity by multiple stimuli. Maximal activation of JNK by lipopolysaccharide requires the MAP3K TAK1. On the other hand, sorbitol requires expression of four MAP3Ks to cause maximal JNK activation. Thus, we demonstrate that specific stimuli use different mechanisms to recruit distinct MAP3Ks to regulate the JNK pathway.     

Activation of cell stress response pathways by Shiga toxins

Shiga toxin-producing bacteria cause widespread outbreaks of bloody diarrhoea that may progress to life-threatening systemic complications. Shiga toxins (Stxs), the main virulence factors expressed by the pathogens, are ribosome-inactivating proteins which inhibit protein synthesis by removing an adenine residue from 28S rRNA. Recently, Stxs were shown to activate multiple stress-associated signalling pathways in mammalian cells. The ribotoxic stress response is activated following the depurination reaction localized to the α-sarcin/ricin loop of eukaryotic ribosomes. The unfolded protein response (UPR) may be initiated by toxin unfolding within the endoplasmic reticulum, and maintained by production of truncated, misfolded proteins following intoxication. Activation of the ribotoxic stress response leads to signalling through MAPK cascades, which appears to be critical for activation of innate immunity and regulation of apoptosis. Precise mechanisms linking ribosomal damage with MAPK activation require clarification but may involve recognition of ribosomal conformational changes and binding of protein kinases to ribosomes, which activate MAP3Ks and MAP2Ks. Stxs appear capable of activating all ER membrane localized UPR sensors. Prolonged signalling through the UPR induces apoptosis in some cell types. The characterization of stress responses activated by Stxs may identify targets for the development of interventional therapies to block cell damage and disease progression.     

Identification of MEKK2/3 serine phosphorylation site targeted by the Toll-like receptor and stress pathways

Members of the mitogen-activated protein kinase kinase kinase (MAP3K) family are crucial for the Toll-like receptor (TLR) signaling and cellular stress responses. However, the molecular mechanisms underlying the TLR- and cellular stress-mediated MAP3K activation remain largely unknown. In this study, we identified a key regulatory phosphorylation site, serine 519 and serine 526, in MAP3K MEKK2 and MEKK3, respectively. Mutation of this serine to an alanine severely impaired MEKK2/3 activation. We generated an anti-p-MEKK2/3 antibody and used this antibody to demonstrate that lipopolysaccharide induced MEKK2 and MEKK3 phosphorylation on their regulatory serine. We found that the serine phosphorylation was crucial for TLR-induced interleukin 6 production and this process is regulated by TRAF6, a key adaptor molecule for the TLR pathway. We further demonstrated that many, but not all, MAPK agonists induced the regulatory serine phosphorylation, suggesting an involvement of different MAP3Ks in activation of the MAPK cascades leading to different cellular responses. In conclusion, this study reveals a novel molecular mechanism for MEKK2/3 activation by the TLR and cellular stress pathways.     

Deciphering the MAP kinase pathway

MAP kinases (MAPK) are serine/threonine kinases which are activated by a dual phosphorylation on threonine and tyrosine residues. Their specific upstream activators, called MAP kinase kinases (MAPKK), constitute a new family of dual-specific threonine/tyrosine kinases, which in turn are activated by upstream MAP kinase kinase kinases (MAPKKK). These three kinase families are successively stimulated in a cascade of activation described in various species such as mammals, frog, fly, worm or yeast.In mammals, the MAP kinase module lies on the signaling pathway triggered by numerous agonists such as growth factors, hormones, lymphokines, tumor promoters, stress factors, etc. Targets of MAP kinase have been characterize tin all subcellular compartments. In yeast, genetic epistasis helped to characterize the presence of several MAP kinase modules in the same system. By complementation tests, the relationships existing between phylogenetically distant members of each kinase family have been described. The roles of the MAP kinase cascade have been analyzed by engineering various mutations in the kinases of the module. The MAP kinase cascade has thus been implicated in higher eukaryotes in cell growth, cell fate and differentiation, and in low eukaryotes, in conjugation, osmotic stress, cell wall constrct and mitosis.     

Mutations in protein kinase subdomain X differentially affect MEKK2 and MEKK1 activity

MAPK/ERK kinase kinase 2 (MEKK2) is a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family of protein kinases. MAP3Ks are components of a three-tiered protein kinase pathway in which a MAP3K phosphorylates and activates a mitogen-activated protein kinase kinase (MAP2K), which in turn activates a mitogen-activated protein kinase (MAPK). We have previously identified residues within protein kinase subdomain X in the MAP3K, MEKK1, that are critical for its interaction with the MAP2K, MKK4, and MEKK1-induced MKK4 activation. We report here that kinase subdomain X also plays a critical role in MEKK2 activity. Select point mutations in subdomain X impair MEKK2 phosphorylation of the MAP2Ks, MKK7 and MEK5, abolish MEKK2-induced activation of the MAPKs, JNK1 and ERK5, and diminish MEKK2-dependent activation of an AP-1 reporter gene. Interestingly, the spectrum of mutations in subdomain X of MEKK2 that affects its activity is overlapping with but not identical to those that have effects on MEKK1. Thus, mutations in subdomain X differentially affect MEKK2 and MEKK1.     

The TGFβ-induced phosphorylation and activation of p38 mitogen-activated protein kinase is mediated by MAP3K4 and MAP3K10 but not TAK1

The signalling pathways downstream of the transforming growth factor beta (TGFβ) family of cytokines play critical roles in all aspects of cellular homeostasis. The phosphorylation and activation of p38 mitogen-activated protein kinase (MAPK) has been implicated in TGFβ-induced epithelial-to-mesenchymal transition and apoptosis. The precise molecular mechanisms by which TGFβ cytokines induce the phosphorylation and activation of p38 MAPK are unclear. In this study, I demonstrate that TGFβ-activated kinase 1 (TAK1/MAP3K7) does not play a role in the TGFβ-induced phosphorylation and activation of p38 MAPK in MEFs and HaCaT keratinocytes. Instead, RNAi-mediated depletion of MAP3K4 and MAP3K10 results in the inhibition of the TGFβ-induced p38 MAPK phosphorylation. Furthermore, the depletion of MAP3K10 from cells homozygously knocked-in with a catalytically inactive mutant of MAP3K4 completely abolishes the TGFβ-induced phosphorylation of p38 MAPK, implying that among MAP3Ks, MAP3K4 and MAP3K10 are sufficient for mediating the TGFβ-induced activation of p38 MAPK.     

Regulation of anoxic death in Caenorhabditis elegans by mammalian apoptosis signal-regulating kinase (ASK) family proteins

Cells and organisms face anoxia in a wide variety of contexts, including ischemia and hibernation. Cells respond to anoxic conditions through multiple signaling pathways. We report that NSY-1, the Caenorhabditis elegans ortholog of mammalian apoptosis signal-regulating kinase (ASK) family of MAP kinase (MAPK) kinase kinases (MAP3Ks), regulates viability of animals in anoxia. Loss-of-function mutations of nsy-1 increased survival under anoxic conditions, and increased survival was also observed in animals with mutations in tir-1 and the MAPK kinase (MAP2K) sek-1, which are upstream and downstream factors of NSY-1, respectively. Consistent with these findings, anoxia was found to activate the p38 MAPK ortholog PMK-1, and this was suppressed in nsy-1 and tir-1 mutant animals. Furthermore, double-mutant analysis showed that the insulin-signaling pathway, which also regulates viability in anoxia, functioned in parallel to NSY-1. These results suggest that the TIR-1-NSY-1-SEK-1-PMK-1 pathway plays important roles in the reponse to anoxia in C. elegans.     

Graded mitogen-activated protein kinase activity precedes switch-like c-Fos induction in mammalian cells

The mitogen-activated protein kinase (MAPK) pathway is an evolutionarily conserved signaling module that controls important cell fate decisions in a variety of physiological contexts. During Xenopus oocyte maturation, the MAPK cascade converts an increasing progesterone stimulus into a switch-like, all-or-nothing response. While the importance of such switch-like behavior is widely discussed in the literature, it is not known whether the MAPK pathway in mammalian cells exhibits a switch-like or graded response. For this study, we used flow cytometry and immunofluorescence to generate single-cell measurements of MAPK signaling in Swiss 3T3 fibroblasts. In contrast to the case in Xenopus oocytes, we found that ERK activation in individual mammalian cells is not ultrasensitive and shows a graded response to changes in agonist concentration. Thus, the conserved MAPK signaling module exhibits different systems-level properties in different cellular contexts. Furthermore, the graded ERK response was converted into a more switch-like behavior at the level of immediate-early gene induction and cell cycle progression. Thus, while MAPK signaling is involved in all-or-nothing cell fate decisions for both Xenopus oocyte maturation and mammalian fibroblast proliferation, the underlying mechanisms responsible for the switch-like nature of the cellular responses are different in these two systems, with the mechanism appearing to lie downstream of the kinase cascade in mammalian fibroblasts.