Mitogen-activated protein kinase kinase 1 (MKK1) is negatively regulated by threonine phosphorylation.

Mitogen-activated protein kinase kinase 1 (MKK1), a dual-specificity tyrosine/threonine protein kinase, has been shown to be phosphorylated and activated by the raf oncogene product as part of the mitogen-activated protein kinase cascade. Here we report the phosphorylation and inactivation of MKK1 by phosphorylation on threonine 286 and threonine 292. MKK1 contains a consensus phosphorylation site for p34cdc2, a serine/threonine protein kinase that regulates the cell division cycle, at Thr-286 and a related site at Thr-292. p34cdc2 catalyzes the in vitro phosphorylation of MKK1 on both of these threonine residues and inactivates MKK1 enzymatic activity. Both sites are phosphorylated in vivo as well. The data presented in this report provide evidence that MKK1 is negatively regulated by threonine phosphorylation.     

Mitogen-activated protein kinase (MAPK) in cardiac tissues

Mitogen-activated protein kinase (MAPK) has recently emerged as a prominent role player in intracellular signalling in the ventricular myocyte with attention being focussed on its possible role in the development of ventricular hypertrophy. It is becoming clear that MAPK is also active in other cells of cardiac origin such as cardiac fibroblasts and possible functions of this signalling pathway in the heart have yet to be explored. In this report the mammalian MAPK pathway is briefly outlined, before reviewing current knowledge of the MAPK pathway in cardiac tissue (ventricular myocytes, vascular smooth muscle cells and cardiac fibroblasts). New data is also presented on the presence and activity of MAPK in two additional cardiac celltypes namely atrial myocytes and vascular endothelial cells from the coronary microcirculation. (Mol Cell Biochem 157: 49–57, 1996)     

Distinct signaling properties of mitogen-activated protein kinase kinases 4 (MKK4) and 7 (MKK7) in embryonic stem cell (ESC) differentiation

Signal transduction pathways are integral components of the developmental regulatory network that guides progressive cell fate determination. MKK4 and MKK7 are upstream kinases of the mitogen-activated protein kinases (MAPKs), responsible for channeling physiological and environmental signals to their cellular responses. Both kinases are essential for survival of mouse embryos, but because of embryonic lethality, their precise developmental roles remain largely unknown. Using gene knock-out mouse ESCs, we studied the roles of MKK4 and MKK7 in differentiation in vitro. While MKK4 and MKK7 were dispensable for ESC self-renewal and pluripotency maintenance, they exhibited unique signaling and functional properties in differentiation. MKK4 and MKK7 complemented each other in activation of the JNK-c-Jun cascades and loss of both led to senescence upon cell differentiation. On the other hand, MKK4 and MKK7 had opposite effects on activation of the p38 cascades during differentiation. Specifically, MKK7 reduced p38 activation, while Mkk7(-/-) ESCs had elevated phosphorylation of MKK4, p38, and ATF2, and increased MEF2C expression. Consequently, Mkk7(-/-) ESCs had higher expression of MHC and MLC and enhanced formation of contractile cardiomyocytes. In contrast, MKK4 was required for p38 activation and Mkk4(-/-) ESCs exhibited diminished p-ATF2 and MEF2C expression, resulting in impaired MHC induction and defective cardiomyocyte differentiation. Exogenous MKK4 expression partially restored the ability of Mkk4(-/-) ESCs to differentiate into cardiomyocytes. Our results uncover complementary and interdependent roles of MKK4 and MKK7 in development, and identify the essential requirement for MKK4 in p38 activation and cardiomyocyte differentiation.     

Parallel regulation of mitogen-activated protein kinase kinase 3 (MKK3) and MKK6 in Gq-signaling cascade

Heterotrimeric G protein G(q) stimulates the activity of p38 mitogen-activated protein kinase (MAPK) in mammalian cells. To investigate the signaling mechanism whereby alpha and betagamma subunits of G(q) activate p38 MAPK, we introduced kinase-deficient mutants of mitogen-activated protein kinase kinase 3 (MKK3), MKK4, and MKK6 into human embryonal kidney 293 cells. The activation of p38 MAPK by Galpha(q) and Gbetagamma was blocked by kinase-deficient MKK3 and MKK6 but not by kinase-deficient MKK4. In addition, Galpha(q) and Gbetagamma stimulated MKK3 and MKK6 activities. The MKK3 and MKK6 activations by Galpha(q), but not by Gbetagamma, were dependent on phospholipase C and c-Src. Galpha(q) stimulated MKK3 in a Rac- and Cdc42-dependent manner and MKK6 in a Rho-dependent manner. On the other hand, Gbetagamma activated MKK3 in a Rac- and Cdc42-dependent manner and MKK6 in a Rho-, Rac-, and Cdc42-dependent manner. Gbetagamma-induced MKK3 and MKK6 activations were dependent on a tyrosine kinase other than c-Src. These results suggest that Galpha(q) and Gbetagamma stimulate the activity of p38 MAPK by regulating MKK3 and MKK6 through parallel signaling pathways.     

Blockade of the translocation and activation of mitogen-activated protein kinase kinase 4 (MKK4) signaling attenuates neuronal damage during later ischemia-reperfusion

Mitogen-activated protein kinase kinase 4 (MKK4), as an upstream activator of c-Jun NH(2)-terminal kinase (JNK), plays a critical role in response to cellular stresses and pro-inflammatory cytokines. In this study, we investigated the subcellular localization and activation of MKK4 in response to global cerebral ischemia. Our results indicated that MKK4 had two activation peaks in both the cytosol and the nucleus, and translocated from the cytosol to the nucleus at 30 min and 6 h of reperfusion. We also detected the interaction of JNK-interacting protein 3 (JIP3) and MKK4, which reached a maximum at 6 h of reperfusion. To elucidate the mechanism of translocation and activation, we administered N-acetylcysteine, an antioxidant reagent, and a glutamate receptor 6 C-terminus-containing peptide (Tat-GluR6-9c) to rats. The data showed that N-acetylcysteine limited the translocation and activation at 30 min of reperfusion; however, the peptide perturbed the subcellular localization and activation at 6 h of reperfusion, and subsequently provided a protective role against delayed neuronal cell death. Taken together, these results demonstrate that the translocation and activation of MKK4 during early reperfusion are closely associated with reactive oxygen species, whereas, at late reperfusion, MKK4 activation may be involved in brain ischemic injury.     

Mitogen-activated protein kinase (MAP kinase) activation in Xenopus oocytes: Roles of MPF and protein synthesis

Mitogen-activated protein kinase (MAP kinase) is a serine/threonine kinase whose enzymatic activity is thought to play a crucial role in mitogenic signal transduction and also in the progesterone-induced meiotic maturation of Xenopus oocytes. We have purified MAP kinase from Xenopus oocytes and have shown that the protein is present in metaphase ll oocytes under two different forms: an inactive 41-kD protein able to autoactivate and to autophosphorylate in vitro, and an active 42-kD kinase resolved into two tyrosine phosphorylated isoforms on 2D gels. During meiotic maturation, MAP kinase becomes tyrosine phosphorylated and activated following the activation of the M-phase promoting factor (MPF), a complex between the p34^cdc2 kinase and cyclin B. In vivo, MAP kinase activity displays a different stability in metaphase l and in metaphase II: protein synthesis is required to maintain MAP kinase activity in metaphase I but not in metaphase II oocytes. Injection of either MPF or cyclin B into prophase oocytes promotes tyrosine phosphorylation of MAP kinase, indicating that its activation is a downstream event of MPF activation. In contrast, injection of okadaic acid, which induces in vivo MPF activation, promotes only a very weak tyrosine phosphorylation of MAP kinase, suggesting that effectors other than MPF are required for the MAP kinase activation. Moreover, in the absence of protein synthesis, cyclin B and MPF are unable to promote in vivo activation of MAP kinase, indicating that this activation requires the synthesis of new protein(s). © 1993 Wiley-Liss, Inc.     

Mitogen-activated protein kinase in human eggs.

Mitogen-activated protein (MAP) kinase in human eggs has been investigated by using immunoblotting with both anti-Active MAPK and anti-ERK2 antibodies. The results showed that the main form of MAP kinase was p42ERK2. It was in a dephosphorylated form in oocytes at the germinal vesicle stage, but fully phosphorylated in unfertilised mature eggs. MAP kinase phosphorylation was significantly decreased when pronuclei were formed after intracytoplasmic sperm injection. Neither MAP kinase expression nor activity was detected in morphologically degenerated eggs. Although MAP kinase still existed in early embryos arrested at the 8-cell or morula stages, little, if any, activity could be detected. These data suggest that MAP kinase may play an important role in the cell cycle regulation of human eggs, as in other mammalian species.     

Mitogen-activated protein kinase cascades in plant signaling

Mitogen-activated protein kinase (MAPK) cascades are key signaling modules downstream of receptors/sensors that perceive either endogenously produced stimuli such as peptide ligands and damage-associated molecular patterns (DAMPs) or exogenously originated stimuli such as pathogen/microbe-associated molecular patterns (P/MAMPs), pathogen-derived effectors, and environmental factors. In this review, we provide a historic view of plant MAPK research and summarize recent advances in the establishment of MAPK cascades as essential components in plant immunity, response to environmental stresses, and normal growth and development. Each tier of the MAPK cascades is encoded by a small gene family, and multiple members can function redundantly in an MAPK cascade. Yet, they carry out a diverse array of biological functions in plants. How the signaling specificity is achieved has become an interesting topic of MAPK research. Future investigations into the molecular mechanism(s) underlying the regulation of MAPK activation including the activation kinetics and magnitude in response to a stimulus, the spatiotemporal expression patterns of all the components in the signaling pathway, and functional characterization of novel MAPK substrates are central to our understanding of MAPK functions and signaling specificity in plants.     

Protein kinase R (PKR) interacts with and activates mitogen-activated protein kinase kinase 6 (MKK6) in response to double-stranded RNA stimulation

The double-stranded RNA (dsRNA)-activated protein kinase R (PKR) has been invoked in different signaling pathways. In cells pre-exposed to the PKR inhibitor 2-aminopurine or in PKR-null cells, the activation of p38 mitogen-activated protein kinase (MAPK) following dsRNA stimulation is attenuated. We found that the p38 MAPK activator MKK6, but not its close relatives MKK3 or MKK4, exhibited an increased affinity for PKR following the exposure of cells to poly(rI:rC), a dsRNA analog. In vitro kinase assays revealed that MKK6 was efficiently phosphorylated by PKR, and this could be inhibited by 2-aminopurine. Expression of kinase-inactive PKR (K296R) in cells inhibited the poly(IC)-induced phosphorylation of MKK3/6 detected by phosphospecific antiserum but did not affect the poly(IC)-induced gel migration retardation of MKK3. This suggests that poly(IC)-mediated in vivo activation of MKK6, but not MKK3, is through PKR. Consistent with this observation, PKR was capable of activating MKK6 as assessed in a coupled kinase assay containing the components of the p38 MAPK pathway. Our results indicate that the interaction of MKK6 and PKR provides a mechanism for regulating p38 MAPK activation in response to dsRNA stimulation.     

Mitogen-activated protein kinase-activated protein (MAPKAP) kinase 2 deficiency protects brain from ischemic injury in mice

Mitogen-activated protein (MAP) kinase-activated protein kinase 2 (MK2) is one of several kinases directly regulated by p38 MAP kinase. A role of p38 MAP kinase in ischemic brain injury has been previously suggested by pharmacological means. In the present study, we provide evidence for a role of MK2 in cerebral ischemic injury using MK2-deficient (MK2(-/-)) mice. MK2(-/-) mice subjected to focal ischemia markedly reduced infarct size by 64 and 76% after transient and permanent ischemia, respectively, compared with wild-type mice. Furthermore, MK2(-/-) mice had significant reduction in neurological deficits. Real-time PCR analysis identified a significantly lower expression in interleukin-1beta mRNA (53% reduction) but not in tumor necrosis factor-alpha mRNA in MK2(-/-) mice over wild-type animals after ischemic injury. The significant reduction in interleukin-1beta was also confirmed in MK2(-/-) mice by enzyme-linked immunosorbent assay. The marked neuroprotection from ischemic brain injury in MK2(-/-) mice was not associated with the alteration of hemodynamic or systemic variables, activation of caspase-3, or apoptosis. Our data provide new evidence for the involvement of MAP kinase pathway in focal ischemic brain injury and suggest that this effect might be associated with the expression of interleukin-1beta in the ischemic brain tissue.     

Mitogen-activated protein kinase kinase kinase kinase 4 as a putative effector of Rap2 to activate the c-Jun N-terminal kinase

Little is known about the specific signaling roles of Rap2, a Ras family small GTP-binding protein. In a search for novel Rap2-interacting proteins by the yeast two-hybrid system, we isolated isoform 3 of the human mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4), a previously described but uncharacterized isoform. Other isoforms of MAP4K4 in humans and mice are known as hematopoietic progenitor kinase (HPK)/germinal center kinase (GCK)-like kinase and Nck-interacting kinase, respectively. MAP4K4 belongs to the STE20 group of protein kinases and regulates c-Jun N-terminal kinase (JNK). MAP4K4 interacted with Rap2 through its C-terminal citron homology domain but did not interact with Rap1 or Ras. Interaction with Rap2 required the intact effector region of Rap2. MAP4K4 interacted preferentially with GTP-bound Rap2 over GDP-bound Rap2 in vitro. In cultured cells, MAP4K4 colocalized with Rap2, while a mutant MAP4K4 lacking the citron homology domain failed to do so. Furthermore, Rap2 enhanced MAP4K4-induced activation of JNK. These results suggest that MAP4K4 is a putative effector of Rap2 mediating the activation of JNK by Rap2.     

The mixed lineage kinase DLK utilizes MKK7 and not MKK4 as substrate

Mixed lineage kinases DLK (dual leucine zipper-bearing kinase) and MLK3 have been proposed to function as mitogen-activated protein kinase kinase kinases in pathways leading to stress-activated protein kinase/c-Jun NH2-terminal kinase activation. Differences in primary protein structure place these MLK (mixed lineage kinase) enzymes in separate subfamilies and suggest that they perform distinct functional roles. Both DLK and MLK3 associated with, phosphorylated, and activated MKK7 in vitro. Unlike MLK3, however, DLK did not phosphorylate or activate recombinant MKK4 in vitro. In confirmatory experiments performed in vivo, DLK both associated with and activated MKK7. The relative localization of endogenous DLK, MLK3, MKK4, and MKK7 was determined in cells of the nervous system. Distinct from MLK3, which was identified in non-neuronal cells, DLK and MKK7 were detected predominantly in neurons in sections of adult rat cortex by immunocytochemistry. Subcellular fractionation experiments of cerebral cortex identified DLK and MKK7 in similar nuclear and extranuclear subcellular compartments. Concordant with biochemical experiments, however, MKK4 occupied compartments distinct from that of DLK and MKK7. That DLK and MKK7 occupied subcellular compartments distinct from MKK4 was confirmed by immunocytochemistry in primary neuronal culture. The dissimilar cellular specificity of DLK and MLK3 and the specific substrate utilization and subcellular compartmentation of DLK suggest that specific mixed lineage kinases participate in unique signal transduction events.     

Molecular cloning and characterization of the mitogen-activated protein kinase kinase gene (MKK4) and its promoter sequence from oilseed rape (Brassica campestris L.)

Mitogen-activated protein kinase (MAPK) cascades are involved in various processes, including plant growth and development as well as biotic and abiotic stress responses. MAPK kinases (MKKs), which link MPKs and MAPKK kinases (MKKKs), are crucial in MAPK cascades because these kinases mediate various stress responses in plants. However, only few MKKs in Brassica campestris (rape) have been functionally characterized. In this study, a novel gene, MKK4 that belongs to a C MKK group, was isolated and characterized from rape. Bioinformatics analysis revealed that the length of cDNA was 1,317 bp with an open reading frame of 993 bp, which encodes a polypeptide containing 330 amino acids, including a putative signal peptide with 27 amino acid residues and a mature protein with 303 amino acids. The obtained MKK4 exhibited a predicted molecular mass of 36.5 kDa and an isoelectric point of 9.01. Quantitative real-time polymerase chain reaction analysis revealed that MKK4 expression could be induced by cold and salt. We also found that the MKK4 protein is localized in the nucleus. In addition, a 999 bp promoter fragment of MKK4 was cloned. Sequence analysis revealed that several putative regulatory elements were found in the MKK4 promoter. Transient expression assay showed that the MKK4 promoter fragments exhibited promoter activity and stimulated GFP expression. The effects of GFP gene expression at different temperatures and in different onion epidermis culture patterns were compared. Results showed that the MKK4 promoter could respond to low temperature and salt stress. These results suggested that MKK4 is possibly important for the regulation of cold- and salt-stress responses in plants.     

MPTP activates c-Jun NH(2)-terminal kinase (JNK) and its upstream regulatory kinase MKK4 in nigrostriatal neurons in vivo

The neuropathology of Parkinson's disease is reflected in experimental animals treated with the selective nigrostriatal dopaminergic neurotoxin MPTP. Neurons exposed to MPTP (MPP(+)) express morphological features of apoptosis, although the intracellular pathways that produce this morphology have not been established. The c-Jun NH(2)-terminal kinase (JNK) signaling cascade has been implicated as a mediator of MPTP-induced apoptotic neuronal death based on the ability of CEP-1347/KT-7515, an inhibitor of JNK activation, to attenuate MPTP-induced nigrostriatal dopaminergic degeneration. In these studies, MPTP-mediated activation of the JNK signaling pathway was assessed in the nigrostriatal system of MPTP-treated mice. MPTP elevated levels of phosphorylated JNK and JNK kinase (MKK4; also known as SEK1 or JNKK), by 2.5- and fivefold, respectively. Peak elevations occurred soon after administration of MPTP and coincided with peak CNS levels of MPP(+). Increased MKK4 phosphorylation, but not JNK phosphorylation, was found in the striatum, suggesting that activation of MKK4 occurs in injured dopaminergic terminals. Both JNK and MKK4 phosphorylations were attenuated by pretreatment with l-deprenyl, indicating that these phosphorylation events were mediated by MPP(+). Moreover, CEP-1347/KT-7515 inhibited MPTP-mediated MKK4 and JNK signaling at a dose that attenuates MPTP-induced dopaminergic loss. These data implicate this signaling pathway in MPTP-mediated nigrostriatal dopaminergic death and suggest that it may be activated in the degenerative process in Parkinson's disease.     

Mitogen-activated protein kinase phosphatase (MKP)-1 in immunology, physiology, and disease

Mitogen-activated protein kinases (MAPKs) are key regulators of cellular physiology and immune responses, and abnormalities in MAPKs are implicated in many diseases. MAPKs are activated by MAPK kinases through phosphorylation of the threonine and tyrosine residues in the conserved Thr-Xaa-Tyr domain, where Xaa represents amino acid residues characteristic of distinct MAPK subfamilies. Since MAPKs play a crucial role in a variety of cellular processes, a delicate regulatory network has evolved to control their activities. Over the past two decades, a group of dual specificity MAPK phosphatases (MKPs) has been identified that deactivates MAPKs. Since MAPKs can enhance MKP activities, MKPs are considered as an important feedback control mechanism that limits the MAPK cascades. This review outlines the role of MKP-1, a prototypical MKP family member, in physiology and disease. We will first discuss the basic biochemistry and regulation of MKP-1. Next, we will present the current consensus on the immunological and physiological functions of MKP-1 in infectious, inflammatory, metabolic, and nervous system diseases as revealed by studies using animal models. We will also discuss the emerging evidence implicating MKP-1 in human disorders. Finally, we will conclude with a discussion of the potential for pharmacomodulation of MKP-1 expression.