Phosphorylation of 1-aminocyclopropane-1-carboxylic acid synthase by MPK6, a stress-responsive mitogen-activated protein kinase, induces ethylene biosynthesis in Arabidopsis

Mitogen-activated protein kinases (MAPKs) are implicated in regulating plant growth, development, and response to the environment. However, the underlying mechanisms are unknown because of the lack of information about their substrates. Using a conditional gain-of-function transgenic system, we demonstrated that the activation of SIPK, a tobacco (Nicotiana tabacum) stress-responsive MAPK, induces the biosynthesis of ethylene. Here, we report that MPK6, the Arabidopsis thaliana ortholog of tobacco SIPK, is required for ethylene induction in this transgenic system. Furthermore, we found that selected isoforms of 1-aminocyclopropane-1-carboxylic acid synthase (ACS), the rate-limiting enzyme of ethylene biosynthesis, are substrates of MPK6. Phosphorylation of ACS2 and ACS6 by MPK6 leads to the accumulation of ACS protein and, thus, elevated levels of cellular ACS activity and ethylene production. Expression of ACS6(DDD), a gain-of-function ACS6 mutant that mimics the phosphorylated form of ACS6, confers constitutive ethylene production and ethylene-induced phenotypes. Increasing numbers of stress stimuli have been shown to activate Arabidopsis MPK6 or its orthologs in other plant species. The identification of the first plant MAPK substrate in this report reveals one mechanism by which MPK6/SIPK regulates plant stress responses. Equally important, this study uncovers a signaling pathway that modulates the biosynthesis of ethylene, an important plant hormone, in plants under stress.     

Multiple roles of the mitogen-activated protein kinase kinase/mitogen-activated protein kinase cascade in Xenopus laevis

Mitogen-activated protein kinase (MAPK) was originally identified as a serine/threonine protein kinase that is rapidly activated in response to various growth factors and tumor promoters in mammalian cultured cells. The kinase cascade including MAPK and its direct activator, MAPK kinase (MAPKK), is now believed to transmit various extracellular signals into their intracellular targets in eukaryotic cells. It has been reported that activation of MAPKK and MAPK occurs during the meiotic maturation of oocytes in several species, including Xenopus laevis . Studies with neutralizing antibodies against MAPKK, MAPK phosphatases and constitutively active MAPKK or MAPK have revealed a crucial role of the MAPKK/MAPK cascade in a number of developmental processes in Xenopus oocytes and embryos.     

Activation of salicylic acid-induced protein kinase, a mitogen-activated protein kinase, induces multiple defense responses in tobacco

The activation of mitogen-activated protein kinases (MAPKs) is one of the earliest responses in plants challenged by avirulent pathogens or cells treated with pathogen-derived elicitors. Expression of a constitutively active MAPK kinase, NtMEK2(DD), in tobacco induces the expression of defense genes and hypersensitive response-like cell death, which are preceded by the activation of two endogenous MAPKs, salicylic acid-induced protein kinase (SIPK) and wounding-induced protein kinase (WIPK). However, the roles that SIPK and WIPK each play in the process are unknown. Here we report that SIPK alone is sufficient to activate these defense responses. In tobacco leaves transiently transformed with SIPK under the control of a steroid-inducible promoter, the induction of SIPK expression after the application of dexamethasone, a steroid, leads to an increase of SIPK activity. The increase of SIPK activity is dependent on the phosphorylation of newly synthesized SIPK by its endogenous upstream kinase. In contrast, the expression of WIPK under the same conditions fails to increase its activity, even though the protein accumulates to a similar level. Studies using chimeras of SIPK and WIPK demonstrated that the C terminus of SIPK contains the molecular determinant for its activation, which is rather surprising because the N termini of SIPK and WIPK are more divergent. SIPK has been implicated previously in the regulation of both plant defense gene activation and hypersensitive response-like cell death based on evidence from pharmacological studies using kinase inhibitors. This gain-of-function study provided more direct evidence for its role in the signaling of multiple defense responses in tobacco.     

Activation of protein kinase C induces mitogen-activated protein kinase dephosphorylation and pronucleus formation in rat oocytes

Mammalian oocytes are arrested at metaphase of the second meiotic division (MII) before fertilization. When oocytes are stimulated by spermatozoa, they exit MII stage and complete meiosis. It has been suggested that an immediate increase in intracellular free calcium concentration and inactivation of maturation promoting factor (MPF) are required for oocyte activation. However, the underlying mechanism is still unclear. In the present study, we investigated the role of protein kinase C (PKC) and mitogen-activated protein (MAP) kinase, and their interplay in rat oocyte activation. We found that MAP kinase became dephosphorylated in correlation with pronucleus formation after fertilization. Protein kinase C activators, phorbol 12-myriatate 13-acetate (PMA) and 1,2-dioctanoyl-rac-glycerol (diC8), triggered dephosphorylation of MAP kinase and pronucleus formation in a dose-dependent and time-dependent manner. Dephosphorylation of MAP kinase was also correlated with pronucleus formation when oocytes were treated with PKC activators. Effects of PKC activators were abolished by the PKC inhibitors, calphostin C and staurosporine, as well as a protein phosphatase blocker, okadaic acid (OA). These results suggest that PKC activation may cause rat oocyte pronucleus formation via MAP kinase dephosphorylation, which is probably mediated by OA-sensitive protein phosphatases. We also provide evidence supporting the involvement of such a process in fertilization.     

Activation of anaplastic lymphoma kinase receptor tyrosine kinase induces neuronal differentiation through the mitogen-activated protein kinase pathway

Anaplastic lymphoma kinase (ALK) is a novel neuronal orphan receptor tyrosine kinase that is essentially and transiently expressed in specific regions of the central and peripheral nervous systems, suggesting a role in its normal development and function. To determine whether ALK could play a role in neuronal differentiation, we established a model system that allowed us to mimic the normal activation of this receptor. We expressed, in PC12 cells, a chimeric protein in which the extracellular domain of the receptor was replaced by the mouse IgG 2b Fc domain. The Fc domain induced the dimerization and oligomerization of the chimeric protein leading to receptor phosphorylation and activation, thus mimicking the effect of ligand binding, whereas the wild type ALK remained as a monomeric nonphosphorylated protein. Expression of the chimera, but not that of the wild type ALK or of a kinase inactive form of the chimera, induced the differentiation of PC12 cells. Analysis of the signaling pathways involved in this process pointed to an essential role of the mitogen-activated protein kinase cascade. These results are consistent with a role for ALK in neuronal differentiation.     

Identification of Cdc37 as a novel regulator of the stress-responsive mitogen-activated protein kinase

Eukaryotic cells utilize multiple mitogen-activated protein kinases (MAPKs) to transmit various extracellular stimuli to the nucleus. A subfamily of MAPKs that mediates environmental stress stimuli is also called stress-activated protein kinase (SAPK), which has crucial roles in cellular survival under stress conditions as well as inflammatory responses. Here we report that Cdc37, an evolutionarily conserved kinase-specific chaperone, is a positive regulator of Spc1 SAPK in the fission yeast Schizosaccharomyces pombe. Through a genetic screen, we have identified cdc37 as a mutation that compromises signaling through Spc1 SAPK. The Cdc37 protein physically interacts with Spc1, and the cdc37 mutation affects both the cellular level of the Spc1 protein and stress-induced Spc1 phosphorylation by Wis1 MAPK kinase (MAPKK). Consistently, expression of the stress response genes regulated by the Spc1 pathway is compromised in cdc37 mutant cells. On the other hand, a mutation in Hsp90, which often cooperates with Cdc37 in chaperoning protein kinases, does not affect Spc1 SAPK. These results suggest that Spc1 SAPK is a novel client protein for the Cdc37 chaperone, and the Cdc37 function is important to maintain the stability of the Spc1 protein and to facilitate stress signaling from Wis1 MAPKK to Spc1 SAPK.     

Activation of p38 mitogen-activated protein kinase/cytosolic phospholipase A2 cascade in hydroperoxide-stressed platelets

12-Hydroperoxy-eicosatetraenoic acid (12-HpETE), the main hydroperoxide formed in platelets from arachidonic acid (AA) by 12-lipoxygenase, has been shown to increase the sensitivity of platelets to agonists resulting in increased aggregation. The aim of the present study was to determine the direct effect of low concentrations of 12-HpETE on the signaling pathways leading to AA release from membrane phospholipids and thromboxane A2 (TxA2) formation. Exogenous 12-HpETE activated platelet p38 mitogen-activated protein kinase (p38 MAPK), as assessed by its phosphorylation, at a concentration as low as 100 nM and was much more potent than hydrogen peroxide. Moreover, the incubation of platelets with 100 nM 12-HpETE for 2 min led to the phosphorylation of cytosolic phospholipase A2 (cPLA2). It was associated with a significant decrease in the concentration of AA esterified in phospholipids and an increased concentration of thromboxane B2, the stable catabolite of TxA2. Additionally, decreasing glutathione peroxidase activity pharmacologically favored endogenous 12-HpETE formation and led to an increase in phosphorylated p38 MAPK, while a thiol-reducing agent such as N-acetyl-cysteine fully prevented it. Finally, significant activation of p38 MAPK was also observed in platelets from type 2 diabetic patients with mild hyperglycemia. In conclusion, our data provide a new insight into the mechanism of 12-HpETE-induced platelet priming, suggesting that hydroperoxide-induced p38 MAPK activation could play a relevant role in the exacerbated platelet activation associated with oxidative stress as found in diabetes.     

A mechanistic study of evolvability using the mitogen-activated protein kinase cascade

Evolvability is a function of the way genetic variation interacts with the mechanisms that produce the phenotype. We explore an explicitly mechanistic way of studying the evolvability of phenotypes that are produced by a relatively simple genetic mechanism, the mitogen-activated protein kinase (MAPK) cascade. We developed a quantitative model of MAPK activation that can be used to study the effects of genetic variation on the various components of this signaling cascade. We show how some standard tools of applied mathematics, such as steady-state formulations and nondimensionalization, can be used to elucidate the relative importance of variation in each gene of this mechanism. We also give insights into non-intuitive patterns of dependence and trade-off among the genes. The mechanism produces several different phenotypes (ultrasensitivity to stimulation, switch-like behavior, amount of MAPK-PP delivered, persistence of MAPK-PP activity), each of which is sensitive to different (but partially overlapping) combinations of genes. We show that the mechanism imposes clear limitations on the evolvability of each of the different phenotypes of the pathway, even in the presence of genetic variation in the components of the mechanism. This approach to the study of evolvability is generally applicable and complements the traditional approach through statistical genetics by providing a mechanistic understanding of the genetic interactions that produce the phenotype.     

Regulation of stress-responsive mitogen-activated protein (MAP) kinase pathways by TAO2

Previous studies demonstrated that in vitro the protein kinase TAO2 activates MAP/ERK kinases (MEKs) 3, 4, and 6 toward their substrates p38 MAP kinase and c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK). In this study, we examined the ability of TAO2 to activate stress-sensitive MAP kinase pathways in cells and the relationship between activation of TAO2 and potential downstream pathways. Over-expression of TAO2 activated endogenous JNK/SAPK and p38 but not ERK1/2. Cotransfection experiments suggested that TAO2 selectively activates MEK3 and MEK6 but not MEKs 1, 4, or 7. Coimmunoprecipitation demonstrated that endogenous TAO2 specifically associates with MEK3 and MEK6 providing one mechanism for preferential recognition of MEKs upstream of p38. Sorbitol, and to a lesser extent, sodium chloride, Taxol, and nocodazole increased TAO2 activity toward itself and kinase-dead MEKs 3 and 6. Activation of endogenous TAO2 during differentiation of C2C12 myoblasts paralleled activation of p38 but not JNK/SAPK, consistent with the idea that TAO2 is a physiological regulator of p38 under certain circumstances.     

Role of a mitogen-activated protein kinase cascade in ion flux-mediated turgor regulation in fungi

Fungi normally maintain a high internal hydrostatic pressure (turgor) of about 500 kPa. In response to hyperosmotic shock, there are immediate electrical changes: a transient depolarization (1 to 2 min) followed by a sustained hyperpolarization (5 to 10 min) prior to turgor recovery (10 to 60 min). Using ion-selective vibrating probes, we established that the transient depolarization is due to Ca²⁺ influx and the sustained hyperpolarization is due to H⁺ efflux by activation of the plasma membrane H⁺-ATPase. Protein synthesis is not required for H⁺-ATPase activation. Net K⁺ and Cl⁻ uptake occurs at the same time as turgor recovery. The magnitude of the ion uptake is more than sufficient to account for the osmotic gradients required for turgor to return to its original level. Two osmotic mutants, os-1 and os-2, homologs of a two-component histidine kinase sensor and the yeast high osmotic glycerol mitogen-activated protein (MAP) kinase, respectively, have lower turgor than the wild type and do not exhibit the sustained hyperpolarization after hyperosmotic treatment. The os-1 mutant does not exhibit all of the wild-type turgor-adaptive ion fluxes (Cl⁻ uptake increases, but net K⁺ flux barely changes and net H⁺ efflux declines) (os-2 was not examined). Both os mutants are able to regulate turgor but at a lower level than the wild type. Our results demonstrate that a MAP kinase cascade regulates ion transport, activation of the H⁺-ATPase, and net K⁺ and Cl⁻ uptake during turgor regulation. Other pathways regulating turgor must also exist.     

Inhibition of mitogen-activated protein kinase kinase induces apoptosis of human chondrocytes

We previously have reported that the mitogen-activated protein kinase (MAPK) pathway is stimulated by adhesion of human chondrocytes to anti-beta(1)-integrin antibodies or collagen type II in vitro. These mechanisms most likely prevent chondrocyte dedifferentiation to fibroblast-like cells and chondrocyte death. To investigate whether this pathway plays an essential role for the differentiation, phenotype, and survival of chondrocytes, we blocked mitogen-activated protein kinase/extracellular signal-regulated kinase (Erk) (MEK), a kinase upstream of the kinase Erk by using U0126. Exposure of chondrocytes to U0126 caused activation of caspase-3 in a dose-dependent manner. Western blot analysis with an antibody specific for dually phosphorylated Erk shows that collagen type II induced phosphorylation of Erk1/2 was specifically blocked by U0126 in a dose-dependent manner. Immunohistochemical analysis showed that treated chondrocytes were caspase-3 positive. In treated chondrocytes, the cleavage of 116-kDa poly(ADP-ribose)polymerase resulted in the 85-kDa apoptosis-related cleavage fragment and was associated with caspase-3 activity. Analysis by electron microscopy showed typical morphological signs of apoptosis, such as crescent-shaped clumps of heterochromatin, and a degraded pericellular matrix. Thus, these results indicate that the MEK/Erk signal transduction pathway is involved in the maintenance of chondrocytes differentiation and survival. These data stimulate further investigations on the role of mitogen-activated protein kinase pathways in human chondrocytes.     

Galphaq-dependent activation of mitogen-activated protein kinase kinase 4/c-Jun N-terminal kinase cascade.

G-protein-coupled receptors (GPCRs) typically activate c-Jun N-terminal kinase (JNK) through the G protein betagamma subunit (Gbetagamma), in a manner dependent on Rho family small GTPases, in mammalian cells. Here we show that JNK activation by the prototypic Gq-coupled alpha1B-adrenergic receptor is mediated by the alpha subunit of Gq (Galphaq), not by Gbetagamma, using a transient transfection system in human embryonic kidney cells. JNK activation by the alpha1B-adrenergic receptor/Galphaq was selectively mediated by mitogen-activated protein kinase kinase 4 (MKK4), but not MKK7. Also, MKK4 activation by the alpha1B-adrenergic receptor/Galphaq required c-Src and Rho family small GTPases. Furthermore, activation of the alpha1B-adrenergic receptor stimulated JNK activity through Src family tyrosine kinases and Rho family small GTPases in hamster smooth muscle cells that natively express the alpha1B-adrenergic receptor. Together, these results suggest that the alpha1B-adrenergic receptor/Galphaq may up-regulate JNK activity through a MKK4 pathway dependent on c-Src and Rho family small GTPases in mammalian cells.