Distinct Mitogen-activated Protein Kinase Research Articles

Cytokine-Specific Activation of Distinct Mitogen-Activated Protein Kinase Subtype Cascades in Human Neutrophils Stimulated by Granulocyte Colony-Stimulating Factor, Granulocyte-Macrophage Colony-Stimulating Factor, and Tumor Necrosis Factor-α

AbstractTo clarify the differences of the signaling pathways used by granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor-α (TNF), we investigated activation of mitogen-activated protein kinase (MAPK) subtype cascades in human neutrophils stimulated by these cytokines. G-CSF exclusively tyrosine-phosphorylated extracellular signal-regulated kinase (ERK). GM-CSF tyrosine-phosphorylated ERK strongly and p38 MAPK weakly, whereas TNF tyrosine-phosphorylated p38 MAPK strongly and ERK weakly. Consistent with these findings, MEK, an upstream kinase of ERK, was phosphorylated by G-CSF, GM-CSF, and TNF, whereas MKK3/MKK6, an upstream kinase of p38 MAPK, was phosphorylated by GM-CSF and TNF, but not by G-CSF. The potency of these cytokines to phosphorylate ERK and MEK was GM-CSF > G-CSF > TNF, whereas that to phosphorylate p38 MAPK and MKK3/MKK6 was TNF > GM-CSF. C-Jun amino-terminal kinase (JNK) was not tyrosine-phosphorylated by any cytokine despite the existence of JNK proteins in human neutrophils, whereas it was tyrosine-phosphorylated by TNF in undifferentiated and all-trans retinoic acid-differentiated HL-60 cells. Increased phosphorylation of ERK or p38 MAPK was detected within 1 to 5 minutes after stimulation with each cytokine and was dependent on the concentrations of cytokines used. MEK inhibitor (PD98059) reduced tyrosine phosphorylation of ERK, but not p38 MAPK, induced by G-CSF, GM-CSF, or TNF. GM-CSF– or TNF-induced superoxide (O2−) release was inhibited by p38 MAPK inhibitor (SB203580) in a dose-dependent manner, suggesting the possible involvement of p38 MAPK in GM-CSF– or TNF-induced O2− release. The results indicate that G-CSF, GM-CSF, and TNF activate the overlapping but distinct MAPK subtype cascades in human neutrophils and suggest that the differential activation of ERK and p38 MAPK cascades may explain the differences of the effects of these cytokines on human neutrophil functions.

PTP-SL and STEP protein tyrosine phosphatases regulate the activation of the extracellular signal-regulated kinases ERK1 and ERK2 by association through a kinase interaction motif.

Protein kinases and phosphatases regulate the activity of extracellular signal-regulated kinases 1 and 2 (ERK1/2) by controlling the phosphorylation of specific residues. We report the physical and functional association of ERK1/2 with the PTP-SL and STEP protein tyrosine phosphatases (PTPs). Upon binding, the N-terminal domains of PTP-SL and STEP were phosphorylated by ERK1/2, whereas these PTPs dephosphorylated the regulatory phosphotyrosine residues of ERK1/2 and inactivated them. A sequence of 16 amino acids in PTP-SL was identified as being critical for ERK1/2 binding and termed kinase interaction motif (KIM) (residues 224-239); it was shown to be required for phosphorylation of PTP-SL by ERK1/2 at Thr253. Co-expression of ERK2 with catalytically active PTP-SL in COS-7 cells impaired the EGF-induced activation of ERK2, whereas a PTP-SL mutant, lacking PTP activity, increased the ERK2 response to EGF. This effect was dependent on the presence of the KIM on PTP-SL. Furthermore, ERK1/2 activity was downregulated in 3T3 cells stably expressing PTP-SL. Our findings demonstrate the existence of a conserved ERK1/2 interaction motif within the cytosolic non-catalytic domains of PTP-SL and STEP, which is required for the regulation of ERK1/2 activity and for phosphorylation of the PTPs by these kinases. Our findings suggest that PTP-SL and STEP act as physiological regulators of the ERK1/2 signaling pathway.

MAP kinase pathways in the yeast Saccharomyces cerevisiae.

A cascade of three protein kinases known as a mitogen-activated protein kinase (MAPK) cascade is commonly found as part of the signaling pathways in eukaryotic cells. Almost two decades of genetic and biochemical experimentation plus the recently completed DNA sequence of the Saccharomyces cerevisiae genome have revealed just five functionally distinct MAPK cascades in this yeast. Sexual conjugation, cell growth, and adaptation to stress, for example, all require MAPK-mediated cellular responses. A primary function of these cascades appears to be the regulation of gene expression in response to extracellular signals or as part of specific developmental processes. In addition, the MAPK cascades often appear to regulate the cell cycle and vice versa. Despite the success of the gene hunter era in revealing these pathways, there are still many significant gaps in our knowledge of the molecular mechanisms for activation of these cascades and how the cascades regulate cell function. For example, comparison of different yeast signaling pathways reveals a surprising variety of different types of upstream signaling proteins that function to activate a MAPK cascade, yet how the upstream proteins actually activate the cascade remains unclear. We also know that the yeast MAPK pathways regulate each other and interact with other signaling pathways to produce a coordinated pattern of gene expression, but the molecular mechanisms of this cross talk are poorly understood. This review is therefore an attempt to present the current knowledge of MAPK pathways in yeast and some directions for future research in this area.

Nerve Growth Factor Up-regulates the Transcriptional Activity of CBP through Activation of the p42/p44MAPK Cascade

Cyclic AMP response element-binding protein-binding protein (CBP) functions as a transcriptional coactivator through interactions with a number of cellular and viral transcription factors. It has been suggested to play a central integrative role in gene regulation. However, little is known about signal cascades that can regulate CBP activity. Here we show that either nerve growth factor (NGF) or cAMP treatment led to enhanced activity of CBP in PC12 cells. The C-terminal glutamine-rich activation domain of CBP was shown to be responsible for induction by NGF and cAMP. NGF-induced enhancement of CBP activity was also observed in protein kinase A (PKA)-deficient PC12 cells, whereas cAMP failed to increase the transcriptional activity of CBP in these cells. Moreover, the specific PKA inhibitor H-89 blocked cAMP-induced but not NGF-induced up-regulation of CBP activity. The up-regulation of CBP transcriptional activity in response to NGF was, however, prevented by the specific inhibitor of mitogen-activated protein kinase (p42/44(MAPK)) activation, PD98059, which had no effect on the up-regulation induced by cyclic AMP, indicating that activation of the mitogen-activated protein kinase signal pathway is specifically involved in the NGF-induced activation of CBP. In addition, expression of a dominant-negative interfering mutant of p42/44(MAPK) can prevent the NGF-mediated induction of the CBP activity, whereas expression of a p42/44(MAPK) constitutively active mutant can enhance the transcriptional activity of CBP. These data indicate that activation of the p42/p44(MAPK) cascade mediates the up-regulation of the transcriptional activity of CBP by NGF, whereas the similar up-regulation induced by cyclic AMP is mediated by PKA activation.

Different protein tyrosine kinases are required for B cell antigen receptor-mediated activation of extracellular signal-regulated kinase, c-Jun NH2-terminal kinase 1, and p38 mitogen-activated protein kinase.

B cell antigen receptor (BCR) cross-linking activates three distinct families of nonreceptor protein tyrosine kinases (PTKs): src-family kinases, Syk, and Btk; these PTKs are responsible for initiating downstream events. BCR cross-linking in the chicken DT40 B cell line also activates three distinct mitogen-activated protein kinases (MAPKs): extracellular signal-regulated kinase (ERK)2, c-jun NH2-terminal kinase (JNK)1, and p38 MAPK. To dissect the functional roles of these PTKs in MAPK signaling, activation of MAPKs was examined in various PTK-deficient DT40 cells. BCR-mediated activation of ERK2, although maintained in Lyn-deficient cells, was abolished in Syk-deficient cells and partially inhibited in Btk-deficient cells, indicating that BCR-mediated ERK2 activation requires Syk and that sustained ERK2 activation requires Btk. BCR-mediated JNK1 activation was maintained in Lyn-deficient cells but abolished in both Syk- and Btk-deficient cells, suggesting that JNK1 is activated via a Syk- and Btk-dependent pathway. Consistent with this, BCR-mediated JNK1 activation was dependent on intracellular calcium and phorbol myristate acetate-sensitive protein kinase Cs. In contrast, BCR-mediated p38 MAPK activation was detected in all three PTK-deficient cells, suggesting that no single PTK is essential. However, BCR-mediated p38 MAPK activation was abolished in Lyn/Syk double deficient cells, demonstrating that either Lyn or Syk alone may be sufficient to activate p38 MAPK. Our data show that BCR-mediated MAPK activation is regulated at the level of the PTKs.

Interleukin-1

Interleukin 1 (IL1) is a primary regulator of inflammatory and immune responses. Via its type I receptor it activates specific protein kinases, including the NFκB inducing kinase (NIK) and three distinct mitogen-activated protein (MAP) kinase cascades. These modulate a number of transcription factors including NFκB, AP1 and CREB each of which regulate a plethora of immediate early genes central to the inflammatory response. Phase I clinical trials of the soluble type I receptor and IRAP indicate that these have potential anti-inflammatory effects.

Suppression of auxin signal transduction by a MAPK cascade in higher plants.

The plant hormone auxin activates many early response genes that are thought to be responsible for diverse aspects of plant growth and development. It has been proposed that auxin signal transduction is mediated by a conserved signalling cascade consisting of three protein kinases: the mitogen-activated protein kinase (MAPK), MAPK kinase (MAPKK) and MAPKK kinase (MAPKKK). Here we show that a specific plant MAPKKK, NPK1, activates a MAPK cascade that leads to the suppression of early auxin response gene transcription. A mutation in the kinase domain abolishes NPK1 activity, and the presence of the carboxy-terminal domain diminishes the kinase activity. Moreover, the effects of NPK1 on the activation of a MAPK and the repression of early auxin response gene transcription are specifically eliminated by a MAPK phosphatase. Transgenic tobacco plants overexpressing the NPK1 kinase domain produced seeds defective in embryo and endosperm development. These results suggest that auxin sensitivity may be balanced by antagonistic signalling pathways that use a distinct MAPK cascade in higher plants.

IGF-I stimulates intestinal muscle cell growth by activating distinct PI 3-kinase and MAP kinase pathways.

Insulin-like growth factor I (IGF-I), acting via its cognate receptor, plays an autocrine role in the regulation of growth of intestinal muscle cells. In the present study the signaling pathways mediating the growth effects of IGF-I were characterized in cultured human intestinal smooth muscle cells. Growth induced by a maximally effective concentration of IGF-I (100 nM), measured as [3H]thymidine incorporation, was only partially inhibited by LY-294002 [phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor] or PD-98059 [mitogen-activated protein (MAP) kinase kinase (MEK) inhibitor] (86 +/- 7% and 35 +/- 6% inhibition, respectively) alone but was abolished by the two combined (114 +/- 18% inhibition), implying the participation of both pathways. IGF-I elicited time- and concentration-dependent increases in PI 3-kinase activity. This effect was inhibited only by LY-294002 (89 +/- 12%). IGF-I elicited time- and concentration-dependent phosphorylation of p44/p42 MAP kinase and increased MAP kinase activity. These effects were inhibited only by PD-98059 (78 +/- 9% and 98 +/- 7%, respectively). We conclude that in human intestinal muscle cells IGF-I activates distinct PI 3-kinase and MAP kinase signaling pathways, which act in conjunction to mediate growth.

Signal transduction by MAP kinase cascades in budding yeast

Budding yeast contain at least four distinct MAPK (mitogen activated protein kinase) cascades that transduce a variety of intracellular signals: mating-pheromone response, pseudohyphal/invasive growth, cell wall integrity, and high osmolarity adaptation. Although each MAPK cascade contains a conserved set of three protein kinases, the upstream activation mechanisms for these cascades are diverse, including a trimeric G protein, monomeric small G proteins, and a prokaryotic-like two-component system. Recently, it became apparent that there is extensive sharing of signaling elements among the MAPK pathways; however, little undesirable cross-talk occurs between various cascades. The formation of multi-protein signaling complexes is probably centrally important for this insulation of individual MAPK cascades.

The Phosphorylation of Eukaryotic Initiation Factor eIF4E in Response to Phorbol Esters, Cell Stresses, and Cytokines Is Mediated by Distinct MAP Kinase Pathways

Initiation factor eIF4E binds to the 5'-cap of eukaryotic mRNAs and plays a key role in the mechanism and regulation of translation. It may be regulated through its own phosphorylation and through inhibitory binding proteins (4E-BPs), which modulate its availability for initiation complex assembly. eIF4E phosphorylation is enhanced by phorbol esters. We show, using specific inhibitors, that this involves both the p38 mitogen-activated protein (MAP) kinase and Erk signaling pathways. Cell stresses such as arsenite and anisomycin and the cytokines tumor necrosis factor-alpha and interleukin-1beta also cause increased phosphorylation of eIF4E, which is abolished by the specific p38 MAP kinase inhibitor, SB203580. These changes in eIF4E phosphorylation parallel the activity of the eIF4E kinase, Mnk1. However other stresses such as heat shock, sorbitol, and H2O2, which also stimulate p38 MAP kinase and increase Mnk1 activity, do not increase phosphorylation of eIF4E. The latter stresses increase the binding of eIF4E to 4E-BP1, and we show that this blocks the phosphorylation of eIF4E by Mnk1 in vitro, which may explain the absence of an increase in eIF4E phosphorylation under these conditions.

Interaction of Transcription Factors with Serum Response Factor: IDENTIFICATION OF THE Elk-1 BINDING SURFACE

Serum response elements (SREs) play important roles in transforming extracellular signals into specific nuclear responses. The SRE-binding protein, serum response factor (SRF), plays a pivotal role in this process. Several transcription factors have been shown to interact with SRF and thereby create distinct complexes with different regulatory potentials. The ETS domain transcription factor Elk-1 is one such protein and serves to integrate distinct mitogen-activated protein kinase cascades at SREs. Elk-1 uses a short hydrophobic surface presented on the surface of an alpha-helix to interact with SRF. In this study we have used site-directed mutagenesis to identify residues in SRF that comprise the Elk-1 binding surface. The Elk-1 binding surface is composed of residues that lie on a hydrophobic surface-exposed groove located at the junction of the MADS box and C-terminal SAM motif. Different residues are implicated in interactions between SRF and the transcription factor Fli-1, indicating that although some overlap with the Elk-1 binding surface occurs, their interaction surfaces on SRF are distinct. Our data are consistent with the hypothesis that the SRF DNA-binding domain acts as docking site for multiple transcription factors that can bind to small surface-exposed patches within this domain.

Nerve growth factor activates extracellular signal-regulated kinase and p38 mitogen-activated protein kinase pathways to stimulate CREB serine 133 phosphorylation.

The mechanisms by which growth factor-induced signals are propagated to the nucleus, leading to the activation of the transcription factor CREB, have been characterized. Nerve growth factor (NGF) was found to activate multiple signaling pathways that mediate the phosphorylation of CREB at the critical regulatory site, serine 133 (Ser-133). NGF activates the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases (MAPKs), which in turn activate the pp90 ribosomal S6 kinase (RSK) family of Ser/Thr kinases, all three members of which were found to catalyze CREB Ser-133 phosphorylation in vitro and in vivo. In addition to the ERK/RSK pathway, we found that NGF activated the p38 MAPK and its downstream effector, MAPK-activated protein kinase 2 (MAPKAP kinase 2), resulting in phosphorylation of CREB at Ser-133. Inhibition of either the ERK/RSK or the p38/MAPKAP kinase 2 pathway only partially blocked NGF-induced CREB Ser-133 phosphorylation, suggesting that either pathway alone is sufficient for coupling the NGF signal to CREB activation. However, inhibition of both the ERK/RSK and the p38/MAPKAP kinase 2 pathways completely abolished NGF-induced CREB Ser-133 phosphorylation. These findings indicate that NGF activates two distinct MAPK pathways, both of which contribute to the phosphorylation of the transcription factor CREB and the activation of immediate-early genes.

Enhancement of Fibroblast Collagenase (Matrix Metalloproteinase-1)Gene Expression by Ceramide Is Mediated by Extracellular Signal-regulated and Stress-activated Protein Kinase Pathways

Inflammatory cytokines tumor necrosis factor-alpha and interleukin-1 trigger the ceramide signaling pathway, initiated by neutral sphingomyelinase-elicited hydrolysis of cell membrane phospholipid sphingomyelin to ceramide, a new lipid second messenger. Here, we show that triggering the ceramide pathway by sphingomyelinase or C2- and C6-ceramide enhances collagenase-1 (matrix metalloproteinase-1; MMP-1) gene expression by fibroblasts. C2-ceramide activates three distinct mitogen-activated protein kinases (MAPKs) in dermal fibroblasts, i.e. extracellular signal-regulated kinase 1/2 (ERK1/2), stress-activated protein kinase/Jun N-terminal-kinase (SAPK/JNK), and p38. Stimulation of MMP-1 promoter activity by C2-ceramide is dependent on the presence of a functional AP-1 cis-element and is entirely inhibited by overexpression of MAPK inhibitor, dual specificity phosphatase CL100 (MAPK phosphatase-1). Activation of MMP-1 promoter by C2-ceramide is also effectively inhibited by kinase-deficient forms of ERK1/2 kinase (MEK1/2) activator Raf-1, ERK1 and ERK2, SAPK/JNK activator SEK1, or SAPKbeta. In addition, ceramide-dependent induction of MMP-1 expression is potently prevented by PD 98059, a selective inhibitor of MEK1 activation, and by specific p38 inhibitor SB 203580. These results show that triggering the ceramide signaling pathway activates MMP-1 gene expression via three distinct MAPK pathways, i.e. ERK1/2, SAPK/JNK, and p38, and suggest that targeted modulation of the ceramide signaling pathway may offer a novel therapeutic approach for inhibiting collagenolytic activity, e.g. in inflammatory disorders.

Butylated Hydroxyanisole and Its Metabolitetert-Butylhydroquinone Differentially Regulate Mitogen-activated Protein Kinases: THE ROLE OF OXIDATIVE STRESS IN THE ACTIVATION OF MITOGEN-ACTIVATED PROTEIN KINASES BY PHENOLIC ANTIOXIDANTS

Phenolic antioxidant butylated hydroxyanisole (BHA) is a commonly used food preservative with broad biological activities, including protection against acute toxicity of chemicals, modulation of macromolecule synthesis and immune response, induction of phase II detoxifying enzymes, and especially its potential tumor-promoting activities. Understanding the molecular basis underlying these diverse biological actions of BHA is thus of great importance. Here we demonstrate that BHA is capable of activating distinct mitogen-activated protein kinases (MAPKs), extracellular signal-regulated protein kinase 2 (ERK2), and c-Jun N-terminal kinase 1 (JNK1). Activation of ERK2 by BHA was rapid and transient, whereas the JNK1 activation was relatively delayed and persistent. A major metabolite of BHA, tert-butylhydroquinone (tBHQ), also activated ERK2 but weakly stimulated JNK1 activity. Furthermore, tBHQ activation of ERK2 was late and prolonged, showing a kinetics different from that induced by BHA. ERK2 activation by both compounds required the involvement of an upstream signaling kinase MAPK/ERK kinase (MEK), as evidenced by the inhibitory effect of a MEK inhibitor, PD98059. Pretreatment with N-acetyl-L-cysteine, glutathione, or vitamin E attenuated ERK2 but not JNK1 activation by BHA and tBHQ. Modulation of intracellular H2O2 levels by direct addition of catalase or pretreatment with a catalase inhibitor, aminotriazole, also affected BHA- and tBHQ-stimulated ERK2 activity but not JNK1, indicating the involvement of oxidative stress in the ERK2 activation by these two compounds. However, we did not observe any generation of H2O2 after exposure of cells to BHA or tBHQ using a H2O2-sensitive fluorescent probe, 2',7'-dichlorofluorescein diacetate. Instead, BHA and tBHQ substantially reduced the amount of intracellular H2O2. Furthermore, BHA and tBHQ activation of ERK2 was strongly inhibited by ascorbic acid and a peroxidase inhibitor, sodium azide, suggesting the potential role of phenoxyl radicals and/or their derivatives. Taken together, our results indicate that (i) BHA and its metabolite tBHQ differentially regulate MAPK pathways, and (ii) oxidative stress due to the generation of reactive intermediates, possibly phenoxyl radicals but not H2O2, is responsible for the ERK2 activation by BHA and tBHQ, whereas the JNK1 activation may require a distinct yet unknown mechanism.

Role of p38 and JNK mitogen-activated protein kinases in the activation of ternary complex factors.

The transcription factors Elk-1 and SAP-1 bind together with serum response factor to the serum response element present in the c-fos promoter and mediate increased gene expression. The ERK, JNK, and p38 groups of mitogen-activated protein (MAP) kinases phosphorylate and activate Elk-1 in response to a variety of extracellular stimuli. In contrast, SAP-1 is activated by ERK and p38 MAP kinases but not by JNK. The proinflammatory cytokine interleukin-1 (IL-1) activates JNK and p38 MAP kinases and induces the transcriptional activity of Elk-1 and SAP-1. These effects of IL-1 appear to be mediated by Rho family GTPases. To examine the relative roles of the JNK and p38 MAP kinase pathways, we examined the effects of IL-1 on CHO and NIH 3T3 cells. Studies of NIH 3T3 cells demonstrated that both the JNK and p38 MAP kinases are required for IL-1-stimulated Elk-1 transcriptional activity, while only p38 MAP kinase contributes to IL-1-induced activation of SAP-1. In contrast, studies of CHO cells demonstrated that JNK (but not the p38 MAP kinase) is required for IL-1-stimulated Elk-1-dependent gene expression and that neither JNK nor p38 MAP kinase is required for IL-1 signaling to SAP-1. We conclude that (i) distinct MAP kinase signal transduction pathways mediate IL-1 signaling to ternary complex transcription factors (TCFs) in different cell types and (ii) individual TCFs show different responses to the JNK and p38 signaling pathways. The differential utilization of TCF proteins and MAP kinase signaling pathways represents a potential mechanism for the determination of cell-type-specific responses to extracellular stimuli.

Structure-Function Studies of p38 Mitogen-activated Protein Kinase: LOOP 12 INFLUENCES SUBSTRATE SPECIFICITY AND AUTOPHOSPHORYLATION, BUT NOT UPSTREAM KINASE SELECTION

Several mitogen-activated protein kinase (MAPK) cascades have been identified in eukaryotic cells. The activation of MAPKs is carried out by distinct MAPK kinases (MEKs or MKKs), and individual MAPKs have different substrate preferences. Here we have examined how amino acid sequences encompassing the dual phosphorylation motif located in the loop 12 linker (L12) between kinase subdomains VII and VIII and the length and amino acid sequence of L12 influence autophosphorylation, substrate specificity, and upstream kinase selectivity for the MAPK p38. Conversion of L12 of p38 to an "ERK-like" structure was accomplished in several ways: (i) by replacing glycine with glutamate in the dual phosphorylation site, (ii) by placing a six-amino acid sequence present in L12 of ERK (but absent in p38) into p38, and (iii) by mutations of amino acid residues in loop 12. Two predominant effects were noted: (i) the Xaa residue in the dual phosphorylation motif Thr-Xaa-Tyr as well as the length of L12 influence p38 substrate specificity, and (ii) the length of L12 plays a major role in controlling autophosphorylation. In contrast, these modifications do not result in any change in the selection of p38 by individual MAPK kinases.

Molecular Cloning and Functional Characterization of a Novel Mitogen-activated Protein Kinase Phosphatase, MKP-4

Extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), and p38/RK/CSBP (p38) mitogen-activated protein (MAP) kinases are target enzymes activated by a wide range of cell-surface stimuli. Recently, a distinct class of dual specificity phosphatase has been shown to reverse activation of MAP kinases by dephosphorylating critical tyrosine and threonine residues. By searching the expressed sequence tag data base (dbEST) for homologues of known dual specificity phosphatases, we identified a novel partial human sequence for which we isolated a full-length cDNA (termed MKP-4). The deduced amino acid sequence of MKP-4 is most similar to MKP-X/PYST2 (61% identity) and MKP-3/PYST1 (57% identity), includes two N-terminal CH2 domains homologous to the cell cycle regulator Cdc25 phosphatase, and contains the extended active site sequence motif VXVHCXAGXSRSXTX3AYLM (where X is any amino acid) conserved in dual specificity phosphatases. MKP-4 produced in Escherichia coli catalyzes vanadate-sensitive breakdown of p-nitrophenyl phosphate as well as in vitro inactivation of purified ERK2. When expressed in COS-7 cells, MKP-4 blocks activation of MAP kinases with the selectivity ERK > p38 = JNK/SAPK. This cellular specificity is similar to MKP-3/PYST1, although distinct from hVH-5/M3-6 (JNK/SAPK = p38 >>> ERK). Northern analysis reveals a highly restricted tissue distribution with a single MKP-4 mRNA species of approximately 2.5 kilobases detected only in placenta, kidney, and embryonic liver. Immunocytochemical analysis showed MKP-4 to be present within cytosol although punctate nuclear staining co-localizing with promyelocytic protein was also observed in a subpopulation (10-20%) of cells. Chromosomal localization by analysis of DNAs from human/rodent somatic cell hybrids and a panel of radiation hybrids assign the human gene for MKP-4 to Xq28. The identification and characterization of MKP-4 highlights the emergence of an expanding family of structurally homologous dual specificity phosphatases possessing distinct MAP kinase specificity and subcellular localization as well as diverse patterns of tissue expression.

Activation of Multiple Mitogen-activated Protein Kinase Signal Transduction Pathways by the Endothelin B Receptor Requires the Cytoplasmic Tail

Endothelin is a 21-amino acid peptide with remarkably diverse biological properties, including potent vasoconstriction, induction of mitogenesis, and a role in the development of blood vessels. In the present study, stimulation of the endothelin B receptor was found to activate three distinct mitogen-activated protein kinase signal transduction pathways, the extracellular regulated kinase (ERK) 2, c-Jun N-terminal kinase 1 (JNK), and p38 kinase. These mitogen-activated protein kinase isozymes are thought to mediate very different biological outcomes, suggesting that the observed pattern of kinases activation may be important for the diverse biological properties of endothelin. The cytoplasmic tail of the endothelin B receptor was found to be required for activation of all three mitogen-activated protein kinases and stimulation of intracellular calcium levels. An endothelin B receptor truncated at the C-terminal tail was not able to stimulate the mitogen-activated protein kinases or increase cytosolic free calcium. Furthermore, ectopic expression of the cytoplasmic tail attenuated signaling through the wild type receptor. The observed ERK activation appeared to be mediated by heterotrimeric G proteins, since ectopic expression of a transducin alpha-subunit inhibited endothelin-stimulated ERK activation. The data suggest that the cytosolic tail of the endothelin B receptor is involved in calcium mobilization and mitogen-activated protein kinase activation via a G protein-dependent mechanism.

The Dual Specificity Phosphatases M3/6 and MKP-3 Are Highly Selective for Inactivation of Distinct Mitogen-activated Protein Kinases

The mitogen-activated protein (MAP) kinase family includes extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38/RK/CSBP (p38) as structurally and functionally distinct enzyme classes. Here we describe two new dual specificity phosphatases of the CL100/MKP-1 family that are selective for inactivating ERK or JNK/SAPK and p38 MAP kinases when expressed in COS-7 cells. M3/6 is the first phosphatase of this family to display highly specific inactivation of JNK/SAPK and p38 MAP kinases. Although stress-induced activation of p54 SAPKbeta, p46 SAPKgamma (JNK1) or p38 MAP kinases is abolished upon co-transfection with increasing amounts of M3/6 plasmid, epidermal growth factor-stimulated ERK1 is remarkably insensitive even to the highest levels of M3/6 expression obtained. In contrast to M3/6, the dual specificity phosphatase MKP-3 is selective for inactivation of ERK family MAP kinases. Low level expression of MKP-3 blocks totally epidermal growth factor-stimulated ERK1, whereas stress-induced activation of p54 SAPKbeta and p38 MAP kinases is inhibited only partially under identical conditions. Selective regulation by M3/6 and MKP-3 was also observed upon chronic MAP kinase activation by constitutive p21(ras) GTPases. Hence, although M3/6 expression effectively blocked p54 SAPKbeta activation by p21(rac) (G12V), ERK1 activated by p21(ras) (G12V) was insensitive to this phosphatase. ERK1 activation by oncogenic p21(ras) was, however, blocked totally by co-expression of MKP-3. This is the first report demonstrating reciprocally selective inhibition of different MAP kinases by two distinct dual specificity phosphatases.

Stimulation of the P-450 side chain cleavage enzyme (CYP11A1) promoter through ras- and Ets-2-signaling pathways.

Expression of the ovine P-450 side-chain cleavage enzyme gene (CYP11A1) is stimulated by epidermal growth factor (EGF) through a pathway that involves c-Jun in JEG-3 placental cells. Growth factor signaling involves ras-dependent and ras-independent signaling pathways, which in turn regulate gene transcription through related but distinct mitogen-activated protein kinase pathways (MAPKs) including the extracellular signal-regulated kinases (ERKs) and the stress-activated protein kinases (SAPKs). We investigated the intracellular signaling pathways governing EGF induction of the CYP11A1 promoter. EGF stimulation of the CYP11A1 promoter (4-fold) was reduced 60% by a dominant negative mutant of ras (N17), and 30-40% by antisense ras. EGF induced both ERK and SAPK activity in JEG-3 cells. EGF-induced CYP11A1 promoter activity was reduced 60% by the MEK1 inhibitor PD098059 and 50% by a dominant negative mutant of the ERK-specific regulator MEK1. In contrast, dominant negative mutants of the SAPK-specific activator, SEK1, induced a further increase in EGF-induced CYP11A1 promoter activity. Constitutively active mutants of ras (V12 or L61) increased CYP11A1 promoter activity 6- to 8-fold. Deletion of the EGF response element (EGF-RE) between -92 and -77 bp reduced ras induction by 60%; however, a residual 3-fold induction remained through the proximal -77 bp. Mutation of the EGF-RE AP-1-like sequence in the context of the native promoter reduced CYP11A1 promoter activation by ras 60%. The EGF-RE sequence was sufficient for 6-fold activation by ras in the context of an heterologous thymidine kinase promoter. Candidate transcription factor targets (c-Jun, c-Ets-2) for the ras-signaling cascade were examined for their effects on CYP11A1 promoter activity. Overexpression of c-Jun induced the CYP11A1 promoter through the EGF-RE; however, c-Ets-2 activation of the CYP11A1 promoter (12-fold) required the proximal ras-responsive promoter sequences that are distinct from the EGF/MEK/c-Jun-responsive element. Induction of the CYP11A1 promoter by EGF involves a ras/MEK1/AP-1-dependent pathway that is distinct from induction by ras/c-Ets-2.