Cloning and alternative splicing of MKK4 and MKK6

Mammalian mitogen-activated protein (MAP) kinases include extracellular signal-regulated protein kinase (ERK), c-Jun amino-terminal kinase (JNK), and p38 subgroups. These MAP kinase isoforms are activated by dual phosphorylation on threonine and tyrosine. Two human MAP kinase kinases (MKK3 and MKK4) were cloned that phosphorylate and activate p38 MAP kinase. These MKK isoforms do not activate the ERK subgroup of MAP kinases, but MKK4 does activate JNK. These data demonstrate that the activators of p38 (MKK3 and MKK4), JNK (MKK4), and ERK (MEK1 and MEK2) define independent MAP kinase signal transduction pathways (Dèrijard, 1995).

A cDNA encoding a novel member of the mitogen-activated protein kinase kinase (MAPKK) family, MAPKK6, was isolated and found to encode a protein of 334 amino acids, with a calculated molecular mass of 37 kDa that is 79% identical to MKK3. MAPKK6 phosphorylates and specifically activate the p38/MPK2 subgroup of the mitogen-activated protein kinase superfamily and could be demonstrated to be phosphorylated and activated in vitro by TAK1, a recently identified MAPKK kinase. MKK3 has also been shown to be a good substrate for TAK1 in vitro. Furthermore, when co-expressed with TAK1 in cells in culture, both MAPKK6 and MKK3 are strongly activated. In addition, co-expression of TAK1 and p38/MPK2 in cells results in activation of p38/MPK2. These results indicate the existence of a novel kinase cascade consisting of TAK1, MAPKK6/MKK3, p38/MPK2 (Moriguchi, 1996a).

A stress-activated, serine/threonine kinase, p38 (also known as HOG1 or MPK2) belongs to a subgroup of mitogen-activated protein kinase (MAPK) superfamily of molecules. The process involved in the activation of p38 (p38 activator activity) as well as p38 activity itself are both greatly stimulated by hyperosmolar media in mouse lymphoma L5178Y cells. The activator activity has been purified by sequential chromatography. A 36-kDa polypeptide that coelutes with the activity in the final chromatography step has been identified as MAPK kinase 6 (MAPKK6) by protein microsequencing analysis. Monoclonal and polyclonal antibodies raised against recombinant MAPKK6 recognize specifically the 36-kDa MAPKK6 protein but do not cross-react with MKK3 proteins. The use of these anti-MAPKK6 antibodies reveals that two major peaks of the p38 activator activity in the first chromatography step reside in the activated MAPKK6. Using a genetic screen in yeast, MKK3b, an alternatively spliced form of MKK3, was isolated. Like MKK3 and MAPKK6, MKK3b is a specific activator for p38 and is activated by osmotic shock when expressed in COS7 cells. MAPKK6 is expressed highly in HeLa and KB cells and scarcely in PC12 cells, whereas MKK3 and MKK3b were expressed in all cells examined. Immunodepletion of MAPKK6 from the extracts obtained from L5178Y cells and KB cells exposed to hyperosmolar media depletes these extracts of almost all of the p38 activator activity, indicating that MAPKK6 is a major activator for p38 in an osmosensing pathway in these cells. In addition, in KB cells MAPKK6 is activated strongly by tumor necrosis factor-alpha, H2O2, and okadaic acid and moderately by cycloheximide. Thus, there are at least three members of p38 activator (MKK3, MKK3b, and MAPKK6) and MAPKK6 may function as a major activator for p38 when expressed (Moriguchi, 1996b).

A novel cDNA species of MKK3 has been obtained, termed MKK3b. MKK3b cDNA differs from its original form, MKK3, at the 5'-end, encoding 29 extra amino acids in the N-terminus. Analysis of MKK3 genomic DNA structure reveals that the MKK3b-unique 5'-end sequence is derived from an exon different from that of MKK3, and that they share identical sequences thereafter. This suggests that the two cDNA forms of MKK3 are either generated by differential splicing of the same gene or derived from differential promoter utilization. Northern blotting analysis has showen that MKK3b mRNA is much more abundant than MKK3. Functional characterization based on the activation of p38 reveals that MKK3b is more efficient than MKK3 in mediating downstream signaling events (Han, 1997).

Signaling immediately upstream of MKK4 and MKK6

Two mammalian mitogen-activated protein kinase (MAPK)/extracellular-regulated kinase (ERK) kinase kinases have been designated MEKK2 and MEKK3. Cotransfection experiments were used to examine the regulation by MEKK2 and MEKK3 of the dual specificity MAP kinase kinases, MKK3 and MKK4. MKK3 specifically phosphorylates and activates p38, whereas MKK4 phosphorylates and activates both p38 and JNK. Coexpression of MEKK2 or MEKK3 with MKK4 in COS-7 cells results in activation of MKK4, as assessed by enhanced autophosphorylation and by MKK4's ability to phosphorylate and activate recombinant JNK1 or p38 in vitro. MKK3 autophosphorylation and activation of p38 is also observed following coexpression of MKK3 with MEKK3, but not with MEKK2. Consistent with these observations, immunoprecipitated MEKK2 directly activates recombinant MKK4 in vitro but fails to activate MKK3. The sites of activating phosphorylation in MKK3 and MKK4 were identified within kinase subdomains VII and VIII. Replacement of Ser189 or Thr193 in MKK3 with Ala abolishes autophosphorylation and activation of MKK3 by MEKK3. Analogous mutations in MKK4 indicates that Ser221 and, to a lesser extent, Thr225 are necessary for MKK4 activation by MEKK2 and MEKK3. These data indicate that MKK3 is preferentially activated by MEKK3, whereas MKK4 is activated both by MEKK2 and MEKK3. Consistent with these observations, MEKK2 and MEKK3 also activate JNK1 in vivo. However, MEKK3 fails to activate p38 when coexpressed in either the absence or presence of MKK3, indicating that MEKK3 is not coupled to p38 activation in vivo. These observations suggest that regulation of p38 and JNK1 pathways by MEKK3 may involve distinct mechanisms to prevent p38 activation but to allow JNK1 activation (Deacon, 1997).

Mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase kinase kinase 3 (MEKK3) activates the c-Jun NH2-terminal kinase (JNK) pathway, although no substrates for MEKK3 have been identified. An examination was carried out of the regulation by MEKK3 of MAPK kinase 7 (MKK7) and MKK6, two novel MAPK kinases specific for JNK and p38, respectively. Coexpression of MKK7 with MEKK3 in COS-7 cells enhances MKK7 autophosphorylation and its ability to activate recombinant JNK1 in vitro. MKK6 autophosphorylation and in vitro activation of p38alpha are also observed following coexpression of MKK6 with MEKK3. MEKK2, a closely related homolog of MEKK3, also activates MKK7 and MKK6 in COS-7 cells. Importantly, immunoprecipitates of either MEKK3 or MEKK2 directly activate recombinant MKK7 and MKK6 in vitro. These data identify MEKK3 as a MAPK kinase kinase specific for MKK7 and MKK6 in the JNK and p38 pathways. Whether MEKK3 or MEKK2 activates p38 in intact cells was examined using MAPK-activated protein kinase-2 (MAPKAPK2) as an affinity ligand and substrate. Anisomycin, sorbitol, or the expression of MEKK3 in HEK293 cells enhances MAPKAPK2 phosphorylation, whereas MEKK2 is less effective. Furthermore, MAPKAPK2 phosphorylation induced by MEKK3 or cellular stress is abolished by the p38 inhibitor SB-203580, suggesting that MEKK3 is coupled to p38 activation in intact cells (Deacon, 1999).

Mixed lineage kinase-3 (MLK-3) is a 97 kDa serine/threonine kinase with multiple interaction domains, including a Cdc42 binding motif. Cdc42 and the related small GTP binding protein Rac1 can activate the SAPK/JNK and p38/RK stress-responsive kinase cascades, suggesting that MLK-3 may have a role in upstream regulation of these pathways. In support of this role, it has been demonstrated that MLK-3 can specifically activate the SAPK/JNK and p38/RK pathways, but has no effect on the activation of ERKs. Immunoprecipitated MLK-3 catalyzes the phosphorylation of SEK1 in vitro, and co-transfected MLK-3 induces phosphorylation of SEK1 and MKK3 at sites required for activation, suggesting direct regulation of these protein kinases. Furthermore, interactions between MLK-3 and SEK and MLK-3 and MKK6 have been observed in co-precipitation experiments. Finally, kinase-dead mutants of MLK-3 block activation of the SAPK pathway by a newly identified mammalian analog of Ste20, germinal center kinase, but not by MEKK, suggesting that MLK-3 functions to activate the SAPK/JNK and p38/RK cascades in response to stimuli transduced by Ste20-like kinases (Tibbles, 1997).

A human homolog of the yeast Ssk2 and Ssk22 mitogen-activated protein kinase kinase kinases (MAPKKK) was cloned by functional complementation of the osmosensitivity of the yeast ssk2delta ssk22delta sho1delta triple mutant. This kinase, termed MTK1 (MAP Three Kinase 1), is 1607 amino acids long and is structurally highly similar to the yeast Ssk2 and Ssk22 MAPKKKs. In mammalian cells (COS-7 and HeLa), MTK1 overexpression stimulates both the p38 and JNK MAP kinase pathways, but not the ERK pathway. MTK1 overexpression also activates the MKK3, MKK6 and SEK1 MAPKKs, but not the MEK1 MAPKK. Furthermore, MTK1 phosphorylates and activates MKK6 and SEK1 in vitro. Overexpression of a dominant-negative MTK1 mutant [MTK1(K/R)] strongly inhibits the activation of the p38 pathway by environmental stresses (osmotic shock, UV and anisomycin), but not the p38 activation by the cytokine TNF-alpha. The dominant-negative MTK1(K/R) has no effect on the activation of the JNK pathway or the ERK pathway. These results indicate that MTK1 is a major mediator of environmental stresses that activate the p38 MAPK pathway, and is also a minor mediator of the JNK pathway (Takekawa, 1997).

Transforming growth factor beta (TGF-beta)-activated kinase (TAK1: see TGF-ß activated kinase 1) is known for its involvement in TGF-beta signaling and its ability to activate the p38-mitogen-activated protein kinase (MAPK) pathway. This report shows that TAK1 is also a strong activator of c-Jun N-terminal kinase (JNK). Both the wild-type and a constitutively active mutant of TAK1 stimulate JNK in transient transfection assays. Mitogen-activated protein kinase kinase 4 (MKK4)/stress-activated protein kinase/extracellular signal-regulated kinase (SEK1), a dual-specificity kinase that phosphorylates and activates JNK, synergizes with TAK1 in activating JNK. Conversely, a dominant-negative (MKK4/SEK1) mutant inhibits TAK1-induced JNK activation. A kinase defective mutant of TAK1 effectively suppresses hematopoietic progenitor kinase-1 (HPK1)-induced JNK activity but has little effect on germinal center kinase activation of JNK. There are two additional MAPK kinase kinases -- MEKK1 and mixed lineage kinase 3 (MLK3) -- that are also downstream of HPK1 and upstream of MKK4/SEK mutant. However, because the dominant-negative mutants of MEKK1 and MLK3 does not inhibit TAK1-induced JNK activity, it has been concluded that activation of JNK1 by TAK1 is independent of MEKK1 and MLK3. In addition to TAK1, TGF-beta also stimulates JNK activity. Taken together, these results identify TAK1 as a regulator in the HPK1 --> TAK1 --> MKK4/SEK1 --> JNK kinase cascade and indicate the involvement of JNK in the TGF-beta signaling pathway. These results also suggest the potential roles of TAK1 not only in the TGF-beta pathway but also in the other HPK1/JNK1-mediated pathways (W. Wang, 1997).

The p38 mitogen-activated protein kinases (MAPK) are activated by cellular stresses and play an important role in regulating gene expression. A cDNA encoding a novel protein kinase has been isolated that has significant homology (57% amino acid identity) to human p38alpha/CSBP. The novel kinase, p38delta, has a nucleotide sequence encoding a protein of 365 amino acids with a putative TGY dual phosphorylation motif. Analysis of p38delta mRNA in 50 human tissues reveals a distribution profile of p38delta that differs from p38alpha. p38delta is highly expressed in salivary gland, pituitary gland, and adrenal gland, whereas p38alpha is highly expressed in placenta, cerebellum, bone marrow, thyroid gland, peripheral leukocytes, liver, and spleen. Like p38alpha, p38delta is activated by cellular stress and proinflammatory cytokines. p38delta phosphorylates ATF-2 and PHAS-I, but not MAPK-activated protein kinase-2 and -3, known in vivo and in vitro substrates of p38alpha. p38delta is strongly activated by MKK3 and MKK6, while p38alpha is preferentially activated by MKK6. A potent p38alpha kinase inhibitor AMG 2372 minimally inhibits the kinase activity of p38delta. Taken together, these data indicate that p38delta is a new member of the p38 MAPK family and that p38delta likely has functions distinct from the function(s) of p38alpha (X. Wang, 1997).

Mitogen-activated protein (MAP) kinase cascades are activated in response to various extracellular stimuli, including growth factors and environmental stresses. A MAP kinase kinase kinase (MAPKKK), termed ASK1, was identified that activated two different subgroups of MAP kinase kinases (MAPKK), SEK1 (or MKK4) and MKK3/MAPKK6 (or MKK6), which in turn activated stress-activated protein kinase (SAPK, also known as JNK; c-Jun amino-terminal kinase) and p38 subgroups of MAP kinases, respectively. Overexpression of ASK1 induces apoptotic cell death, and ASK1 is activated in cells treated with tumor necrosis factor-alpha (TNF-alpha). Moreover, TNF-alpha-induced apoptosis is inhibited by a catalytically inactive form of ASK1. ASK1 may be a key element in the mechanism of stress- and cytokine-induced apoptosis (Ichijo, 1997).

The stress-activated p38 mitogen-activated protein kinase (p38 MAPK), a member of the subgroup of mammalian kinases, appears to play an important role in regulating inflammatory responses, including cytokine secretion and apoptosis. The upstream mediators that link extracellular signals with the p38 MAPK signaling pathway are currently unknown. pp125 focal adhesion kinase-related tyrosine kinase RAFTK (also known as PYK2, CADTK) is activated specifically by methylmethane sulfonate (MMS) and hyperosmolarity but not by ultraviolet radiation, ionizing radiation, or cis-platinum. Overexpression of RAFTK leads to the activation of p38 MAPK. Furthermore, overexpression of a dominant-negative mutant of RAFTK (RAFTK K-M) inhibits MMS-induced p38 MAPK activation. MKK3 and MKK6 are known potential constituents of p38 MAPK signaling pathway, whereas SEK1 and MEK1 are upstream activators of SAPK/JNK and ERK pathways, respectively. The dominant-negative mutant of MKK3 but not of MKK6, SEK1, or MEK1 inhibits RAFTK-induced p38 MAPK activity. Furthermore, the results demonstrate that treatment of cells with a membrane-permeable calcium chelator, inhibits MMS-induced activation of RAFTK and p38 MAPK. Taken together, these findings indicate that RAFTK represents a stress-sensitive mediator of the p38 MAPK signaling pathway in response to certain cytotoxic agents (Pandey, 1999).

TRAF6 is a signal transducer that activates IkappaB kinase (IKK) and Jun amino-terminal kinase (JNK) in response to pro-inflammatory mediators such as interleukin-1 (IL-1) and lipopolysaccharides (LPS). IKK activation by TRAF6 requires two intermediary factors, TRAF6-regulated IKK activator 1 (TRIKA1) and TRIKA2. TRIKA1 is a dimeric ubiquitin-conjugating enzyme complex composed of Ubc13 and Uev1A (or the functionally equivalent Mms2). This Ubc complex, together with TRAF6, catalyses the formation of a Lys 63 (K63)-linked polyubiquitin chain that mediates IKK activation through a unique proteasome-independent mechanism. TRIKA2 is composed of TAK1, TAB1 and TAB2, a protein kinase complex previously implicated in IKK activation through an unknown mechanism. TAK1 kinase complex phosphorylates and activates IKK in a manner that depends on TRAF6 and Ubc13-Uev1A. Moreover, the activity of TAK1 to phosphorylate MKK6, which activates the JNK-p38 kinase pathway, is directly regulated by K63-linked polyubiquitination. Evidence is provided that TRAF6 is conjugated by the K63 polyubiquitin chains. These results indicate that ubiquitination has an important regulatory role in stress response pathways, including those of IKK and JNK (Wang, 2001).

TAK1 was thought to activate IKK through the intermediary kinase NIK; however, genetic studies have shown that NIK is not involved in IKK activation by most stimuli, including IL-1ß. Thus, it is unlikely that NIK is the link between TAK1 and IKK. Instead, the data strongly suggest that TAK1 is an IKKK that phosphorylates and activates IKK in the TRAF6 pathway. How TAK1 itself is activated has been unknown. Although recombinant TAK1 can be activated by TAB1, endogenous TAK1 is inactive even though it is always associated with TAB1 and TAB2. Thus, association of TAB1 with TAK1 per se cannot account for the activation of the endogenous TAK1 complex. The results indicating that TRAF6 is ubiquitinated, and that the formation of K63-linked polyubiquitin chains directly activates the endogenous TAK1 complex in a biochemically well defined system provide a new mechanism for the activation of TAK1. According to this mechanism, stimulation of cells with IL-1ß leads to the oligomerization of TRAF6, which triggers its ubiquitination through the action of Ubc13-Uev1A. IL-1ß treatment also triggers the release of TAB2 from a membraneous location to cytoplasm, where it binds to ubiquitinated TRAF6 as well as TAK1. Exactly how TAK1 is activated by ubiquitinated TRAF6 is unclear. One possibility is that polyubiquitination may convert the TRAF6-TAB2 complex into an activator of TAK1 (for example, through a conformational change). Alternatively, the K63-linked polyubiquitin chains attached to TRAF6 may reach into TAK1 to activate it as a result of complex formation among TRAF6, TAB2 and TAK1. It is also formally possible that there is a transient but undetectable level of polyubiquitination on TAK1, and that this dynamic ubiquitination of TAK1 leads directly to its activation. Notwithstanding this uncertainty concerning the detailed mechanism of TAK1 activation, these results demonstrate a crucial role of K63-linked polyubiquitination in TAK1 activation (Wang, 2001).

Ubiquitin-dependent activation of TAK1 offers a possible solution to a conceptual problem inherent in kinase cascades: if kinase C is activated by kinase B, which is in turn activated by kinase A, how is kinase A initially activated? By definition, the first kinase cannot be activated by another kinase, so the initial activation has to be a kinase-independent mechanism. The formation of K63-linked polyubiquitin chains may provide such a mechanism, at least in the case of TAK1 activation. Activated TAK1 then phosphorylates IKK and MKK6, leading to the activation of NF-kappaB and JNK-p38 kinase pathways, respectively. These results raise the possibility that polyubiquitination through K63 of ubiquitin may be a general mechanism for the regulation of stress kinase pathways, including those of IKK and JNK (Wang, 2001).

Signaling downstream from MKK4 and MKK6

A cDNA has been cloned that encodes human stress-activated protein kinase-4 (SAPK4), a novel MAP kinase family member whose amino acid sequence is approximately 60% identical to that of the other three SAP kinases that contain a TGY motif in their activation domain. The mRNA encoding SAPK4 is widely distributed in human tissues. When expressed in KB cells, SAPK4 is activated in response to cellular stresses and pro-inflammatory cytokines, in a manner similar to other SAPKs. SAPK4 is activated in vitro by SKK3 (also called MKK6) or when co-transfected with SKK3 into COS cells. SKK3 is the only activator of SAPK4 that is induced when KB cells are exposed to a cellular stress or stimulated with interleukin-1. These findings indicate that SKK3 mediates the activation of SAPK4. The substrate specificity of SAPK4 in vitro is similar to that of SAPK3. Both enzymes phosphorylate the transcription factors ATF2, Elk-1 and SAP-1 at similar rates, but are far less effective than SAPK2a (also called RK/p38) or SAPK2b (also called p38beta) in activating MAPKAP kinase-2 and MAPKAP kinase-3. Unlike SAPK1 (also called JNK), SAPK3 and SAPK4 do not phosphorylate the activation domain of c-Jun. Unlike SAPK2a and SAPK2b, SAPK4 and SAPK3 are not inhibited by the drugs SB 203580 and SB 202190. These results suggest that cellular functions previously attributed to SAPK1 and/or SAPK2 may be mediated by SAPK3 or SAPK4 (Goedert, 1997).

Stress-activated protein kinase-3 (SAPK3), a recently described MAP kinase family member with a wide-spread tissue distribution, was transfected into several mammalian cell lines and shown to be activated in response to cellular stresses, interleukin-1 (IL-1) and tumour necrosis factor (TNF) in a similar manner to SAPK1 (also termed JNK) and SAPK2 (also termed p38, RK, CSBP and Mxi2). SAPK3 and SAPK2 are activated at similar rates in vitro by SAPKK3 (also termed MKK6), and SAPKK3 is the only activator of SAPK3 that is induced when KB or 293 cells are exposed to cellular stresses or stimulated with IL-1 or TNF. Co-transfection with SAPKK3 induces SAPK3 activity and greatly enhances activation in response to osmotic shock. These experiments indicate that SAPKK3 mediates the activation of SAPK3 in several mammalian cells. SAPK3 and SAPK2 phosphorylate a number of proteins at similar rates, including the transcription factors ATF2, Elk-1 and SAP1, but SAPK3 is far less effective than SAPK2 in activating MAPKAP kinase-2 and MAPKAP kinase-3. Unlike SAPK2, SAPK3 is not inhibited by the drug SB 203580. SAPK3 phosphorylates ATF2 at Thr69, Thr71 and Ser90, the same residues phosphorylated by SAPK1, whereas SAPK2 only phosphorylates Thr69 and Thr71. These results suggest that cellular functions previously attributed to SAPK1 and/or SAPK2 may be mediated by SAPK3 (Cuenda, 1997).

The cellular response to treatment with proinflammatory cytokines or exposure to environmental stress is mediated, in part, by the p38 group of mitogen-activated protein (MAP) kinases. A novel isoform of p38 MAP kinase, p38 beta 2 has been isolated. This p38 MAP kinase, like p38 alpha, is inhibited by the pyridinyl imidazole drug SB203580. The p38 MAP kinase kinase MKK6 is identified as a common activator of p38 alpha, p38 beta 2, and p38 gamma MAP kinase isoforms, while MKK3 activates only p38 alpha and p38 gamma MAP kinase isoforms. The MKK3 and MKK6 signal transduction pathways are therefore coupled to distinct, but overlapping, groups of p38 MAP kinases (Enslen, 1998).

In various cell types certain stresses can stimulate p38 mitogen-activated protein kinase (p38 MAPK), leading to the transcriptional activation of genes that contribute to appropriate compensatory responses. In this report the mechanism of p38-activated transcription was studied in cardiac myocytes where this MAPK is a key regulator of the cell growth and the cardiac-specific gene induction that occurs in response to potentially stressful stimuli. In the cardiac atrial natriuretic factor (ANF) gene, a promoter-proximal serum response element (SRE), which binds serum response factor (SRF), has been shown to be critical for ANF induction in primary cardiac myocytes transfected with the selective p38 MAPK activator, MKK6 (Glu). This ANF SRE does not possess sequences typically required for the binding of the Ets-related ternary complex factors (TCFs), such as Elk-1, indicating that p38-mediated induction through this element may take place independent of such TCFs. Although p38 did not phosphorylate SRF in vitro, it efficiently phosphorylates ATF6, a newly discovered SRF-binding protein that is believed to serve as a co-activator of SRF-inducible transcription at SREs. Expression of an ATF6 antisense RNA blocks p38-mediated ANF induction through the ANF SRE. Moreover, when fused to the Gal4 DNA-binding domain, an N-terminal 273-amino acid fragment of ATF6 is sufficient to support trans-activation of Gal4/luciferase expression in response to p38 but not the other stress kinase, N-terminal Jun kinase (JNK); p38-activating cardiac growth promoters also stimulate ATF6 trans-activation. These results indicate that through ATF6, p38 can augment SRE-mediated transcription independent of Ets-related TCFs, representing a novel mechanism of SRF-dependent transcription by MAP kinases (Thuerauf, 1998).

The p38 mitogen-activated protein (MAP) kinase signal transduction pathway is activated by proinflammatory cytokines and environmental stress. The detection of p38 MAP kinase in the nucleus of activated cells suggests that p38 MAP kinase can mediate signaling to the nucleus. To test this hypothesis, expression vectors were constructed for activated MKK3 and MKK6, two MAP kinase kinases that phosphorylate and activate p38 MAP kinase. Expression of activated MKK3 and MKK6 in cultured cells causes a selective increase in p38 MAP kinase activity. Cotransfection experiments demonstrate that p38 MAP kinase activation causes increased reporter gene expression mediated by the transcription factors ATF2 and Elk-1. These data demonstrate that the nucleus is one target of the p38 MAP kinase signal transduction pathway (Raingeaud, 1996).

The p38 mitogen-activated protein kinase (MAPK) is activated in vitro by three different protein kinases: MKK3, MKK4, and MKK6. To examine the relative roles of these protein kinases in the mechanism of p38 MAP kinase activation in vivo, the effect was examined of disruption of the murine Mkk3, Mkk4, and Mkk6 genes on the p38 MAPK signaling pathway. MKK3 and MKK6 are essential for tumor necrosis factor-stimulated p38 MAPK activation. In contrast, ultraviolet radiation-stimulated p38 MAPK activation is mediated by MKK3, MKK4, and MKK6. Loss of p38 MAPK activation in the mutant cells is associated with defects in growth arrest and increased tumorigenesis. These data indicate that p38 MAPK is regulated by the coordinated and selective actions of three different protein kinases in response to cytokines and exposure to environmental stress (Kelkar, 2003).

Negative regulation of MKK4 and MKK6 by phosphatases

Because phosphorylation is essential for the activation of both MAPKKs and MAPKs, protein phosphatases are likely to be important regulators of signaling through MAPK cascades. To identify protein phosphatases that negatively regulate the stress-responsive p38 and JNK MAPK cascades, human cDNA libraries were screened for genes that down-regulate the yeast HOG1 MAPK pathway (which shares similarities with the p38 and JNK pathways) using a hyperactivating yeast mutant. In this screen, the human protein phosphatase type 2Calpha (PP2Calpha) was found to negatively regulate the HOG1 pathway in yeast. Moreover, when expressed in mammalian cells, PP2Calpha inhibits the activation of the p38 and JNK cascades induced by environmental stresses. Both in vivo and in vitro observations indicate that PP2Calpha dephosphorylates and inactivates MAPKKs (MKK6 and SEK1) and a MAPK (p38) in the stress-responsive MAPK cascades. Furthermore, a direct interaction of PP2Calpha and p38 was demonstrated by a co-immunoprecipitation assay. This interaction is observed only when cells are stimulated with stresses or when a catalytically inactive PP2Calpha mutant is used, suggesting that only the phosphorylated form of p38 interacts with PP2Calpha (Takakawa, 1998)

MKK4 and MKK6 and stress

licorne Evolutionary homologs part 2/2 |

licorne: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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