Mutsuhiro Takekawa

A Family of Stress-Inducible GADD45-like Proteins Mediate Activation of the Stress-Responsive MTK1/MEKK4 MAPKKK

The stress-responsive p38 and JNK MAPK pathways regulate cell cycle and apoptosis. A human MAPKKK, MTK1 (= MEKK4), mediates activation of both p38 and JNK in response to environmental stresses. Using a yeast two-hybrid method, three related proteins, GADD45alpha (= GADD45), GADD45, (= MyD118), and GADD45gamma, were identified that bound to an N-terminal domain of MTK1. These proteins activated MTK1 kinase activity, both in vivo and in vitro. The GADD45-like genes are induced by environmental stresses, including MMS, UV, and gamma irradiation. Expression of the GADD45-like genes induces p38/JNK activation and apoptosis, which can be partially suppressed by coexpression of a dominant inhibitory MTK1 mutant protein. We propose that the GADD45-like proteins mediate activation of the p38/JNK pathway, via MTK1/ MEKK4, in response to environmental stresses.

NF-κB inhibits TNF-induced accumulation of ROS that mediate prolonged MAPK activation and necrotic cell death

NF‐κB downregulates tumor necrosis factor (TNF)‐induced c‐Jun N‐terminal kinase (JNK) activation that promotes cell death, but the mechanism is not yet fully understood. By using murine embryonic fibroblasts (MEFs) that are deficient in TNF receptor‐associated factor (TRAF) 2 and TRAF5 (DKO) or p65 NF‐κB subunit (p65KO), we demonstrate here that TNF stimulation leads to accumulation of reactive oxygen species (ROS), which is essential for prolonged mitogen‐activated protein kinase (MAPK) activation and cell death. Interestingly, dying cells show necrotic as well as apoptotic morphological changes as assessed by electron microscopy and flow cytometry, and necrotic, but not apoptotic, cell death is substantially inhibited by antioxidant. Importantly, TNF does not induce ROS accumulation or prolonged MAPK activation in wild‐type MEFs, indicating that TRAF‐mediated NF‐κB activation normally suppresses the TNF‐induced ROS accumulation that subsequently induces prolonged MAPK activation and necrotic cell death

p53-inducible Wip1 phosphatase mediates a negative feedback regulation of p38 MAPK-p53 signaling in response to UV radiation

The stress‐responsive p38 MAPK, when activated by genotoxic stresses such as UV radiation, enhances p53 activity by phosphorylation and leads to cell cycle arrest or apoptosis. Here we report that a member of the protein phosphatase type 2C family, Wip1, has a role in down‐regulating p38‐p53 signaling during the recovery phase of the damaged cells. Wip1 was originally identified as a gene whose expression is induced following γ or UV radiation in a p53‐dependent manner. We found that Wip1 is also inducible by other environmental stresses, such as anisomycin, H2O2 and methyl methane sulfonate. UV‐induction of Wip1 requires p38 activity in addition to the wild‐type p53. Wip1 selectively inactivates p38 by specific dephosphorylation of its conserved threonine residue. Furthermore, Wip1 expression attenuates UV‐induced p53 phosphorylation at Ser33 and Ser46, residues previously reported to be phosphorylated by p38. Wip1 expression also suppresses both p53‐mediated transcription and apoptosis in response to UV radiation. These results suggest that p53‐dependent expression of Wip1 mediates a negative feedback regulation of p38‐p53 signaling and contributes to suppression of the UV‐induced apoptosis.

Formation of stress granules inhibits apoptosis by suppressing stress-responsive MAPK pathways

When confronted with environmental stress, cells either activate defence mechanisms to survive, or initiate apoptosis, depending on the type of stress. Certain types of stress, such as hypoxia, heatshock and arsenite (type 1 stress), induce cells to assemble cytoplasmic stress granules (SGs), a major adaptive defence mechanism. SGs are multimolecular aggregates of stalled translation pre-initiation complexes that prevent the accumulation of mis-folded proteins. Type 2 stress, which includes X-rays and genotoxic drugs, induce apoptosis through the stress-activated p38 and JNK MAPK (SAPK) pathways. A functional relationship between the SG and SAPK responses is unknown. Here, we report that SG formation negatively regulates the SAPK apoptotic response, and that the signalling scaffold protein RACK1 functions as a mediator between the two responses. RACK1 binds to the stress-responsive MTK1 MAPKKK and facilitates its activation by type 2 stress; however, under conditions of type 1 stress, RACK1 is sequestered into SGs. Thus, type 1 conditions suppress activation of the MTK1–SAPK pathway and apoptosis induced by type 2 stress. These findings may be relevant to the problem of hypoxia-induced resistance to cancer chemotherapy.

Protein phosphatase 2Cα inhibits the human stress‐responsive p38 and JNK MAPK pathways

MAPK (mitogen‐activated protein kinase) cascades are common eukaryotic signaling modules that consist of a MAPK, a MAPK kinase (MAPKK) and a MAPKK kinase (MAPKKK). 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, we screened human cDNA libraries for genes that down‐regulated 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 2Cα (PP2Cα) was found to negatively regulate the HOG1 pathway in yeast. Moreover, when expressed in mammalian cells, PP2Cα inhibited the activation of the p38 and JNK cascades induced by environmental stresses. Both in vivo and in vitro observations indicated that PP2Cα dephosphorylated and inactivated MAPKKs (MKK6 and SEK1) and a MAPK (p38) in the stress‐responsive MAPK cascades. Furthermore, a direct interaction of PP2Cα and p38 was demonstrated by a co‐immunoprecipitation assay. This interaction was observed only when cells were stimulated with stresses or when a catalytically inactive PP2Cα mutant was used, suggesting that only the phosphorylated form of p38 interacts with PP2Cα.

A human homolog of the yeast Ssk2/Ssk22 MAP kinase kinase kinases, MTK1, mediates stress-induced activation of the p38 and JNK pathways.

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 ssk2Δ ssk22Δ sho1Δ 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 stimulated both the p38 and JNK MAP kinase pathways, but not the ERK pathway. MTK1 overexpression also activated the MKK3, MKK6 and SEK1 MAPKKs, but not the MEK1 MAPKK. Furthermore, MTK1 phosphorylated and activated MKK6 and SEK1 in vitro. Overexpression of a dominant‐negative MTK1 mutant [MTK1(K/R)] strongly inhibited the activation of the p38 pathway by environmental stresses (osmotic shock, UV and anisomycin), but not the p38 activation by the cytokine TNF‐α. The dominant‐negative MTK1(K/R) had 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.

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.

Smad‐dependent GADD45β expression mediates delayed activation of p38 MAP kinase by TGF‐β

Transforming growth factor‐β (TGF‐β), when bound to its specific receptor, activates the transcription factor Smad by phosphorylation. TGF‐β also activates the p38 MAPK pathway, but there seem to be disparate mechanisms for the early p38 activation and delayed p38 activation. In this report, we demonstrate that Smad‐dependent expression of GADD45β is responsible for the delayed activation of p38 by TGF‐β. The GADD45β protein binds and activates MTK1 (= MEKK4), which is a member of the MAPKKK family kinases and an upstream activator of the p38 MAPK cascade. Both TGF‐β‐induced GADD45β expression and the delayed p38 activation require functional Smad proteins. Antisense inhibition of GADD45β expression suppresses the TGF‐β‐induced delayed p38 activation, whereas overexpression of GADD45β activates the p38 MAPK via MTK1. Expression of the angiogenesis inhibitor thrombospondin‐1 (TSP‐1) is induced by TGF‐β via Smad‐dependent p38 activation. Thus TGF‐β‐induced p38 activation, mediated by GADD45β expression, may play an important role in the biological effects of TGF‐β.

Regulation of MTK1/MEKK4 Kinase Activity by Its N-Terminal Autoinhibitory Domain and GADD45 Binding

ABSTRACT A variety of cellular stresses activate the stress-responsive mitogen-activated protein (MAP) kinases p38 and JNK. In this study, we studied the activation mechanism of a human MAP kinase kinase kinase, MTK1 (also known as MEKK4), which mediates activation of both p38 and JNK. MTK1 has an extensive N-terminal noncatalytic domain composed of ∼1,300 amino acids. Full-length or near full-length MTK1 is catalytically inactive when expressed in Saccharomyces cerevisiae cells, as it is in mammalian cells. Deletion of a segment including positions 253 to 553 activates kinase, indicating that this segment contains the autoinhibitory domain. In the autoinhibited conformation, the MTK1 kinase domain cannot interact with its substrate, MKK6. By a functional complementation screening with yeast cells, GADD45 proteins (GADD45α, β, and γ) were identified as MTK1 activators. GADD45 proteins bind a site in MTK1 near the inhibitory domain and relieve autoinhibition. Mutants of full-length MTK1 were isolated that can interact with MKK6 in the absence of the activator GADD45 proteins. These MTK1 mutants are constitutively active, in both yeast and mammalian cells. A model of MTK1 autoinhibition by the N-terminal inhibitory domain and activation by GADD45 binding is presented.

GADD45β/GADD45γ and MEKK4 comprise a genetic pathway mediating STAT4-independent IFNγ production in T cells

The stress‐inducible molecules GADD45β and GADD45γ have been implicated in regulating IFNγ production in CD4 T cells. However, how GADD45 proteins function has been controversial. MEKK4 is a MAP kinase kinase kinase that interacts with GADD45 in vitro. Here we generated MEKK4‐deficient mice to define the function and regulation of this pathway. CD4 T cells from MEKK4−/− mice have reduced p38 activity and defective IFNγ synthesis. Expression of GADD45β or GADD45γ promotes IFNγ production in MEKK4+/+ T cells, but not in MEKK4−/− cells or in cells treated with a p38 inhibitor. Thus, MEKK4 mediates the action of GADD45β and GADD45γ on p38 activation and IFNγ production. During Th1 differentiation, the GADD45β/GADD45γ/MEKK4 pathway appears to integrate upstream signals transduced by both T cell receptor and IL12/STAT4, leading to augmented IFNγ production in a process independent of STAT4.