Regulation of MTK1/MEKK4 kinase activity by its N-terminal autoinhibitory domain and GADD45 binding

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 approximately 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 (GADD45alpha, beta, and gamma) 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.     

Loss of oncogenic H-ras-induced cell cycle arrest and p38 mitogen-activated protein kinase activation by disruption of Gadd45a

The activation of p53 is a guardian mechanism to protect primary cells from malignant transformation; however, the details of the activation of p53 by oncogenic stress are still incomplete. In this report we show that in Gadd45a(-/-) mouse embryo fibroblasts (MEF), overexpression of H-ras activates extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) but not p38 kinase, and this correlates with the loss of H-ras-induced cell cycle arrest (premature senescence). Inhibition of p38 mitogen-activated protein kinase (MAPK) activation correlated with the deregulation of p53 activation, and both a p38 MAPK chemical inhibitor and the expression of a dominant-negative p38alpha inhibited p53 activation in the presence of H-ras in wild-type MEF. p38, but not ERK or JNK, was found in a complex with Gadd45 proteins. The region of interaction was mapped to amino acids 71 to 96, and the central portion (amino acids 71 to 124) of Gadd45a was required for p38 MAPK activation in the presence of H-ras. Our results indicate that this Gadd45/p38 pathway plays an important role in preventing oncogene-induced growth at least in part by regulating the p53 tumor suppressor.     

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 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 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-alpha. 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.     

Regulation of Gadd45a mRNA expression in vascular smooth muscle under growth and stress conditions

In order to identify differentially expressed genes under growth conditions, quiescent vascular smooth muscle cells (VSMCs) were stimulated with foetal calf serum (FCS) or platelet-derived growth factor-BB (PDGF-BB) for different time periods. Analysing the gene expression by the differential display (DD) method, we identified the cDNA of the growth arrest and DNA damage inducible gene 45a (Gadd45a, also known as gadd45 and gadd45a). Treatment with FCS or PDGF-BB led to a transient down-regulating of Gadd45a expression during the G0/G1 phase and maximal expression when cells had completed division. We found that expression of p53 and BRCA1 mRNA precedes Gadd45a mRNA expression with a maximal induction in the S phase. As in smooth muscle cells, a similar pattern of the Gadd45a mRNA expression was observed in knockout Gadd45a(-/-) cultured mouse embryonic fibroblasts (MEFs). However, no differences between Gadd45a(+/+) and Gadd45a(-/-) cell lines were observed regarding their kinetics of cell division. These experiments suggest a function of Gadd45a when cells exit the cell cycle rather than when regulating the entry into the S phase.     

Activation of MTK1/MEKK4 by GADD45 through induced N-C dissociation and dimerization-mediated trans autophosphorylation of the MTK1 kinase domain

The mitogen-activated protein kinase (MAPK) module, composed of a MAPK, a MAPK kinase (MAPKK), and a MAPKK kinase (MAPKKK), is a cellular signaling device that is conserved throughout the eukaryotic world. In mammalian cells, various extracellular stresses activate two major subfamilies of MAPKs, namely, the Jun N-terminal kinases and the p38/stress-activated MAPK (SAPK). MTK1 (also called MEKK4) is a stress-responsive MAPKKK that is bound to and activated by the stress-inducible GADD45 family of proteins (GADD45alpha/beta/gamma). Here, we dissected the molecular mechanism of MTK1 activation by GADD45 proteins. The MTK1 N terminus bound to its C-terminal segment, thereby inhibiting the C-terminal kinase domain. This N-C interaction was disrupted by the binding of GADD45 to the MTK1 N-terminal GADD45-binding site. GADD45 binding also induced MTK1 dimerization via a dimerization domain containing a coiled-coil motif, which is essential for the trans autophosphorylation of MTK1 at Thr-1493 in the kinase activation loop. An MTK1 alanine substitution mutant at Thr-1493 has a severely reduced activity. Thus, we conclude that GADD45 binding induces MTK1 N-C dissociation, dimerization, and autophosphorylation at Thr-1493, leading to the activation of the kinase catalytic domain. Constitutively active MTK1 mutants induced the same events, but in the absence of GADD45.     

ASK1 regulates influenza virus infection-induced apoptotic cell death

Apoptosis occurs in influenza virus (IV)-infected cells. There are a number of mechanisms for the regulation of apoptosis. However, the molecular mechanism of IV infection-induced apoptosis is still controversial. Apoptosis signal-regulating kinase1 (ASK1) is a ubiquitously expressed mitogen-activated protein kinase kinase kinase (MAPKKK) that activates the SEK1-c-Jun N-terminal kinase (JNK) and MKK3/MKK6-p38 MAPK signaling cascades. ASK1 has been implicated in cytokine- and stress-induced apoptosis. Here, we show the following: (1) IV infection activated ASK1 and concomitantly phosphorylated JNK and p38 MAPK in human bronchial epithelial cells; (2) the activation of JNK and p38 MAPK but not extracellular-regulated kinase (ERK) in embryonic fibroblasts (MEFs) derived from ASK1 knockout mice (ASK1(-/-) MEFs) was depressed compared to MEFs derived from wild type mice (ASK1(+/+) MEFs); and (3) ASK1(-/-) MEFs were defective in IV infection-induced caspase-3 activation and cell death. These results indicate that apoptosis in IV-infected BEC is mediated through ASK1-dependent cascades.     

Gadd45alpha regulates p38-dependent dendritic cell cytokine production and Th1 differentiation

Gadd45alpha inhibits the activation of p38 by the T cell alternative pathway involving phosphorylation of p38 Tyr(323). Given that T cell p38 may play a role in Th1 development, the response to Th-skewing Ags was analyzed in Gadd45alpha(-/-) mice. Despite constitutively increased p38 activity in Gadd45alpha(-/-) T cells, the Th1 immune response to Toxoplasma gondii Ag (STAg), was diminished. In contrast to T cells, dendritic cells (DC) lacked the alternative p38 activation pathway. Gadd45alpha(-/-) DCs responded to STAg with low levels of MAP kinase cascade-dependent p38 activation, IL-12 production, and CD40 expression. Wild-type T cells transferred into Gadd45alpha(-/-) recipients had a diminished Th1 response to STAg, whereas Gadd45alpha(-/-) T cells transferred into wild-type hosts behaved normally. Therefore, Gadd45alpha has tissue-specific and opposing functions on p38 activity, and Gadd45alpha-regulated p38 activation in DCs is a critical event in Th1 polarization in vivo.     

Gadd45a interacts with aurora-A and inhibits its kinase activity

Centrosome stability is required for successful mitosis in mammalian cells. Amplification of the centrosome leads to chromosomal missegregation and generation of aneuploidy, which are closely associated with cell transformation and tumorigenesis (Doxsey, S. J. (2001) Nat. Cell Biol. 3, E105-E108; Hinchcliffe, E. H., and Sluder, G. (2001) Genes Dev. 15, 1167-1181; Pihan, G. A., Purohit, A., Wallace, J., Malhotra, R., Liotta, L., and Doxsey, S. J. (2001) Cancer Res. 61, 2212-2219). However, there are currently limited insights into mechanism(s) for this critical biological event. Here we show that Gadd45a, a DNA damage-inducible protein that is regulated by tumor suppressors p53 and BRCA1, participates in the maintenance of centrosome stability. Mouse embryonic fibroblasts derived from gadd45a knock-out mice exhibit centrosome amplification (designated as increased centrosome numbers). Introduction of exogenous Gadd45a into mouse embryonic fibroblasts isolated from gadd45a-null mice substantially restored the normal centrosome profile. In contrast to p21(waf1/cip1), which ensures coordinated initiation of centrosome, Gadd45a had no significant effect on centrosome duplication in S phase. Interestingly Gadd45a was found to physically associate with Aurora-A protein kinase, whose deregulated expression results in centrosome abnormality. Furthermore Gadd45a was demonstrated to strongly inhibit Aurora-A kinase activity and to antagonize Aurora-A-induced centrosome amplification. These findings identify a novel mechanism for Gadd45a in the maintenance of centrosome stability and broaden understandings of p53- and BRCA1-regulated signaling pathways in maintaining genomic fidelity.     

Gadd45a and Gadd45b modulate innate immune functions of granulocytes and macrophages by differential regulation of p38 and JNK signaling

Gadd45 proteins function as stress sensors in response to various physiological and environmental stressors, interacting with other cellular proteins implicated in cellular stress responses, including p38 and JNK. This study shows that mice lacking either Gadd45a or Gadd45b are defective in the recruitment of granulocytes and macrophages to the intra-peritoneal cavity following intra-peritoneal administration of the bacterial cell wall pathogen-associated molecular pattern lipopolysaccharide (LPS). Bone marrow derived granulocytes and macrophages lacking either Gadd45a or Gadd45b are shown to be impaired in their chemotactic response to LPS, as well as other inflammatory stimuli such as N-formyl-methionine-leucine-phenylalanine and IL-8. Evidence was obtained also implicating Gadd45a and Gadd45b in other myeloid innate immune functions, including reactive oxygen species production, phagocytosis, and adhesion. Gadd45a and Gadd45b activation of p38 kinase was implicated in the response of granulocytes to LPS mediated chemotaxis, whereas Gadd45a and Gadd45b curtailment of JNK activation was linked to chemotaxis of macrophages in response to LPS. Collectively, these data highlight a novel role for both Gadd45a and Gadd45b in myeloid innate immune functions by differential modulation of p38 and JNK signaling in granulocytes compared to macrophages.     

Oligomerization of human Gadd45a protein

Gadd45a is an 18-kDa acidic protein that is induced by genotoxic and certain other cellular stresses. The exact function of this protein is not known. However, there is evidence for its involvement in growth control, maintenance of genomic stability, DNA repair, cell cycle control, and apoptosis. Consistently, Gadd45a has previously been shown to interact in vitro and/or in vivo with a number of proteins playing central roles in these cellular processes: proliferating cell nuclear antigen, p21(Cip1/Waf1), Cdc2-CyclinB complex, MTK1, and histones. Adding to this complexity, we have found that Gadd45a self-associates in solution, both in vitro and when expressed in the cell. Moreover, Gadd45a can complex with the two other members of the Gadd45 family of stress-induced proteins, human Gadd45b (MyD118) and Gadd45g (CR6). Gel-exclusion chromatography, native gel electrophoretic analysis, enzyme-linked immunosorbent assay, and chemical cross-linking showed that recombinant Gadd45a forms dimeric, trimeric, and tetrameric species in vitro, the dimers being the predominant form. Deletion mutant and peptide scanning analyses suggest that Gadd45a has two self-association sites: within N-terminal amino acids 33-61 and within 40 C-terminal amino acids. Despite the low abundance of Gadd45a in the cell, oligomer-forming concentrations can probably be achieved in the foci-like nuclear structures formed by the protein upon overexpression. Evidence for a potential role of Gadd45a self-association in altering DNA accessibility on damaged nucleosomes is presented.     

Gadd45b Mediates Fas-induced Apoptosis by Enhancing the Interaction between p38 and Retinoblastoma Tumor Suppressor

Gadd45b has been known as a positive mediator of apoptosis induced by certain cytokines and oncogenes. Here, we identified Gadd45b as an effector of Fas-induced apoptosis and found that p38-mediated Rb hyperphosphorylation is one of the mechanisms of Fas-induced apoptosis in murine hepatocyte AML12 cells. Gadd45b has been shown to activate p38 through its physical interaction with MTK1 and induce apoptosis. However, in this study, we have showed that the function of Gadd45b during Fas-induced apoptosis in AML12 cells is different from that reported in previous studies. Depletion of Gadd45b expression did not inhibit the phosphorylation of p38, but it suppressed p38-mediated Rb phosphorylation and apoptosis in response to Fas stimulation by reducing the interaction between p38 and Rb. Ectopic expression of Gadd45b was sufficient to enhance this interaction. These findings suggest that Gadd45b mediates p38-induced Rb phosphorylation by enhancing the interaction between p38 and Rb during Fas-induced apoptosis in murine hepatocytes.     

Stress induces mitochondria-mediated apoptosis independent of SAPK/JNK activation in embryonic stem cells

SAPK/JNK, which belongs to the family of mitogen-activated protein kinase (MAPK), is activated by many types of cellular stresses or extracellular signals and is involved in embryonic development, immune responses, and cell survival or apoptosis. However, the physiological roles of SAPK/JNK in the signaling of stress-induced apoptosis are still controversial. To evaluate the precise function, SAPK/JNK-inactivated mouse embryonic stem (ES) cells were generated by disrupting genes of the MAPK activators, SEK1 and MKK7. Although SAPK/JNK activation by various stresses was completely abolished in sek1(-/-) mkk7(-/-) ES cells, apoptotic responses including DNA fragmentation and caspase 3 activation still occurred normally, which displays a sharp contrast to apaf1(-/-) ES cells exhibiting profound defects in the mitochondria-dependent apoptosis. These normal apoptotic responses without SAPK/JNK activation were also observed in fibroblasts derived from sek1(-/-) mkk7(-/-) ES cells. Instead, interleukin-1 beta (IL-1 beta)-induced IL-6 gene expression was greatly suppressed in sek1(-/-) mkk7(-/-) fibroblasts. These results clearly show that SAPK/JNK activation is responsible for the inflammatory cytokine-induced gene expression but not essentially required for the mitochondria-dependent apoptosis at least in ES or fibroblast-like cells, which are prototypes of all cell lineages.     

Gadd45a, a p53- and BRCA1-regulated stress protein, in cellular response to DNA damage

Mammalian cells exhibit complex, but intricate cellular responses to genotoxic stress, including cell cycle checkpoints, DNA repair and apoptosis. Inactivation of these important biological events may result in genomic instability and cell transformation, as well as alterations of therapeutic sensitivity. Gadd45a, a p53- and BRCA1-regulated stress-inducible gene, has been characterized as one of the important players that participate in cellular response to a variety of DNA damage agents. Interestingly, the signaling machinery that regulates Gadd45a induction by genotoxic stress involves both p53-dependent and -independent pathways; the later may employ BRCA1-related or MAP kinase-mediated signals. Gadd45a protein has been reported to interact with multiple important cellular proteins, including Cdc2 protein kinase, proliferating cell nuclear antigen (PCNA), p21Waf1/Cip1 protein, core histone protein and MTK/MEKK4, an up-stream activator of the JNK/SAPK pathway, indicating that Gadd45a may play important roles in the control of cell cycle checkpoint, DNA repair process, and signaling transduction. The importance of Gadd45a in maintaining genomic integrity is well manifested by the demonstration that disruption of endogenous Gadd45a in mice results in genomic instability and increased carcinogenesis. Therefore, Gadd45a appears to be an important component in the cellular defense network that is required for maintenance of genomic stability.