As the global environment deteriorates alarmingly rapidly by pollution, it is becoming critically vital to learn more about the cellular responses to environmental stresses caused by exposures to, for example, ultraviolet radiation, genotoxins, and oxidants. There is, however, only a rudimentary understanding of the basic mechanisms by which cells respond to these environmental stresses. A conspicuous cellular stress response is activation of the stress-responsive MAP kinases (JNK and p38), which are conserved throughout the eukaryotic world, indicating their fundamental importance in cellular survival and adaptation. Our primary research goal is to elucidate the molecular mechanism of activation of the stress-responsive MAP kinase cascades, using both yeast and human cells as model systems.
1. Regulation of MTK1/MEKK4 kinase activity by its N-terminal autoinhibitory domain and GADD45 binding
Hiroaki Mita1, Junichiro Tsutsui1, Mutsuhiro Takekawa, Elizabeth A. Witten1, and Haruo Saito: 1Harvard Medical School
In eukaryotes from yeast to mammals, various cellular stresses generate intracellular signals that converge on the stress-responsive MAP kinases (MAPKs). In mammalian cells, two families of stress-responsive MAPKs, JNK and p38, are activated by stimuli such as UV radiation, ionizing radiation, hyperosmolarity, oxidative stress, and translation inhibitors, as well as cytokines such as IL-1, TNFα, and TGF-β. These MAPKs are activated by their cognate MAPK kinases (MAPKKs) through phosphorylation of conserved threonine and tyrosine residues in the kinase domain activation loop. A MAPKK is activated by specific MAPKK kinases (MAPKKKs) through phosphorylation of conserved threonine and/or serine residues. An interacting set of MAPKKKs, MAPKKs, and MAPKs constitutes a specific MAP kinase cascade. Numerous MAPKKKs are now known to function upstream of the JNK and p38 MAPKs, undoubtedly reflecting the diverse stimuli that lead to JNK and p38 activation. While it is believed that different MAPKKKs are activated by disparate mechanisms, individual mechanisms are either unknown or only vaguely understood. Human MTK1 (and its mouse homolog MEKK4) is one of MAPKKKs that act upstream of JNK and p38. To gain more insight into general principles of MAPKKK activation, we made use of our previous observation that MTK1 can be expressed functionally in yeast cells.
MTK1 has an extensive N-terminal non-catalytic domain composed of ~1300 amino acids. Full-length or near full-length MTK1 is catalytically inactive when expressed in yeast cells as is in mammalian cells. Deletion of a segment including position 253-553 activates kinase, indicating that this segment contains the auto-inhibitory domain. In the auto-inhibited conformation, the MTK1 kinase domain cannot interact with its substrate, MKK6. By a functional complementation screening using yeast, GADD45 proteins (GADD45α, β, and γ) were identified as MTK1 activators. GADD45 proteins bind a site in MTK1 near the inhibitory domain, and relieves auto-inhibition. Furthermore, 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, both in yeast and in mammalian cells. From these results, we concluded the followings: 1) the MTK1 N-terminal region contains an auto-inhibitory domain that interacts with the C-terminal kinase domain, inhibits kinase activity and prevents interaction with its substrate MKK6; 2) GADD45 proteins are MTK1 activators; and 3) Binding of GADD45 to the N-terminal region of MTK1 eliminates inhibition of the kinase domain by the auto-inhibitory domain. The isolation of constitutively active MTK1 mutants, many of which are located in the non-catalytic domain, further supports these conclusions.
2. Role of the GADD45 proteins in activation of the p38 SAPK pathway by Transforming Growth Factor-β
Mutsuhiro Takekawa, Kazuo Tatebayashi, Fumio Itoh1, Masaaki Adachi1, Kohzoh Imai1 and Haruo Saito: 1First Department of Internal Medicine, Sapporo Medical University, School of Medicine
Transforming growth factor-β (TGF-β) belongs to a family of multifunctional cytokines that regulate cell adhesion, angiogenesis, cell proliferation and apoptosis. TGF-β expression and responsiveness also regulate tumor development. TGF-β initiates its pleiotropic effects by binding a heteromeric cell surface receptor complex composed of type I and II transmembrane S/T kinase receptors. Upon ligand binding, the type II receptor phosphorylates and activates the type I receptor. Activated type I receptor initiates intracellular signaling through activation of specific Smad proteins that relay signals into the nucleus where they direct transcriptional responses. TGF-β has also been found to activate the stress-responsive p38 MAPK cascade in a variety of cell systems. The mechanism by which TGF-β activates the p38 pathway, however, yet remains to be elucidated. In order to clarify the molecular mechanisms of TGF-β-induced p38 activation, we first investigated the p38 MAPK activity in TGF-β responsive and unresponsive cell lines. We found that TGF-β-induced p38 activation did not occur in Smad4-deficient cell lines, but that reintroduction of Smad4 in these cell lines restored the p38 activation. Furthermore, expression of the constitutively active TGFβRI activated the p38 pathway, and this activation was further enhanced by co-expression of Smad proteins. Perhaps more important, overexpression of Smad proteins alone was capable of activating the p38 pathway. In addition, the p38 activation induced by the constitutively active TGFƒÀRI was strongly inhibited by expression of the dominant-negative Smad4 mutant. These findings suggest that Smad-dependent gene expression mediates the activation of the p38 pathway in response to TGF-β. In order to identify the TGF-β-inducible gene whose expression activates the p38 pathway, we investigated expression of the GADD45 family proteins (GADD45α, β, and γ), which bind to and activate MTK1 MAPKKK. We found that expression of GADD45β mRNA was specifically and efficiently induced by TGF-β in a Smad-dependent manner, and that the timing of the TGF-ƒÀ-induced p38 activation was almost parallel to that of GADD45β induction. Overexpression of GADD45β was sufficient, even in Smad4-deficient cell lines, to enhance the p38 activity, presumably through the activation of MTK1. Moreover, TGF-β-induced p38 activation was strongly inhibited by expression of dominant-negative MTK1 or anti-sense GADD45β. These findings thus suggest that TGF-β activates the p38 pathway, at least in part, through Smad-dependent transcription of GADD45β. To clarify physiological roles of TGF-β-induced p38 activation, we further screened for genes whose expression were regulated by both TGF-β-mediated signaling and the p38 pathway using a cDNA array system. We identified that expression of thrombospondin 1, a potent inhibitor of tumor cell growth and angiogenesis, is induced by TGF-β via Smad-dependent p38 activation, suggesting that the p38 pathway may play an important role in tumor suppression by TGF-β.
3. Usage of tautomycetin, a novel PP1 specific inhibitor, reveals that PP1 is a positive regulator of Raf-1 in vivo
Shinya Mitsuhashi1, Hiroshi Shima1, Nobuhiro Tanuma1, Nobuyasu Matsuura2, Mutsuhiro Takekawa, Takeshi Urano3, Tohru Kataoka4, Makoto Ubukata2, and Kunimi Kikuchi1: 1Division of Biochemical Oncology and Immunology, Institute of Genetic Medicine, Hokkaido University. 2Laboratory of Biofunctional Chemistry, Biotechnology Research Center, Toyama Prefectural University. 3Department of Biochemistry II, Nagoya University School of Medicine. 4Division of Molecular Biology, Department of Molecular and Cellular Biology, Kobe University Graduate School of Medicine.
Protein phosphatase type1 (PP1), together with protein phosphatase 2A(PP2A), is a major eukaryotic serine/threonine protein phosphatase (PP) involved in regulation of numerous cellular functions. Although roles of PP2A have been extensively studied using okadaic acid (OA), a well-known inhibitor of PP2A, biological analysis of PP1 has remained restricted due to lack of a specific inhibitor. We have recently found that tautomycetin (TC) acts as a highly specific inhibitor of PP1 phosphatase. To elucidate the biological effects of TC, we demonstrated in preliminary experiments that treatment of COS-7 cells with 5mM TC inhibited endogenous PP1 activity by more than 90% without affecting PP2A activity. Therefore, using TC as a specific PP1-inhibitor, the biological effect of PP1 on the MAP kinase (MAPK) signaling pathways was examined. First, we found that inhibition of PP1 in COS-7 cells by TC selectively suppresses activation of ERK, but not stress-responsive MAP kinases (JNK and p38). TC-mediated inhibition of PP1 also suppressed activation of Raf-1, resulting in inhibition of MEK-ERK signaling. To further examine the role of PP1 in regulation of Raf-1, we overexpressed the PP1 catalytic subunit (PP1C) in COS-7 cells. Ectopic expression of PP1C enhanced Raf-1 kinase activity, whereas a phosphatase-dead PP1C mutant blocked Raf-1 activation in vivo. Furthermore, a physical interaction between PP1C and Raf-1 was observed. These data strongly suggest that PP1 positively regulates Raf-1 in vivo.
Publications (2002-2003)