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gadd45 Is Not Required for Activation of c-Jun N-terminal Kinase or p38 during Acute Stress

1999; Elsevier BV; Volume: 274; Issue: 42 Linguagem: Inglês

10.1074/jbc.274.42.29599

1083-351X

Xiantao Wang, Myriam Gorospe, Nikki J. Holbrook,

Computational Drug Discovery Methods

Cells respond to environmental stress with activation of c-Jun N-terminal kinase (JNK) and p38. Recent studies have implicated Gadd45 and two related proteins, MyD118/Gadd45β and CR6/Gadd45γ, as initiators of JNK/p38 signaling via their interaction with an upstream kinase MTK1. It was proposed that stress-induced expression of the Gadd45-related proteins leads to MTK1 activation and subsequent JNK/p38 activation. Using embryo fibroblasts fromgadd45-null mice, we have addressed the requirement for Gadd45 in mediating JNK/p38 activation during acute stress. Comparison of JNK/p38 activities in response to methyl methanesulfonate, hydrogen peroxide, UVC irradiation, sorbitol, and anisomycin treatment ofgadd45 +/+ andgadd45 −/− fibroblasts revealed no deficiency in JNK/p38 activation in gadd45 −/−fibroblasts. In addition, in wild type cells, JNK and p38 activation significantly preceded gadd45 induction with all stresses. Examination of myd118/gadd45β andcr6/gadd45γ expression ingadd45 +/+ andgadd45 −/− fibroblasts revealed similar induction patterns in the two cell types, which, likegadd45 expression, was delayed relative to JNK/p38 activation. We conclude that gadd45 expression is not required for activation of JNK/p38 by environmental stresses, nor are stress-induced increases in myd118/gadd45β andcr6/gadd45γ expression necessary for kinase activation in response to such insults. Cells respond to environmental stress with activation of c-Jun N-terminal kinase (JNK) and p38. Recent studies have implicated Gadd45 and two related proteins, MyD118/Gadd45β and CR6/Gadd45γ, as initiators of JNK/p38 signaling via their interaction with an upstream kinase MTK1. It was proposed that stress-induced expression of the Gadd45-related proteins leads to MTK1 activation and subsequent JNK/p38 activation. Using embryo fibroblasts fromgadd45-null mice, we have addressed the requirement for Gadd45 in mediating JNK/p38 activation during acute stress. Comparison of JNK/p38 activities in response to methyl methanesulfonate, hydrogen peroxide, UVC irradiation, sorbitol, and anisomycin treatment ofgadd45 +/+ andgadd45 −/− fibroblasts revealed no deficiency in JNK/p38 activation in gadd45 −/−fibroblasts. In addition, in wild type cells, JNK and p38 activation significantly preceded gadd45 induction with all stresses. Examination of myd118/gadd45β andcr6/gadd45γ expression ingadd45 +/+ andgadd45 −/− fibroblasts revealed similar induction patterns in the two cell types, which, likegadd45 expression, was delayed relative to JNK/p38 activation. We conclude that gadd45 expression is not required for activation of JNK/p38 by environmental stresses, nor are stress-induced increases in myd118/gadd45β andcr6/gadd45γ expression necessary for kinase activation in response to such insults. mitogen-activated protein kinase c-Jun N-terminal kinase MAPK kinase MAPKK kinase hemagglutinin methyl methanesulfonate short-wavelength ultraviolet light glutathione S-transferase mouse embryo fibroblasts 4-morpholinepropanesulfonic acid gadd45 (also referred to as gadd45α) was first described by Fornace et al. (1Fornace Jr., A.J. Alamo Jr., I. Hollander M.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8800-8804Crossref PubMed Scopus (560) Google Scholar, 2Fornace Jr., A.J. Nebert D.W. Hollander M.C. Luethy J.D. Papathanasiou M. Fargnoli J. Holbrook N.J. Mol. Cell. Biol. 1989; 9: 4196-4203Crossref PubMed Scopus (647) Google Scholar) as showing increased expression in response to DNA-damaging agents and other stresses associated with growth arrest. Although its precise function remains unclear, Gadd45 has been implicated in a variety of growth regulatory mechanisms, including DNA replication, DNA repair, G2/M checkpoint control, and apoptosis (3Kastan M.B. Zhan Q. El-Deiry W.S. Carrier F. Jacks T. Walsh W.V. Plunkett B.S. Vogelstein B. Fornace Jr., A.J. Cell. 1992; 71: 587-597Abstract Full Text PDF PubMed Scopus (2927) Google Scholar, 4Smith M.L. Chen I. Zhan Q. Bae I. Chen C. Gilmer T. Kastan M.B. O'Connor P.M. Fornace A.J. Science. 1994; 266: 1376-1380Crossref PubMed Scopus (895) Google Scholar, 5Kearsey J.M. Coates P.J. Prescott A.R. Warbrick E. Hall P.A. Oncogene. 1995; 11: 1675-1683PubMed Google Scholar, 6Wang X.W. Zhan Q. Coursen J.D. Khan M. Kontny U., Yu, L. Hollander C.M. O'Connor P.M. Fornace Jr., A.J. Harris C.C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3706-3711Crossref PubMed Scopus (532) Google Scholar, 7Zhan Q. Antinore M.J. Wang X.W. Carrier F. Harris C.C. Fornace Jr., A.J. Oncogene. 1999; 18: 2892-2900Crossref PubMed Scopus (395) Google Scholar, 8Amundson S.A. Myers T.G. Fornace Jr., A.J. Oncogene. 1998; 17: 3287-3300Crossref PubMed Scopus (401) Google Scholar). Indeed, Gadd45 has been shown to bind to several proteins involved in these processes, including proliferating cell nuclear antigen (4Smith M.L. Chen I. Zhan Q. Bae I. Chen C. Gilmer T. Kastan M.B. O'Connor P.M. Fornace A.J. Science. 1994; 266: 1376-1380Crossref PubMed Scopus (895) Google Scholar), p21Waf1/Cip1 (5Kearsey J.M. Coates P.J. Prescott A.R. Warbrick E. Hall P.A. Oncogene. 1995; 11: 1675-1683PubMed Google Scholar), and Cdc2 (7Zhan Q. Antinore M.J. Wang X.W. Carrier F. Harris C.C. Fornace Jr., A.J. Oncogene. 1999; 18: 2892-2900Crossref PubMed Scopus (395) Google Scholar). Two other genes with homology to gadd45 have also been described: myd118 (also referred to as gadd45β) (9Abdollahi A. Lord K.A. Hoffman-Liebermann B. Liebermann D.A. Oncogene. 1991; 6: 165-167PubMed Google Scholar), implicated in myeloid differentiation, and the recently described gadd45γ (also referred to as cr6) (10Takekawa M. Saito H. Cell. 1998; 95: 521-530Abstract Full Text Full Text PDF PubMed Scopus (649) Google Scholar, 11Zhang W. Bae I. Krishnaraju K. Azam N. Fan W. Smith K. Hoffman B. Liebermann D.A. Oncogene. 1999; 18: 4899-4907Crossref PubMed Scopus (130) Google Scholar). The mitogen-activated protein kinase (MAPK)1 signaling cascades leading to the activation of c-Jun N-terminal kinase (JNK) and p38 play important roles in mediating cellular responses to stressful stimuli (reviewed in Refs. 12Kyriakis J.M. Avruch J. J. Biol. Chem. 1996; 271: 24313-24316Abstract Full Text Full Text PDF PubMed Scopus (1025) Google Scholar, 13Karin M. Ann. N. Y. Acad. Sci. 1998; 851: 139-146Crossref PubMed Scopus (289) Google Scholar, 14Davis R.J. Biochem. Soc. Symp. 1999; 64: 1-12PubMed Google Scholar). JNK and p38 are phosphorylated via a group of related kinases (MAPKK), which are in turn activated by another set of kinases, designated MAPKKK. While some of the upstream kinases involved in JNK and p38 activation are selective for one or the other pathway, others function in the activation of both signaling cascades. The human MAPKKK MTK1 (its mouse homologue is referred to as MEKK4) has been implicated in the activation of both JNK and p38 (15Gerwins P. Blak J.L. Johnson G.L. J. Biol. Chem. 1997; 272: 8288-8295Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, 16Takekawa M. Posas F. Saito H. EMBO J. 1997; 16: 4973-4982Crossref PubMed Scopus (157) Google Scholar). Using a yeast two-hybrid screen to detect proteins that interact with MTK1, Takekawa and Saito (10Takekawa M. Saito H. Cell. 1998; 95: 521-530Abstract Full Text Full Text PDF PubMed Scopus (649) Google Scholar) recently identified the Gadd45-related proteins as MTK1-binding proteins. Additional experiments demonstrated that the Gadd45-related proteins were capable of enhancing MTK1 activityin vitro and that their overexpression led to activation of JNK and p38. These findings suggest a pivotal role for the Gadd45-related proteins in regulating JNK and p38 activities and Takekawa and Saito (10Takekawa M. Saito H. Cell. 1998; 95: 521-530Abstract Full Text Full Text PDF PubMed Scopus (649) Google Scholar) proposed that stress-induced expression of Gadd45-like proteins is important in initiating the activation of JNK and p38. One concern with this hypothesis is the apparent discordance between the kinetics of JNK and p38 activation and gadd45induction (2Fornace Jr., A.J. Nebert D.W. Hollander M.C. Luethy J.D. Papathanasiou M. Fargnoli J. Holbrook N.J. Mol. Cell. Biol. 1989; 9: 4196-4203Crossref PubMed Scopus (647) Google Scholar, 10Takekawa M. Saito H. Cell. 1998; 95: 521-530Abstract Full Text Full Text PDF PubMed Scopus (649) Google Scholar, 17Liu Y. Gorospe M. Yang C. Holbrook N.J. J. Biol. Chem. 1995; 270: 8377-8380Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar, 18Liu Z.-G. Baskaran R. Lea-Chou E.T. Wood L.D. Chen Y. Karin M. Wang J.Y.J. Nature. 1996; 384: 273-276Crossref PubMed Scopus (346) Google Scholar), but there are no studies in which JNK/p38 activation and gadd45 expression have been simultaneously examined. The recent generation of gadd45-null mice (19Hollander M.C. Sheikh M.S. Bulavin D. Lundgren K. Augeri- Henmueller L. Shehee R. Molinaro T. Kim K. Tolosa E. Ashwell J.D. Rosenberg M.D. Zhan Q. Fernandez-Salguero P.M. Morgan W.F. Deng C.X. Fornace Jr., A.J. Nat. Genet. 1999; 23: 176-184Crossref PubMed Scopus (442) Google Scholar) has provided us the opportunity to directly address its role in activating JNK and p38 during stress. The results of experiments described here, comparing kinase activities of embryo fibroblasts derived fromgadd45 +/+ and gadd45 −/−mice, indicate that Gadd45 is not required for JNK or p38 activation during stress, nor does absence of Gadd45 result in any deficiency in JNK or p38 activity. From additional experiments, in which we have systematically compared the kinetics and magnitude of activation of JNK and p38 with those for expression of mRNAs encodinggadd45, myd118/gadd45β, andcr6/gadd45γ, we conclude that stress-induced increases in the expression of these gadd45-related genes are also not involved in the acute activation of JNK or p38 during stress. Primary wild type andgadd45-null mouse embryo fibroblasts (MEF), and spontaneously immortalized MEF cell lines were kindly provided by Dr. Albert J. Fornace, Jr. (National Cancer Institute). NIH3T3, COS-7, and HeLa cells were obtained from American Type Culture Collection (Manassas, VA). The cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (10% fetal calf serum for NIH3T3), penicillin, streptomycin, and 2 mmglutamine and cultured at 37 °C in an atmosphere of 5% CO2, 95% air. pCMV3, Gadd45/pCMV3, Gadd45/HA-pCEP4, pCS2MT, and Gadd45/pCS2MT were provided by Dr. Albert J. Fornace, Jr. Vectors expressing hemagglutinin-tagged JNK1 (HA-JNK1) and ΔMEKK1 were provided by Dr. Michael Karin, the HA-p38 vector was from Dr. Silvio Gutkind, and the vector expressing the activated form of MKK6 (Glu) was obtained from Dr. Roger J. Davis. Cells were plated into 60-mm dishes and transfected with the appropriate expression vectors using LipofectAMINE (Life Technologies, Inc.) following the manufacturer's instructions. The total amount of DNA added to cells was kept constant through addition of the appropriate control plasmid DNA. Following ∼30-h incubation, cells were lysed, and protein extracts were used for kinase assays or Western analysis as described below. Cells were lysed in 0.5 ml of lysis buffer (20 mm Hepes, pH 7.4, 2 mm EGTA, 50 mm β-glycerol phosphate, 1% Triton X-100, 10% glycerol, 1 mm dithiothreitol, 1 mm phenylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 1 mm Na2VO4, and 5 mm NaF). Samples containing equal amounts of protein were immunoprecipitated at 4 °C overnight with 1 μg/ml of either anti-JNK1 (Santa Cruz Biotechnology, Santa Cruz, CA) or anti-HA antibody (Roche Molecular Biochemicals) with the addition of 30 μl of 50% slurry protein A-Sepharose. The beads were pelleted by centrifugation and washed three times each in lysis buffer and kinase assay buffer (20 mm MOPS, pH 7.2, 2 mm EGTA, 10 mm MgCl2, 1 mm dithiothreitol, and 0.1% Triton X-100). JNK kinase assays were performed as described previously using GST-c-Jun as a substrate (17Liu Y. Gorospe M. Yang C. Holbrook N.J. J. Biol. Chem. 1995; 270: 8377-8380Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar). Samples were separated on a 12% SDS-PAGE gel, and after drying, were subjected to autoradiography. Quantitation was performed using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). To assess the activation of endogenous p38, total cell lysates were immunoblotted with an anti-phospho-p38 antibody (New England Biolabs, Inc., Beverly, MA). In the transient overexpression studies examining HA-p38 activity, cell lysates were first immunoprecipitated with an anti-HA antibody, and phosphorylated HA-p38 was subsequently examined by Western blot analysis (see below) using the anti-phospho-p38-specific antibody. Total cell proteins were extracted from cell monolayers, and 30 μg of protein was size-separated by SDS-PAGE. After electrophoresis, the proteins were transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA) and incubated with the appropriate antibodies. The anti-Gadd45 and anti-Myc antibodies were obtained from Santa Cruz Biotechnology. Specific proteins were detected with the enhanced chemiluminescence (ECL) system (Amersham Pharmacia Biotech). Total RNA was isolated and Northern blot analysis carried out as described (17Liu Y. Gorospe M. Yang C. Holbrook N.J. J. Biol. Chem. 1995; 270: 8377-8380Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar). For detection of gadd45, andmyd118/gadd45β mRNAs, random primer-labeled probes were prepared from plasmids pCMV3Gadd45 and pCMV3MyD118, both obtained from Dr. Albert J. Fornace, Jr. Expression of cr6/gadd45γmRNA was detected using an end-labeled oligonucleotide (5′-tgaaagcattgcccgggatccgttttttgaaagagcagtg-3′) complementary to a unique region in the 3′-untranslated portion of the mRNA (11Zhang W. Bae I. Krishnaraju K. Azam N. Fan W. Smith K. Hoffman B. Liebermann D.A. Oncogene. 1999; 18: 4899-4907Crossref PubMed Scopus (130) Google Scholar). Normalization of Northern signals was performed using an oligomer recognizing the 18 S rRNA (17Liu Y. Gorospe M. Yang C. Holbrook N.J. J. Biol. Chem. 1995; 270: 8377-8380Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar). Radioactive signals were visualized and quantitated with a PhosphorImager. To determine whether Gadd45 was necessary for JNK activation during stress, we examined the relative activation of JNK occurring in response to a panel of stresses in primary embryo fibroblasts (MEF) obtained from wild type andgadd45-null mice (Fig.1 A). With the exception of γ irradiation, all of the stresses examined significantly elevated JNK activity. No differences in either basal or stress-induced JNK activity were observed between gadd45 +/+ andgadd45 −/− MEF. These results were further confirmed with spontaneously immortalized cultures ofgadd45 +/+ (109T) andgadd45 −/− (132T) MEF, where kinase activities were monitored over a 5-h time period (Fig. 1 B). Although the kinetics of activation varied for the different stresses, in all cases JNK activation was apparent at the earliest time point examined (30 min) and occurred in the absence of detectable gadd45expression. Similar assessment of p38 activity also revealed no deficiency in its stress-induced activation in gadd45 −/− cells (Fig. 2). In fact, in most cases, p38 activation was actually higher in thegadd45 −/− cells relative togadd45 +/+ MEF. Consistent with our observation in primary MEF, we detected no significant activation of JNK or p38 in response to γ irradiation in immortalized lines of eithergadd45 +/+ or gadd45 −/−MEF (data not shown). Gadd45 protein levels were examined in the same immortal MEF populations analyzed for JNK and p38 activities above (Fig.3). As expected, no Gadd45 expression was evident in gadd45 −/− MEF, but in keeping with previous studies in other cell types, MMS was a potent inducer of Gadd45 expression in gadd45 +/+ cells. Importantly, however, induction of Gadd45 was significantly delayed relative to JNK and p38 activation, as no Gadd45 expression was evident until 2 h after addition of MMS (Fig. 3 A). Kinetics and relative magnitude of Gadd45 expression seen with other stresses revealed similar inconsistencies with the activation of JNK and p38 (Fig. 3, A and B); hydrogen peroxide resulted in significantly lower induction of Gadd45 protein compared with MMS treatment, and no induction of Gadd45 protein was seen following UVC irradiation. However, both treatments strongly induced JNK and p38. Although the experiments above rule out a requirement for Gadd45 in stress-induced activation of JNK and p38, they do not preclude the involvement of other Gadd45-related proteins. To investigate this possibility, we examinedgadd45, myd118/gadd45β, andcr6/gadd45γ mRNA expression in gadd45+/+and gadd45−/− MEF. All three mRNAs showed low expression in wild type cells in the absence of stress, but each showed enhanced expression following MMS treatment (Fig.4 A). Importantly, none of the mRNAs were significantly increased until at least 2 h posttreatment. These findings are consistent with the kinetics for Gadd45 protein expression seen above and indicate that stress-induced expression of these genes cannot account for the early activation of JNK and p38 by MMS. Similar observations were made with several other stresses (Fig. 4 B, upper panel). As reported previously (10Takekawa M. Saito H. Cell. 1998; 95: 521-530Abstract Full Text Full Text PDF PubMed Scopus (649) Google Scholar), the three transcripts appear to be differentially affected by stresses. cr6/gadd45γ was elevated to a much lesser degree by MMS than either gadd45 ormyd118/gadd45β, but showed greater induction than these with anisomycin treatment. Examination of myd118/gadd45βand gadd45γ/cr6 mRNA expression in gadd45−/− MEF revealed kinetics of induction and relative fold increases similar to those seen in the wild type cells (Fig.4 B, lower panel). However, basal expression ofmyd118/gadd45β was higher in gadd45-null cells than in wild type MEF. Analysis of MyD118/Gadd45β and CR6/Gadd45γ protein expression could not be performed due to the lack of suitable antibodies. Although the studies above rule out a requirement for Gadd45 in the activation of JNK and p38, they do not exclude the possibility that high levels of Gadd45 could result in JNK or p38 activation. To address this possibility, three different Gadd45 expression vectors were utilized in co-transfection studies with HA-tagged JNK (HA-JNK). HA-JNK was immunoprecipitated from cells on the following day and assayed for kinase activity. In none of three cell lines examined, HeLa, NIH3T3, or COS-7, could we see evidence for the ability of Gadd45 to activate JNK (Fig.5 A). In contrast, overexpression of a constitutively active mutant form of MEKK1 (a known activator of JNK) (20Yan M. Dai T. Deak J.C. Kyriakis J.M. Zon L.I. Woodgett J.R. Templeton D.J. Nature. 1994; 372: 798-800Crossref PubMed Scopus (658) Google Scholar) markedly increased JNK activity. As shown for NIH3T3 cells, expression of HA- and Myc-tagged Gadd45 was verified using anti-HA and anti-Myc antibodies, respectively. Similar co-transfection studies were performed with the Myc-Gadd45 expression vector and HA-tagged p38, after which, activation of HA-p38 was assessed by Western analysis using a phosphospecific antibody. No increase in phosphorylated p38 was evident with Gadd45 overexpression, but phosphorylation was evident with overexpression of an active mutant form of MKK6, a known p38 activator (21Derijard B. Raingeaud J. Barrett T. Wu I.H. Han J. Ulevitch R.J. Davis R.J. Science. 1995; 267: 682-685Crossref PubMed Scopus (1409) Google Scholar) (Fig. 5 B). NIH3T3 cells contained a considerable amount of phosphorylated p38 even in the absence of stress, but this did not increase with Gadd45 overexpression. We have obtained similar results in several experiments using varying amounts of expression vector DNA. These findings contrast with those reported by Takekawa and Saito (10Takekawa M. Saito H. Cell. 1998; 95: 521-530Abstract Full Text Full Text PDF PubMed Scopus (649) Google Scholar), in which overexpression of all three Gadd45-related proteins led to increased JNK and p38 activities. However, Gadd45 was the least effective of the Gadd45-related proteins in activating the kinases. The recent finding that Gadd45 and the Gadd45-related proteins, MyD118/Gadd45β and CR6/Gadd45γ, can bind directly to MTK1 and enhance its kinase activity has implicated these proteins in the early signaling events leading to JNK and p38 activation. In particular, it was proposed that DNA damage and other environmental stresses result in the induction of Gadd45-related proteins, which in turn activate MTK1 leading to activation of JNK and p38. Our observation that JNK and p38 activation in response to environmental stresses clearly precedes induction of all threegadd45-related genes argues strongly against the hypothesis that stress-induced levels of the Gadd45-related proteins are involved in the acute activation of JNK and p38. However, it remains possible that stress-elevated levels of the Gadd45-related proteins could contribute to the activation of JNK or p38 in other stress paradigms in which there is a later onset and sustained activation. Such a scenario was suggested in a recent report where BRCA1 overexpression was found to result in both induction of GADD45 and JNK-dependent apoptosis, but these effects have not been functionally linked (22Harkin P.D. Bean J.M. Miklos D. Song Y.H. Truong V.B. Englert C. Christians F.C. Ellisen L.W. Maheswaran S. Oliner J.D. Haber D.H. Cell. 1999; 97: 575-586Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar). It is also possible that the Gadd45-related proteins serve a role in regulating MTK1 activity that can be satisfied by basal levels of the proteins, but that MTK1 activation in cells requires some additional factor(s) that is influenced by stress. It is important to emphasize that while MTK1 is localized within the cytoplasm, Gadd45 is present mostly in the nucleus and has been implicated in a variety of nuclear-associated processes (e.g. replication, DNA repair, and cell cycle arrest). Its enhanced expression during stress is likely related to these important functions. Finally, our studies with gadd45-null MEF demonstrate that at least this member of the Gadd45-related gene group is not required for acute activation of JNK or p38. However, our findings do not exclude the possibility that a function for Gadd45 in regulating JNK/p38 signaling could be compensated for by basal expression of Gadd45β and/or Gadd45γ in the gadd45-null MEF. Even in such a case, stress-induced increases in these proteins are clearly not necessary for the acute activation of JNK and p38 following stress. Obviously, further studies, including targeted disruption of the other Gadd45-related genes will be necessary to address whether any of the Gadd45-related proteins serve an obligatory function in regulating the activities of these stress-activated kinases. We thank Dr. Albert J. Fornace, Jr. for his generosity in providing to us the gadd45 +/+ andgadd45 −/− MEF prior to their publication. We also thank Drs. Eitan Shaulian and Michael Karin for helpful discussions and sharing of unpublished data, and Dr. Dan Longo for critical review of this work.