MAP/ERK Kinase Kinase 1 (MEKK1) Mediates Transcriptional Repression by Interacting with Polycystic Kidney Disease-1 (PKD1) Promoter-bound p53 Tumor Suppressor Protein
2010; Elsevier BV; Volume: 285; Issue: 50 Linguagem: Inglês
10.1074/jbc.m110.145284
ISSN1083-351X
AutoresMR Islam, Tamara Jiménez, Christopher J. Pelham, Marianna Rodova, Sanjeev Puri, Brenda S. Magenheimer, Robin L. Maser, Christian Widmann, James P. Calvet,
Tópico(s)Microtubule and mitosis dynamics
ResumoMitogen-activated protein kinase (MAPK) cascades regulate a wide variety of cellular processes that ultimately depend on changes in gene expression. We have found a novel mechanism whereby one of the key MAP3 kinases, Mekk1, regulates transcriptional activity through an interaction with p53. The tumor suppressor protein p53 down-regulates a number of genes, including the gene most frequently mutated in autosomal dominant polycystic kidney disease (PKD1). We have discovered that Mekk1 translocates to the nucleus and acts as a co-repressor with p53 to down-regulate PKD1 transcriptional activity. This repression does not require Mekk1 kinase activity, excluding the need for an Mekk1 phosphorylation cascade. However, this PKD1 repression can also be induced by the stress-pathway stimuli, including TNFα, suggesting that Mekk1 activation induces both JNK-dependent and JNK-independent pathways that target the PKD1 gene. An Mekk1-p53 interaction at the PKD1 promoter suggests a new mechanism by which abnormally elevated stress-pathway stimuli might directly down-regulate the PKD1 gene, possibly causing haploinsufficiency and cyst formation. Mitogen-activated protein kinase (MAPK) cascades regulate a wide variety of cellular processes that ultimately depend on changes in gene expression. We have found a novel mechanism whereby one of the key MAP3 kinases, Mekk1, regulates transcriptional activity through an interaction with p53. The tumor suppressor protein p53 down-regulates a number of genes, including the gene most frequently mutated in autosomal dominant polycystic kidney disease (PKD1). We have discovered that Mekk1 translocates to the nucleus and acts as a co-repressor with p53 to down-regulate PKD1 transcriptional activity. This repression does not require Mekk1 kinase activity, excluding the need for an Mekk1 phosphorylation cascade. However, this PKD1 repression can also be induced by the stress-pathway stimuli, including TNFα, suggesting that Mekk1 activation induces both JNK-dependent and JNK-independent pathways that target the PKD1 gene. An Mekk1-p53 interaction at the PKD1 promoter suggests a new mechanism by which abnormally elevated stress-pathway stimuli might directly down-regulate the PKD1 gene, possibly causing haploinsufficiency and cyst formation. IntroductionAutosomal dominant polycystic kidney disease (ADPKD) 3The abbreviations used are: ADPKDautosomal dominant polycystic kidney diseasePKDpolycystic kidney diseaseMekk1MAP/ERK kinase kinase 1MAP3KMAP kinase kinase kinaseCA-Mekk1constitutively active Mekk1DMkinase-dead Mekk1DCkinase-dead CA-Mekk1CEcytosolic extractNEnuclear extractRLUrelative light unitDMSOdimethyl sulfoxideAP-1activator protein-1. is an inherited disorder that affects ∼1 in 500–1,000 individuals, and accounts for ∼1 in 10 cases of end stage kidney failure (1Grantham J.J. N. Engl. J. 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Approximately 85% of ADPKD cases are caused by mutations in the PKD1 gene, with the remainder being caused by mutations in the PKD2 gene. PKD1 encodes polycystin-1, a large membrane protein that regulates a number of signaling pathways involved in cell cycle control, cell differentiation, and apoptotic cell death.PKD1 loss-of-function and/or decreased expression leading to haploinsufficiency have been shown to cause renal cyst formation in ADPKD and Pkd1 mutant mice (8Boulter C. Mulroy S. Webb S. Fleming S. Brindle K. Sandford R. Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 12174-12179Crossref PubMed Scopus (249) Google Scholar, 9Brasier J.L. Henske E.P. J. Clin. Invest. 1997; 99: 194-199Crossref PubMed Scopus (225) Google Scholar, 10Kim K. Drummond I. Ibraghimov-Beskrovnaya O. Klinger K. Arnaout M.A. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 1731-1736Crossref PubMed Scopus (264) Google Scholar, 11Lantinga-van Leeuwen I.S. Dauwerse J.G. Baelde H.J. Leonhard W.N. van de Wal A. Ward C.J. 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The p53 protein is widely known for its role in cell cycle control, DNA repair, and programmed cell death; and its inactivation by genetic mutation is the most frequent alteration in human cancers (18Boehme K.A. Blattner C. Crit. Rev. Biochem. Mol. Biol. 2009; 44: 367-392Crossref PubMed Scopus (93) Google Scholar, 19Kruse J.P. Gu W. Cell. 2009; 137: 609-622Abstract Full Text Full Text PDF PubMed Scopus (1259) Google Scholar, 20Vousden K.H. Prives C. Cell. 2009; 137: 413-431Abstract Full Text Full Text PDF PubMed Scopus (2270) Google Scholar). p53 regulates transcription of target genes by directly binding DNA elements and functioning as an activator or repressor depending on the target gene (21Menendez D. Inga A. Resnick M.A. Nat. Rev. Cancer. 2009; 9: 724-737Crossref PubMed Scopus (435) Google Scholar). The mechanisms of p53 transcriptional repression are not well understood (e.g. Ref. 22Marks J. Saifudeen Z. Dipp S. El-Dahr S.S. J. Biol. Chem. 2003; 278: 34158-34166Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar).The mammalian mitogen-activated protein kinases (MAPKs) include at least three subgroups: ERKs (extracellular signal-regulated kinases), p38 MAPKs, and JNKs (the c-Jun N-terminal kinases, also known as stress-activated protein kinases or SAPKs) (23Hagemann C. Blank J.L. Cell Signal. 2001; 13: 863-875Crossref PubMed Scopus (244) Google Scholar, 24Takekawa M. Tatebayashi K. Saito H. Mol. Cell. 2005; 18: 295-306Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 25Krishna M. Narang H. Cell Mol. Life Sci. 2008; 65: 3525-3544Crossref PubMed Scopus (307) Google Scholar). Each is activated through a phosphorylation cascade initiated by activation of a MAP kinase kinase kinase (MAPKKK or MAP3K), which phosphorylates a MAP kinase kinase (MAPKK), which in turn phosphorylates a MAPK. Following activation, the MAPKs translocate to the nucleus to regulate the activity of transcription factors controlling a wide range of genes. One of the key MAP3K components of the stress-activated JNK pathway is Mekk1. Upon stimulation, Mekk1 phosphorylates Mkk4/Mkk7 (MAPKKs), which then phosphorylate and activate JNK (26Healy Z.R. Zhu F. Stull J.D. Konstantopoulos K. Am. J. Physiol. Cell Physiol. 2008; 294: C1146-1157Crossref PubMed Scopus (33) Google Scholar, 27Lee C.K. Lee E.Y. Kim Y.G. Mun S.H. Moon H.B. Yoo B. Int. Immunopharmacol. 2008; 8: 362-370Crossref PubMed Scopus (35) Google Scholar, 28Takatori A. Geh E. Chen L. Zhang L. Meller J. Xia Y. Development. 2008; 135: 23-32Crossref PubMed Scopus (38) Google Scholar, 29Chaveroux C. Jousse C. Cherasse Y. Maurin A.C. Parry L. Carraro V. Derijard B. Bruhat A. Fafournoux P. Mol. Cell. Biol. 2009; 29: 6515-6526Crossref PubMed Scopus (49) Google Scholar, 30Das K.C. Muniyappa H. Mol. Cell Biochem. 2010; 337: 53-63Crossref PubMed Scopus (20) Google Scholar, 31Maillet M. Lynch J.M. Sanna B. York A.J. Zheng Y. Molkentin J.D. J. Clin. Invest. 2009; 119: 3079-3088Crossref PubMed Scopus (57) Google Scholar). Signaling initiated with the typically membrane-associated Mekk1 ends with activation of the transcription factor AP-1 (activator protein-1), which is a homo- or heterodimer of c-Jun with c-Fos or ATF2; or with other Mekk1-JNK responsive transcription factors including p53. The small GTPases, Ras, Rac, and cdc42, and their downstream effectors JNK and AP-1 have been linked to the PKD renal cystic phenotype and polycystin-1 function (32Arnould T. Kim E. Tsiokas L. Jochimsen F. Grüning W. Chang J.D. Walz G. J. Biol. Chem. 1998; 273: 6013-6018Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 33Parnell S.C. Magenheimer B.S. Maser R.L. Zien C.A. Frischauf A.M. Calvet J.P. J. Biol. Chem. 2002; 277: 19566-19572Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar) suggesting that the Mekk1 pathway may be involved in the regulation of PKD1 expression.In the present study, we discovered that Mekk1 directly represses transcription of the PKD1 gene. However, Mekk1 kinase activity is not required for this repression, suggesting a novel mechanism. Multiple lines of evidence revealed that the Mekk1 effect is mediated in the nucleus through interaction with the tumor suppressor protein p53 via an atypical p53 DNA binding motif in the PKD1 proximal promoter. A physical association between Mekk1 and p53 has not been reported previously. Mekk1 was found to reduce endogenous mRNA levels for the PKD1, bradykinin receptor B2, and IEX-1 genes, suggesting a general mechanism. As such, these results have identified a new transcriptional mechanism involving the nuclear localization of Mekk1 and its kinase-independent regulation of p53 target gene transcription.DISCUSSIONIn this study, we report that the PKD1 gene is a target of Mekk1, thus establishing a formal link between a MAPK pathway component and PKD1 gene regulation. This connection was not entirely unexpected as MAPK pathways are involved in many cellular processes including cell proliferation, differentiation, migration, and apoptosis, all considered functions of PKD1. Mekk1 is at the top of a phosphorylation cascade that leads to activation of JNK, and Mekk1 can activate the ERK (50Li H. Ung C.Y. Ma X.H. Li B.W. Low B.C. Cao Z.W. Chen Y.Z. Bioinformatics. 2009; 25: 358-364Crossref PubMed Scopus (26) Google Scholar) and p38 pathways and NFκB in response to pro-inflammatory, growth stimulatory, or stress-response signals (51Kopp E. Medzhitov R. Carothers J. Xiao C. Douglas I. Janeway C.A. Ghosh S. 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What was unexpected was the discovery that Mekk1 repression of the PKD1 promoter is mediated by a kinase-independent binding interaction with p53 at the PKD1 promoter.Mekk1 Targets the PKD1 PromoterEvidence that the PKD1 gene is a target of Mekk1 regulation came from the following observations: 1) PKD1 promoter-reporter assays demonstrated that transfected Mekk1 constructs repressed transcriptional activity of the 200-bp PKD1 proximal promoter in three different cell lines: human HEK293T, monkey COS-1, and mouse M-1 cells. 2) Ectopic Mekk1 expression markedly reduced endogenous PKD1 gene expression in human and mouse cells. 3) ChIP-PCR assays showed the presence of Mekk1 at the endogenous PKD1 promoter within a 135-bp fragment located within 169 bp of the start site of transcription.Mekk1 Targets the PKD1 Promoter by an Unconventional MechanismIt is widely known that signals from Mekk1 are propagated by sequential activation of Mek4/7 and JNK, which then activates or inhibits a number of transcription factors. However, we found that pharmacological inhibitors of the three major MAPK pathways: SP600125 and JBDIP (JNK pathway), PD98059 (MEK-ERK pathway), and SB202190 (p38 pathway), failed to reverse Mekk1 repression of the PKD1 promoter. We also showed that Mekk1 kinase activity, which is necessary for phosphorylation of its downstream targets, the key mechanism by which Mekk1 exerts its effects, was dispensable. This was shown by using kinase-dead mutants of Mekk1, which were found to be just as inhibitory as their kinase-active counterparts. These results suggested that classical MAPK signaling and substrate phosphorylation are not involved in Mekk1 repression of PKD1, thus pointing to a novel mechanism of transcriptional regulation. Although it has been shown that Mekk1 can function as an E3 ligase to regulate ERK (54Lu Z. Xu S. Joazeiro C. Cobb M.H. Hunter T. Mol. Cell. 2002; 9: 945-956Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar) and c-Jun (55Xia Y. Wang J. Xu S. Johnson G.L. Hunter T. Lu Z. Mol. Cell. Biol. 2007; 27: 510-517Crossref PubMed Scopus (64) Google Scholar) protein levels through ubiquitation and proteasomal degradation, we have no evidence supporting such a mechanism in the regulation of PKD1 promoter activity.Mekk1 Targets the p53 Binding Site in the PKD1 PromoterMutation of the p53 binding site within the 200-bp PKD1 proximal promoter prevented Mekk1 repression of the promoter. Further support was obtained by DNA pulldown assays, whereby Mekk1 was brought down by a DNA fragment harboring the p53 binding site from the PKD1 proximal promoter. These findings demonstrated that the p53 site in the 200-bp PKD1 promoter is involved in Mekk1-mediated transcriptional repression.Mekk1 Repression Requires p53As shown earlier (17Van Bodegom D. Saifudeen Z. Dipp S. Puri S. Magenheimer B.S. Calvet J.P. El-Dahr S.S. J. Biol. Chem. 2006; 281: 31234-31244Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar) and more recently by Van Bodegom et al. (56van Bodegom D. Roessingh W. Pridjian A. El Dahr S.S. Biochim. Biophys. Acta. 2010; 1799: 502-509Crossref PubMed Scopus (14) Google Scholar), the PKD1 gene is a physiological target of p53, whereby p53 directly binds the proximal-most p53 element in the PKD1 promoter to repress transcription. Thus, one possibility was that Mekk1 and p53 might function together to repress the promoter. This idea was supported by multiple lines of evidence that made use of a dominant-negative p53, siRNA knockdown of p53, and p53-null cells. Similar increases in basal activity of the 200-bp promoter observed with either p53 siRNA or Mekk1 siRNA suggest an interdependent mechanism for the two.Mekk1 and p53 InteractOur data suggest that Mekk1 binds the PKD1 promoter through p53 by acting as a co-repressor. Although p53 is a known DNA-binding protein, Mekk1 is not. Co-immunoprecipitation of Mekk1 and p53 from nuclear lysates suggest their close, physical interaction and thus the possibility that Mekk1 binds promoter-bound p53. This was supported by ChIP analysis, which showed that Mekk1 cannot bind the endogenous PKD1 promoter in the absence of p53 binding. It also appears that p53 does not function on the PKD1 promoter without Mekk1, because p53 was unable to repress promoter activity following Mekk1 siRNA knockdown. The IEX-1 (49Im H.J. Pittelkow M.R. Kumar R. J. Biol. Chem. 2002; 277: 14612-14621Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar) and BDRB2 (22Marks J. Saifudeen Z. Dipp S. El-Dahr S.S. J. Biol. Chem. 2003; 278: 34158-34166Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar) genes are also repressed by p53. Here we showed a marked reduction in the mRNA levels for these two genes following Mekk1 expression. Thus, a similar Mekk1-p53 repression mechanism may be operational for other genes as well.Mekk1 Regulation of p53 and PKD1Our studies place Mekk1 in a prominent position as a major upstream regulator of p53 function (Fig. 9). Others have shown Mekk1-mediated, JNK-dependent p53 protein stabilization, and JNK-dependent p53 phosphorylation and transcriptional activation of p53 target genes (Fig. 9, left) (47Buschmann T. Potapova O. Bar-Shira A. Ivanov V.N. Fuchs S.Y. Henderson S. Fried V.A. Minamoto T. Alarcon-Vargas D. Pincus M.R. Gaarde W.A. Holbrook N.J. Shiloh Y. Ronai Z. Mol. Cell. Biol. 2001; 21: 2743-2754Crossref PubMed Scopus (248) Google Scholar, 48Fuchs S.Y. Adler V. Pincus M.R. Ronai Z. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 10541-10546Crossref PubMed Scopus (452) Google Scholar). We now show that Mekk1 can directly regulate p53 target genes by translocating to the nucleus to act as an essential p53 co-repressor in close, physical association with DNA-bound p53 (Fig. 9, right). The kinase-dead mutants of Mekk1 were seen to increase p53 protein levels (Fig. 7E and supplemental Fig. S2). But because these increases in p53 were somewhat greater with kinase-active Mekk1 (supplemental Fig. S2), it is possible that both JNK-dependent and JNK-independent pathways regulate p53 protein levels (Fig. 9, left and right). In p53-positive HCT116 cells, γ-irradiation was found to cause significant reductions in PKD1 mRNA, as compared with irradiated isogenic p53-null cells (17Van Bodegom D. Saifudeen Z. Dipp S. Puri S. Magenheimer B.S. Calvet J.P. El-Dahr S.S. J. Biol. Chem. 2006; 281: 31234-31244Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). γ-Irradiation, which in many cells is a poor activator of JNK, is thought to increase p53 through a JNK-independent pathway (48Fuchs S.Y. Adler V. Pincus M.R. Ronai Z. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 10541-10546Crossref PubMed Scopus (452) Google Scholar, 57Maki C.G. Howley P.M. Mol. Cell. Biol. 1997; 17: 355-363Crossref PubMed Scopus (298) Google Scholar). As shown in Fig. 7E, an increase in p53 was seen in cells stably expressing kinase-dead CA-Mekk1, and this was associated with a significant decrease in PKD1 mRNA. Thus, the Mekk1 repressive effect may involve a combination of increased p53 protein and Mekk1-p53 transcriptional repression at the promoter. Importantly, extracellular stimuli thought to target the stress-response pathway (H2O2, PMA, and TNFα) were shown to recruit Mekk1 to the PKD1 promoter and to down-regulate PKD1 mRNA levels in a p53-dependent fashion.p53 Regulation of Target Gene ExpressionThe p53 protein can function as a transcriptional activator or repressor depending on the target gene. In fact, PKD1 and BDRB2, which have multiple p53 sites, can be activated or repressed by p53 (22Marks J. Saifudeen Z. Dipp S. El-Dahr S.S. J. Biol. Chem. 2003; 278: 34158-34166Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 56van Bodegom D. Roessingh W. Pridjian A. El Dahr S.S. Biochim. Biophys. Acta. 2010; 1799: 502-509Crossref PubMed Scopus (14) Google Scholar). The mechanisms of p53-mediated repression are not well understood and vary widely. They include: complex formation with the co-repressor mSin3a and histone deacetylases (for the PKD1, Map4, and stathmin genes (56van Bodegom D. Roessingh W. Pridjian A. 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Acta. 2010; 1799: 502-509Crossref PubMed Scopus (14) Google Scholar). Our data now suggest that signals acting on Mekk1, such as oxidative stress or TNFα, could tip the balance at the PKD1 gene, resulting in sufficient down-regulation of gene expression to cause PKD1 haploinsufficiency and cyst formation (Fig. 9). Stress-pathway signals have been implicated in cyst formation in ADPKD (1Grantham J.J. N. Engl. J. Med. 2008; 359: 1477-1485Crossref PubMed Scopus (401) Google Scholar, 2Harris P.C. J. Am. Soc. Nephrol. 2009; 20: 1188-1198Crossref PubMed Scopus (49) Google Scholar, 3Harris P.C. Torres V.E. Annu. Rev. Med. 2009; 60: 321-337Crossref PubMed Scopus (581) Google Scholar, 4Patel V. Chowdhury R. Igarashi P. Curr. Opin. Nephrol. Hypertens. 2009; 18: 99-106Crossref PubMed Scopus (103) Google Scholar, 5Sandford R.N. Kidney Int. 2009; 75: 765-767Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar, 6Torres V.E. Harris P.C. Kidney Int. 2009; 76: 149-168Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar, 7Zhou J. Annu. Rev. Physiol. 2009; 71: 83-113Crossref PubMed Scopus (165) Google Scholar, 64Li X. Magenheimer B.S. Xia S. Johnson T. Wallace D.P. Calvet J.P. Li R. Nat. Med. 2008; 14: 863-868Crossref PubMed Scopus (114) Google Scholar), including TNFα, which has been found in human cyst fluid (64Li X. Magenheimer B.S. Xia S. Johnson T. Wallace D.P. Calvet J.P. Li R. Nat. Med. 2008; 14: 863-868Crossref PubMed Scopus (114) Google Scholar). Notably, TNFα has been shown to cause cyst formation in embryonic kidney organ cultures and in vivo through a Pkd2-dependent mechanism, and has been considered a potential target for PKD therapy (64Li X. Magenheimer B.S. Xia S. Johnson T. Wallace D.P. Calvet J.P. Li R. Nat. 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IntroductionAutosomal dominant polycystic kidney disease (ADPKD) 3The abbreviations used are: ADPKDautosomal dominant polycystic kidney diseasePKDpolycystic kidney diseaseMekk1MAP/ERK kinase kinase 1MAP3KMAP kinase kinase kinaseCA-Mekk1constitutively active Mekk1DMkinase-dead Mekk1DCkinase-dead CA-Mekk1CEcytosolic extractNEnuclear extractRLUrelative light unitDMSOdimethyl sulfoxideAP-1activator protein-1. is an inherited disorder that affects ∼1 in 500–1,000 individuals, and accounts for ∼1 in 10 cases of end stage kidney failure (1Grantham J.J. N. Engl. J. Med. 2008; 359: 1477-1485Crossref PubMed Scopus (401) Google Scholar, 2Harris P.C. J. Am. Soc. Nephrol. 2009; 20: 1188-1198Crossref PubMed Scopus (49) Google Scholar, 3Harris P.C. Torres V.E. Annu. Rev. Med. 2009; 60: 321-337Crossref PubMed Scopus (581) Google Scholar, 4Patel V. Chowdhury R. Igarashi P. Curr. Opin. Nephrol. Hypertens. 2009; 18: 99-106Crossref PubMed Scopus (103) Google Scholar, 5Sandford R.N. 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PKD1 encodes polycystin-1, a large membrane protein that regulates a number of signaling pathways involved in cell cycle control, cell differentiation, and apoptotic cell death.PKD1 loss-of-function and/or decreased expression leading to haploinsufficiency have been shown to cause renal cyst formation in ADPKD and Pkd1 mutant mice (8Boulter C. Mulroy S. Webb S. Fleming S. Brindle K. Sandford R. Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 12174-12179Crossref PubMed Scopus (249) Google Scholar, 9Brasier J.L. Henske E.P. J. Clin. Invest. 1997; 99: 194-199Crossref PubMed Scopus (225) Google Scholar, 10Kim K. Drummond I. Ibraghimov-Beskrovnaya O. Klinger K. Arnaout M.A. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 1731-1736Crossref PubMed Scopus (264) Google Scholar, 11Lantinga-van Leeuwen I.S. Dauwerse J.G. Baelde H.J. Leonhard W.N. van de Wal A. Ward C.J. Verbeek S. Deruiter M.C. Breuning M.H. de Heer E. Peters D.J. Hum. Mol. Genet. 2004; 13: 3069-3077Crossref PubMed Scopus (252) Google Scholar, 12Lu W. Fan X. Basora N. Babakhanlou H. Law T. Rifai N. Harris P.C. Perez-Atayde A.R. Rennke H.G. Zhou J. Nat. Genet. 1999; 21: 160-161Crossref PubMed Scopus (139) Google Scholar, 13Lu W. Peissel B. Babakhanlou H. Pavlova A. Geng L. Fan X. Larson C. Brent G. Zhou J. Nat. Genet. 1997; 17: 179-181Crossref PubMed Scopus (375) Google Scholar). Because cyst formation may be caused by decreased expression of the PKD1 gene, it is important to identify transcriptional mechanisms that up-regulate gene expression, as they may ameliorate the disease; and that down-regulate gene expression as they may initiate or exacerbate the disease.We have demonstrated that the human PKD1 gene can be up-regulated by β-catenin via a TCF/LEF consensus motif (14Rodova M. Islam M.R. Maser R.L. Calvet J.P. J. Biol. Chem. 2002; 277: 29577-29583Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), and by retinoic acid via Sp1-binding GC-box motifs in the PKD1 proximal promoter (15Islam M.R. Puri S. Rodova M. Magenheimer B.S. Maser R.L. Calvet J.P. Am. J. Physiol. Renal Physiol. 2008; 295: F1845-1854Crossref PubMed Scopus (12) Google Scholar). Conversely, we and others have shown that the PKD1 promoter is negatively regulated by Ets-1/Fli-1 (16Puri S. Rodova M. Islam M.R. Magenheimer B.S. Maser R.L. Calvet J.P. Biochem. Biophys. Res. Commun. 2006; 342: 1005-1013Crossref PubMed Scopus (10) Google Scholar) and by the tumor suppressor protein p53 (17Van Bodegom D. Saifudeen Z. Dipp S. Puri S. Magenheimer B.S. Calvet J.P. El-Dahr S.S. J. Biol. Chem. 2006; 281: 31234-31244Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). The p53 protein is widely known for its role in cell cycle control, DNA repair, and programmed cell death; and its inactivation by genetic mutation is the most frequent alteration in human cancers (18Boehme K.A. Blattner C. Crit. Rev. Biochem. Mol. Biol. 2009; 44: 367-392Crossref PubMed Scopus (93) Google Scholar, 19Kruse J.P. Gu W. Cell. 2009; 137: 609-622Abstract Full Text Full Text PDF PubMed Scopus (1259) Google Scholar, 20Vousden K.H. Prives C. Cell. 2009; 137: 413-431Abstract Full Text Full Text PDF PubMed Scopus (2270) Google Scholar). p53 regulates transcription of target genes by directly binding DNA elements and functioning as an activator or repressor depending on the target gene (21Menendez D. Inga A. Resnick M.A. Nat. Rev. Cancer. 2009; 9: 724-737Crossref PubMed Scopus (435) Google Scholar). The mechanisms of p53 transcriptional repression are not well understood (e.g. Ref. 22Marks J. Saifudeen Z. Dipp S. El-Dahr S.S. J. Biol. Chem. 2003; 278: 34158-34166Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar).The mammalian mitogen-activated protein kinases (MAPKs) include at least three subgroups: ERKs (extracellular signal-regulated kinases), p38 MAPKs, and JNKs (the c-Jun N-terminal kinases, also known as stress-activated protein kinases or SAPKs) (23Hagemann C. Blank J.L. Cell Signal. 2001; 13: 863-875Crossref PubMed Scopus (244) Google Scholar, 24Takekawa M. Tatebayashi K. Saito H. Mol. Cell. 2005; 18: 295-306Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 25Krishna M. Narang H. Cell Mol. Life Sci. 2008; 65: 3525-3544Crossref PubMed Scopus (307) Google Scholar). Each is activated through a phosphorylation cascade initiated by activation of a MAP kinase kinase kinase (MAPKKK or MAP3K), which phosphorylates a MAP kinase kinase (MAPKK), which in turn phosphorylates a MAPK. Following activation, the MAPKs translocate to the nucleus to regulate the activity of transcription factors controlling a wide range of genes. One of the key MAP3K components of the stress-activated JNK pathway is Mekk1. Upon stimulation, Mekk1 phosphorylates Mkk4/Mkk7 (MAPKKs), which then phosphorylate and activate JNK (26Healy Z.R. Zhu F. Stull J.D. Konstantopoulos K. Am. J. Physiol. Cell Physiol. 2008; 294: C1146-1157Crossref PubMed Scopus (33) Google Scholar, 27Lee C.K. Lee E.Y. Kim Y.G. Mun S.H. Moon H.B. Yoo B. Int. Immunopharmacol. 2008; 8: 362-370Crossref PubMed Scopus (35) Google Scholar, 28Takatori A. Geh E. Chen L. Zhang L. Meller J. Xia Y. Development. 2008; 135: 23-32Crossref PubMed Scopus (38) Google Scholar, 29Chaveroux C. Jousse C. Cherasse Y. Maurin A.C. Parry L. Carraro V. Derijard B. Bruhat A. Fafournoux P. Mol. Cell. Biol. 2009; 29: 6515-6526Crossref PubMed Scopus (49) Google Scholar, 30Das K.C. Muniyappa H. Mol. Cell Biochem. 2010; 337: 53-63Crossref PubMed Scopus (20) Google Scholar, 31Maillet M. Lynch J.M. Sanna B. York A.J. Zheng Y. Molkentin J.D. J. Clin. Invest. 2009; 119: 3079-3088Crossref PubMed Scopus (57) Google Scholar). Signaling initiated with the typically membrane-associated Mekk1 ends with activation of the transcription factor AP-1 (activator protein-1), which is a homo- or heterodimer of c-Jun with c-Fos or ATF2; or with other Mekk1-JNK responsive transcription factors including p53. The small GTPases, Ras, Rac, and cdc42, and their downstream effectors JNK and AP-1 have been linked to the PKD renal cystic phenotype and polycystin-1 function (32Arnould T. Kim E. Tsiokas L. Jochimsen F. Grüning W. Chang J.D. Walz G. J. Biol. Chem. 1998; 273: 6013-6018Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 33Parnell S.C. Magenheimer B.S. Maser R.L. Zien C.A. Frischauf A.M. Calvet J.P. J. Biol. Chem. 2002; 277: 19566-19572Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar) suggesting that the Mekk1 pathway may be involved in the regulation of PKD1 expression.In the present study, we discovered that Mekk1 directly represses transcription of the PKD1 gene. However, Mekk1 kinase activity is not required for this repression, suggesting a novel mechanism. Multiple lines of evidence revealed that the Mekk1 effect is mediated in the nucleus through interaction with the tumor suppressor protein p53 via an atypical p53 DNA binding motif in the PKD1 proximal promoter. A physical association between Mekk1 and p53 has not been reported previously. Mekk1 was found to reduce endogenous mRNA levels for the PKD1, bradykinin receptor B2, and IEX-1 genes, suggesting a general mechanism. As such, these results have identified a new transcriptional mechanism involving the nuclear localization of Mekk1 and its kinase-independent regulation of p53 target gene transcription.
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