EPAS1 trans-Activation during Hypoxia Requires p42/p44 MAPK

Hypoxia is a common environmental stress that regulates gene expression and cell function. A number of hypoxia-regulated transcription factors have been identified and have been shown to play critical roles in mediating cellular responses to hypoxia. One of these is the endothelial PAS-domain protein 1 (EPAS1/HIF2-α/HLF/HRF). This protein is 48% homologous to hypoxia-inducible factor 1-α (HIF1-α). To date, virtually nothing is known about the signaling pathways that lead to either EPAS1 or HIF1-α activation. Here we show that EPAS1 is phosphorylated when PC12 cells are exposed to hypoxia and that p42/p44 MAPK is a critical mediator of EPAS1 activation. Pretreatment of PC12 cells with the MEK inhibitor, PD98059, completely blocked hypoxia-inducedtrans-activation of a hypoxia response element (HRE) reporter gene by transfected EPAS1. Likewise, expression of a constitutively active MEK1 mimicked the effects of hypoxia on HRE reporter gene expression. However, pretreatment with PD98059 had no effect on EPAS1 phosphorylation during hypoxia, suggesting that MAPK targets other proteins that are critical for thetrans-activation of EPAS1. We further show that hypoxia-induced trans-activation of EPAS1 is independent of Ras. Finally, pretreatment with calmodulin antagonists nearly completely blocked both the hypoxia-induced phosphorylation of MAPK and the EPAS1 trans-activation of HRE-Luc. These results demonstrate that the MAPK pathway is a critical mediator of EPAS1 activation and that activation of MAPK and EPAS1 occurs through a calmodulin-sensitive pathway and not through the GTPase, Ras. These results are the first to identify a specific signaling pathway involved in EPAS1 activation. Hypoxia is a common environmental stress that regulates gene expression and cell function. A number of hypoxia-regulated transcription factors have been identified and have been shown to play critical roles in mediating cellular responses to hypoxia. One of these is the endothelial PAS-domain protein 1 (EPAS1/HIF2-α/HLF/HRF). This protein is 48% homologous to hypoxia-inducible factor 1-α (HIF1-α). To date, virtually nothing is known about the signaling pathways that lead to either EPAS1 or HIF1-α activation. Here we show that EPAS1 is phosphorylated when PC12 cells are exposed to hypoxia and that p42/p44 MAPK is a critical mediator of EPAS1 activation. Pretreatment of PC12 cells with the MEK inhibitor, PD98059, completely blocked hypoxia-inducedtrans-activation of a hypoxia response element (HRE) reporter gene by transfected EPAS1. Likewise, expression of a constitutively active MEK1 mimicked the effects of hypoxia on HRE reporter gene expression. However, pretreatment with PD98059 had no effect on EPAS1 phosphorylation during hypoxia, suggesting that MAPK targets other proteins that are critical for thetrans-activation of EPAS1. We further show that hypoxia-induced trans-activation of EPAS1 is independent of Ras. Finally, pretreatment with calmodulin antagonists nearly completely blocked both the hypoxia-induced phosphorylation of MAPK and the EPAS1 trans-activation of HRE-Luc. These results demonstrate that the MAPK pathway is a critical mediator of EPAS1 activation and that activation of MAPK and EPAS1 occurs through a calmodulin-sensitive pathway and not through the GTPase, Ras. These results are the first to identify a specific signaling pathway involved in EPAS1 activation. hypoxia-inducible factor cyclic-AMP response element-binding protein endothelial PAS-domain protein HIF-like factor HIF-related factor hypoxia response element pheochromocytoma mitogen-activated protein kinase stress-activated protein kinase Dulbecco's modified Eagle's medium nerve growth factor calmidazolium chloride CREB-binding protein von Hippel Lindau polyacrylamide gel electrophoresis Regulation of gene expression is a primary response by which cells adapt to changes in the environment. The mechanisms involved in regulation of gene expression in response to hypoxia are beginning to be understood. Transcription factors that are activated by hypoxia include the hypoxia-inducible factor (HIF1-α),1c-fos, and CREB (1Wang G.L. Jiang B.H. Rue E.A. Semenza G.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5510-5514Crossref PubMed Scopus (5059) Google Scholar, 2Norris M.L. Millhorn D.E. J. Biol. Chem. 1995; 270: 23774-23779Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 3Beitner-Johnson D. Millhorn D.E. J. Biol. Chem. 1998; 273: 19834-19839Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 4Mishra R.R. Adhikary G. Simonson M.S. Cherniack N.S. Prabhakar N.R. Brain Res. Mol. Brain Res. 1998; 15: 74-83Crossref Scopus (54) Google Scholar). HIF1-α has been shown to be critical for hypoxia-induced regulation of a number of genes, including glycolytic enzymes, vascular endothelial growth factor, and erythropoieitin (5Wang G.L. Semenza G.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4304-4308Crossref PubMed Scopus (1205) Google Scholar, 6Semenza G.L. Roth P.H. Fang H.M. Wang G.L. J. Biol. Chem. 1994; 269: 23757-23763Abstract Full Text PDF PubMed Google Scholar, 7Forsythe J.A. Jiang B.H. Iyer N.V. Agani F. Leung S.W. Koos R.D. Semenza G.L. Mol. Cell. Biol. 1996; 16: 4604-4613Crossref PubMed Scopus (3217) Google Scholar). Recently, endothelial PAS-domain protein 1 (EPAS1, also known as HIF2-α, HLF, and HRF) was identified as a hypoxia-inducible transcription factor (8Tian H. McKnight S.L. Russell D.W. Genes Dev. 1997; 11: 72-82Crossref PubMed Scopus (1073) Google Scholar, 9Ema M. Taya S. Yokotani N. Sogawa K. Matsuda Y. Fujii-Kuriyama Y. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4273-4278Crossref PubMed Scopus (844) Google Scholar, 10Flamme I. Frohlich T. von Reutern M. Kappel A. Damert A. Risau W. Mech. Dev. 1997; 63: 51-60Crossref PubMed Scopus (299) Google Scholar). EPAS1 is a basic helix-loop-helix transcription factor, which shares 48% sequence identity with HIF1-α (8Tian H. McKnight S.L. Russell D.W. Genes Dev. 1997; 11: 72-82Crossref PubMed Scopus (1073) Google Scholar). EPAS1 protein levels, like HIF1-α levels, are relatively low under basal conditions and accumulate upon exposure of cells to hypoxia (11Wiesner M.S. Turley H. Allen W.E. William C. Eckardt K.-U. Talks K.L. Wood S.M. Gatter K.C. Harris A.L. Pugh C.W. Ratcliffe P.J. Maxwell P.H. Blood. 1998; 92: 2260-2268Crossref PubMed Google Scholar). These factors then translocate to the nucleus and trans-activate target genes containing the sequence 5′-GCCCTACGTGCTGTCTCA-3′, which is commonly referred to as the hypoxia response element (HRE) (8Tian H. McKnight S.L. Russell D.W. Genes Dev. 1997; 11: 72-82Crossref PubMed Scopus (1073) Google Scholar, 12Semenza G.L. Wang G.L. Mol. Cell. Biol. 1992; 12: 5447-5454Crossref PubMed Scopus (2209) Google Scholar). EPAS1 is expressed in many tissues and is particularly abundant in the type I oxygen-sensing cells of the carotid body (13Tian H. Hammer R.E. Matsumoto A.M. Russell D.W. McKnight S.L. Genes Dev. 1998; 12: 3320-3324Crossref PubMed Scopus (520) Google Scholar). Type I cells act as the primary O2 sensors in mammals and are responsible for matching changes in arterial pO2 with appropriate changes in respiration (14Purves M.J. J. Physiol (Lond.). 1966; 185: 60-77Crossref Scopus (23) Google Scholar). Our laboratory has used PC12 cells as a model system to study the biophysical and molecular properties of oxygen-sensing cells (15Millhorn D.E. Conforti L. Beitner-Johnson D. Zhu W. Raymond R. Filisko T. Kobayashi S. Peng M. Genter M.B. Adv. Exp. Med. Biol. 1996; 410: 135-142Crossref PubMed Scopus (14) Google Scholar). There are a number of phenotypic similarities between type I and PC12 cells, including the presence of O2-sensitive K+ channels, which are inhibited by hypoxia (16Lopez-Barneo J. Lopez-Lopez J.R. Urena J. Gonzalez C. Science. 1988; 241: 580-582Crossref PubMed Scopus (455) Google Scholar, 17Conforti L. Millhorn D.E. J. Physiol. (Lond.). 1997; 502: 293-305Crossref Scopus (123) Google Scholar). In addition, both PC12 cells and type I cells respond to hypoxia with an increase in tyrosine hydroxylase gene expression (18Czyzk-Krzeska M.F. Bayliss D.A. Lawson E.E. Millhorn D.E. J. Neurochem. 1992; 58: 1538-1546Crossref PubMed Scopus (171) Google Scholar, 19Czyzk-Krzeska M.F. Furnari B.A. Lawson E.E. Millhorn D.E. J. Biol. Chem. 1994; 269: 760-764Abstract Full Text PDF PubMed Google Scholar). Finally, both cell types depolarize and secrete the neurotransmitter dopamine in response to hypoxia (20Krammer E.B. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 2507-2511Crossref PubMed Scopus (27) Google Scholar, 21Kumar G.K. Overholt J.L. Bright G.R. Hui K.Y. Lu H. Gratzl M. Prabhakar N.R. Am. J. Physiol. 1998; 274: C1592-C1600Crossref PubMed Google Scholar, 22Taylor S.C. Peers C. Biochem. Biophys. Res. Commun. 1998; 9: 13-17Crossref Scopus (56) Google Scholar). We have therefore utilized PC12 cells to study the regulation of EPAS1. The specific signaling pathways that are involved in HIF1-α and EPAS1 activation are almost completely unknown. In our previous studies, we measured the effects of hypoxia on the mitogen and stress-activated protein kinase pathways (MAPKs and SAPKs) (23Conrad P.W. Rust R.T. Han J. Millhorn D.E. Beitner-Johnson D. J. Biol. Chem. 1999; 274: 23570-23576Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). We found that moderate hypoxia (5% O2) activates p42/p44 MAPK, two closely related protein kinases that can lead to the phosphorylation and activation of a number of transcription factors (24Garrington T.P. Johnson G.L. Curr. Opin. Cell Biol. 1999; 11: 211-218Crossref PubMed Scopus (1133) Google Scholar). We therefore hypothesized that the MAPK pathway may be important for EPAS1 activation during hypoxia. Results from the current study show that the MAPK pathway is critical for EPAS1 activation, as the specific MEK1 inhibitor, PD98059, prevents EPAS1 trans-activation of the HRE. Interestingly, PD98059 had no effect on EPAS1 protein levels, suggesting that the MAPK pathway is involved in theactivation of EPAS1, rather than the accumulationof EPAS1. We also show, for the first time, that EPAS1 itself is phosphorylated during hypoxia. However, EPAS1 is not directly phosphorylated by MAPK, suggesting that MAPK mediates its effects indirectly, possibly by recruiting other proteins critical for EPAS1 trans-activation. Finally, we show that MAPK-activation of EPAS1 during hypoxia occurs via a calmodulin-sensitive pathway and not through a Ras-dependent mechanism. PC12 cells were cultured in Dulbecco's modified Eagle's medium (DMEM)/Ham's F-12 (Life Technologies, Inc.) supplemented with 20 mm HEPES, pH 7.4, 10% fetal bovine serum (Life Technologies, Inc.), and with penicillin (100 units/ml) and streptomycin (100 μg/ml). Prior to experimentation, cells were grown to approximately 85% confluence in 35- or 60-mm tissue culture dishes (Corning), or in 24-well plates for luciferase assays, in an environment of 21% O2, 5% CO2, balanced with N2. Hypoxia was achieved by exposing cells to various levels (10, 5, and 1%) of O2, 5% CO2, balanced with N2 for various times in an O2-regulated incubator (Forma Scientific, Marietta, OH). PD98059 was obtained from New England Biolabs (Beverly, MA). EPAS1 polyclonal antibody, the HRE-Luc reporter gene, and the EPAS1 cDNA were generous gifts from Dr. Steven L. McKnight (University of Texas Southwestern, Dallas, TX). pFC-MEK1 was obtained from Stratagene (La Jolla, CA). Additional EPAS1 polyclonal antibody was obtained from Novus Biologicals (Littleton, CO), and similar results were obtained with both antibodies. RasN-17 was a gift from Dr. J. Silvio Gutkind (National Institutes of Health, NIDR, Bethesda, MD). A c-fos-luciferase fusion reporter gene (fos-Luc) was constructed from a c-fos-β-galactosidase fusion gene construct, kindly provided by Dr. Tom Curran (St. Jude's Childrens Research Hospital, Memphis, TN). The β-galactosidase coding region was excised from the fos-lacZ plasmid (26Schilling K. Luk D. Morgan J.I. Curran T. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5665-5669Crossref PubMed Scopus (75) Google Scholar) withNcoI and BamHI and replaced with the luciferase coding region from the pGL3-basic plasmid (Promega, Madison, WI). W13 was obtained from RBI (Natick, MA). Calmidazolium chloride was obtained from Calbiochem. PC12 cells were transfected with the hypoxia response element-luciferase (HRE-Luc) reporter gene using the Transfast transfection reagent according to the manufacturers recommended conditions (Promega). This reporter gene has been described previously (8Tian H. McKnight S.L. Russell D.W. Genes Dev. 1997; 11: 72-82Crossref PubMed Scopus (1073) Google Scholar, 12Semenza G.L. Wang G.L. Mol. Cell. Biol. 1992; 12: 5447-5454Crossref PubMed Scopus (2209) Google Scholar). PC12 cells seeded in 24-well plates confluence were transfected in with of Transfast and of HRE-Luc In of EPAS1, or RasN-17 was with the HRE-Luc. In was to the of to 1 of were to medium for to the of the The PC12 cells were exposed to or hypoxia O2) for In other the effect of on a reporter gene was In these cells were with of the c-fos-luciferase reporter gene and of an expression plasmid in 24-well cells were with nerve growth factor for To luciferase assays, cells were with and in of cell culture reagent (Promega). Cell were for 1 with a cell of cell were then (Promega). of was to and were in a We found previously that hypoxia expression of and reporter Beitner-Johnson and D. as in previous studies, luciferase was to of protein D. Millhorn D.E. J. Biol. Chem. 1998; 273: 19834-19839Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). by than between was as described previously D. Millhorn D.E. J. Biol. Chem. 1998; 273: 19834-19839Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, P.W. Rust R.T. Han J. Millhorn D.E. Beitner-Johnson D. J. Biol. Chem. 1999; 274: 23570-23576Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). were with kinase New England EPAS1 protein expression was using a polyclonal antibody of the EPAS1 protein a of were as described by E.A. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus (75) Google Scholar). PC12 cells dishes were with and then in (Life Technologies, Inc.) for medium containing 1 of and either or PD98059 was to the for cells were exposed to or hypoxia O2, were by with and in 1 of a containing mm pH 7.4, mm mm 1 mm mm 1 and cell were with of and of a 10% of protein EPAS1 was using of an EPAS1 polyclonal antibody by the of of a 10% of protein The was to for were times with and then to The gel was and using a CA). a first the regulation of EPAS1 in PC12 cells, we EPAS1 protein levels exposure to hypoxia. 1 A that exposure to hypoxia O2) for in a increase in EPAS1 protein has been previously that EPAS1 can trans-activate an HRE-Luc reporter gene (8Tian H. McKnight S.L. Russell D.W. Genes Dev. 1997; 11: 72-82Crossref PubMed Scopus (1073) Google Scholar). We found that the of hypoxia from 21% O2 to O2 in a increase in We have shown that hypoxia regulates of the and MAPK (23Conrad P.W. Rust R.T. Han J. Millhorn D.E. Beitner-Johnson D. J. Biol. Chem. 1999; 274: 23570-23576Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). We that moderate hypoxia (5% O2) a phosphorylation of A results obtained when PC12 cells were exposed to hypoxia which a phosphorylation of p42/p44 the MAPK pathway is known to a number of transcription including and D. A. M. Genes Dev. 1994; PubMed Scopus Google Scholar, J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar, J. Science. 1996; 273: PubMed Scopus Google we hypothesized that the MAPK pathway be important for EPAS1 activation during hypoxia. To PC12 cells were with the HRE-Luc reporter gene and a plasmid the EPAS1 cDNA or the expression were then with either PD98059 or and exposed to or hypoxia O2) for by (8Tian H. McKnight S.L. Russell D.W. Genes Dev. 1997; 11: 72-82Crossref PubMed Scopus (1073) Google Scholar) we found that expression of EPAS1 HRE-Luc under both and conditions We also found that of by PD98059, completely blocked the effect of hypoxia on both basal and HRE-Luc These results that the signaling pathway is critical for mediating EPAS1 activation of gene expression. To we measured the effect of a constitutively active MEK1 on basal and hypoxia-induced HRE-Luc MEK1 is a protein kinase that directly and activates MAPK (24Garrington T.P. Johnson G.L. Curr. Opin. Cell Biol. 1999; 11: 211-218Crossref PubMed Scopus (1133) Google Scholar). that expression of basal HRE-Luc during both and hypoxia. However, when with EPAS1, pFC-MEK1 a increase in the trans-activation of the HRE-Luc The increase in HRE-Luc in the presence of pFC-MEK1 and EPAS1 was than cells transfected with EPAS1 and exposed to In transfection with EPAS1 by hypoxia, in a increase in HRE-Luc The increase in EPAS1 by hypoxia was by a in the of the EPAS1 protein suggesting that EPAS1 itself be phosphorylated during hypoxia. To PC12 cells were with either PD98059 or then with or EPAS1 was from cell and its phosphorylation was by and A that hypoxia phosphorylation of EPAS1. However, EPAS1 phosphorylation was not blocked by PD98059, in to the effects of hypoxia of the HRE-Luc reporter gene by EPAS1. We also MAPK was involved in the of EPAS1 by hypoxia. In these PC12 cells were with or to exposure to hypoxia. cell were then for EPAS1. hypoxia a increase in EPAS1 protein levels, of MEK1 with PD98059 had no effect on the hypoxia-induced of EPAS1. is an of MAPK (24Garrington T.P. Johnson G.L. Curr. Opin. Cell Biol. 1999; 11: 211-218Crossref PubMed Scopus (1133) Google Scholar, M. D. Biol. 1994; Google Scholar). In to was involved in the EPAS1 trans-activation of the PC12 cells were with the EPAS1 expression the HRE-Luc and of a expression A that of RasN-17 had no effect on the of HRE-Luc. However, of the of RasN-17 activation of a gene by nerve growth factor in PC12 cells EPAS1 activation by hypoxia occurs via a mechanism. Hypoxia results in and PC12 cells during hypoxia L. Millhorn D.E. J. Physiol. (Lond.). 1997; 502: 293-305Crossref Scopus (123) Google Scholar, Conforti L. M.F. Millhorn D.E. Am. J. Physiol. 1996; Google Scholar). with these J. C. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar, J. C. J. Neurochem. 1998; PubMed Scopus Google Scholar) have shown that of PC12 cells results in MAPK activation via a mechanism. we hypothesized that calmodulin be involved in the activation of MAPK and EPAS1 during hypoxia. A that pretreatment of PC12 cells with the calmodulin W13 a in hypoxia-induced MAPK These results are shown in We also found that with either or calmidazolium chloride 1 calmodulin inhibited both HRE as as the of the HRE reporter gene MAPK activation of EPAS1 occurs via a rather than through the Ras. Regulation of gene expression by hypoxia is by a number of pathways (15Millhorn D.E. Conforti L. Beitner-Johnson D. Zhu W. Raymond R. Filisko T. Kobayashi S. Peng M. Genter M.B. Adv. Exp. Med. Biol. 1996; 410: 135-142Crossref PubMed Scopus (14) Google Scholar, M. Biol. Chem. 1997; Google Scholar). MAPK is known to be critical for the trans-activation of many genes and mediates its effects through the of transcription factors (24Garrington T.P. Johnson G.L. Curr. Opin. Cell Biol. 1999; 11: 211-218Crossref PubMed Scopus (1133) Google Scholar, R. 1994; 9: Google Scholar, M. D. Biol. 1994; Google Scholar). The current study for the first time, that EPAS1 is phosphorylated during hypoxia and that the MAPK pathway is critical for EPAS1 trans-activation during hypoxia in PC12 our that phosphorylation is an important for EPAS1 activation, have also shown that changes are critical to the of the EPAS1 M. K. J. H. J. Sogawa K. L. Fujii-Kuriyama Y. J. 1999; PubMed Google Scholar). is that EPAS1 activation results from the of and that the of specific in a cell We found that MAPK is for EPAS1 trans-activation of the HRE-Luc reporter gene, as was completely blocked by a of MEK1 A. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google and by constitutively activated However, hypoxia-induced phosphorylation of EPAS1 protein was inhibited by MAPK is critical for regulation of EPAS1 is not the kinase that EPAS1 during hypoxia. These results that and are for EPAS1 activation. One to of the EPAS1 by of S. J. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). A to the phosphorylation of EPAS1. by that are involved in regulation of EPAS1 and two of EPAS1 that are for its activation during hypoxia. One of the critical EPAS1 is an that from and shares with the of HIF1-α M. K. J. H. J. Sogawa K. L. Fujii-Kuriyama Y. J. 1999; PubMed Google Scholar, L. M. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar, Y. Ratcliffe P.J. Pugh C.W. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). the EPAS1 and the HIF1-α were identified as critical for the of proteins during hypoxia. The important EPAS1 is a activation which is the of in EPAS1 during hypoxia M. K. J. H. J. Sogawa K. L. Fujii-Kuriyama Y. J. 1999; PubMed Google Scholar). is therefore to that phosphorylation of EPAS1 occurs the activation of the However, the of EPAS1 phosphorylation are and further The of activation of EPAS1 is unknown. The that EPAS1 phosphorylation in the presence of PD98059 that the MAPK pathway not directly target EPAS1, targets other that are critical for the of the EPAS1 have shown that CREB-binding protein with HIF1-α and EPAS1 and the activation of these proteins M. K. J. H. J. Sogawa K. L. Fujii-Kuriyama Y. J. 1999; PubMed Google Scholar, R. S. Jiang C. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar). R. A. Biochem. Biophys. Res. Commun. 1996; PubMed Scopus Google Scholar) have shown that of can be phosphorylated by MAPK in Y. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar) that MAPK can directly the of in PC12 be a target of MAPK, which then EPAS1 to the In to the von Hippel Lindau gene has been shown to be involved in the regulation of HIF1-α and EPAS1 protein levels P.W. M.S. G. S.C. Pugh C.W. Ratcliffe P.J. 1999; PubMed Scopus Google Scholar). Interestingly, was also shown to be in the HIF1-α P.W. M.S. G. S.C. Pugh C.W. 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Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar) to our study that was critical to the activation of MAPK and that activation from phosphorylation of the growth factor In to these we have found that MAPK activation by hypoxia was and we were to demonstrate phosphorylation of the growth factor by hypoxia. L. These results that may be important between hypoxia-induced and In our results show that MAPK and calmodulin are critical of hypoxia-induced and transcription factor activation. The of pathway is to be to PC12 cells and other cells, as cells not depolarize when exposed to a environment. These results the first to a specific signaling pathway that to EPAS1 activation. We show that the MAPK pathway is a critical mediator of EPAS1 activation and that activation of MAPK and EPAS1 occurs through a calmodulin-sensitive not through the GTPase, Ras. are the molecular by which MAPK regulates EPAS1 and the kinase that EPAS1. our of cells adapt and respond to low We also show, for the first time, that EPAS1 is phosphorylated during hypoxia and that phosphorylation is independent of We Dr. S. L. McKnight for and for We also Dr. J. W. and Dr. T. for critical on the and G. for of