Molecular Pharmacology Fast Forward. Published on September 9, 2005 as DOI: 10.1124/mol.105.015271 Molecular PharmacologyThis article Fasthas not Forward. been copyedited Published and formatted. on TheSeptember final version 9,may 2005 differ asfrom doi:10.1124/mol.105.015271 this version. MOL(15271) Regulation of PHD2 activity by MYND-Zn finger Inhibition of the catalytic activity of HIF-1α-prolyl hydroxylase 2 by a MYND-type zinc finger Downloaded from molpharm.aspetjournals.org Kyung-Ok Choi, Taekyong Lee, Naery Lee, Ji-Hyun Kim, Eun Gyeong Yang, Jung Min Yoon, Jin Hwan Kim, Tae-Gyu Lee, and Hyunsung Park Department of Life Science, University of Seoul, Seoul 130-743, Korea (K.-O. C., T. at ASPET Journals on September 26, 2021 L., N. L., H. P.), Life Science Division, Korea Institute of Science and Technology, Seoul 130-650, Korea (J.-H. K., E. G. Y.), and The Division of Drug Discovery, CrystalGenomics, Inc., Daejeon City, 305-390, Korea (J. M. Y, J. H. K., T.-G. L.) 1 Copyright 2005 by the American Society for Pharmacology and Experimental Therapeutics. Molecular Pharmacology Fast Forward. Published on September 9, 2005 as DOI: 10.1124/mol.105.015271 This article has not been copyedited and formatted. The final version may differ from this version. MOL(15271) Regulation of PHD2 activity by MYND-Zn finger Running title page a) Running title : Regulation of PHD2 activity by MYND-Zn finger b) Corresponding author. (Hyunsung Park) Mailing address: Department of Life Science, University of Seoul, 90 Cheonnong-dong, Tongdaemun-gu, Seoul 130-743, Downloaded from Korea. Phone: 82-2-2210-2622. Fax: 82-2-2210-2888. E-mail:[email protected]. c) The number of text pages (19), molpharm.aspetjournals.org number of tables (0), number of figures (7), references (31), at ASPET Journals on September 26, 2021 the number of words in the Abstract ( 191) the number of words in the Introduction ( 555) the number of words in the Discussion ( 671) d) A list of nonstandard abbreviations used : bHLH, basic helix-loop-helix; HIF-1α, hypoxia-inducible factor-1α; HRE, hypoxia-responsive element; GST, glutathione-S- transferase; MYND, Myeloid translocation protein 8, Nervy, and DEAF1; ODD, Oxygen dependent degradation domain; PAS, Per-Arnt-Sim; PHD, HIF-1α-specific prolyl hydroxylase; TPEN, N, N, N', N'-tetrakis (2-pyridylmethyl) ethylenediamine; VHL, von Hippel Lindau. 2 Molecular Pharmacology Fast Forward. Published on September 9, 2005 as DOI: 10.1124/mol.105.015271 This article has not been copyedited and formatted. The final version may differ from this version. MOL(15271) Regulation of PHD2 activity by MYND-Zn finger Abstract Hypoxia-induced gene expression is initiated when the hypoxia-inducible factor-1α (HIF-1α) subunit is stabilized in response to a lack of oxygen. An HIF-1α-specific prolyl hydroxylase (PHD) catalyzes hydroxylation of the proline-564 and/or 402 residues of HIF-1α by an oxygen molecule. The hydroxyproline then interacts with the Downloaded from ubiquitin E3 ligase, von Hippel Lindau (VHL) protein, and is degraded by an ubiquitin- dependent proteasome. PHD2 is the most active of three PHD isoforms in hydroxylating molpharm.aspetjournals.org HIF-1α. Structural analysis showed that the N-terminal region of PHD2 contains a MYND-type zinc finger domain whereas the catalytic domain is located in its C- terminal region. We found that deletion of the MYND domain increased the activity of both recombinant PHD2 protein and in vitro translated PHD2. The zinc chelator, N, N, at ASPET Journals on September 26, 2021 N', N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) augmented the activity of wild type PHD2-F but not that of PHD2 lacking the MYND domain, confirming that the zinc finger domain is inhibitory. Overexpression of PHD2 lacking the MYND domain caused a greater reduction in the stability and function of HIF-1α than did overexpression of wild type PHD2, indicating that the MYND domain also inhibits the catalytic activity of PHD2 in vivo. 3 Molecular Pharmacology Fast Forward. Published on September 9, 2005 as DOI: 10.1124/mol.105.015271 This article has not been copyedited and formatted. The final version may differ from this version. MOL(15271) Regulation of PHD2 activity by MYND-Zn finger Introduction Hypoxia is the most common type of cell injury in various human diseases including myocardial infarction, stroke, acute renal failure and solid tumors. However, organisms have evolved mechanisms for adapting to hypoxia. Thus, hypoxia leads to up-regulation of the transcription of genes involved in anaerobic ATP production and Downloaded from oxygen delivery. Hypoxia-inducible factor-1(HIF-1) is a widespread transcription factor that promotes expression of hypoxia-inducible genes such as vascular endothelial molpharm.aspetjournals.org growth factor, erythropoietin, glucose transporters and glycolytic enzymes (Masson and Ratcliffe, 2003; Seagroves et al., 2001). It consists of HIF-1α and HIF-1β subunits, both of which belong to the basic helix-loop-helix (bHLH) Per-Arnt-Sim (PAS) family. Arnt (HIF-1β) is a partner of the aryl hydrocarbon receptor (AhR), as well as of HIF-1α and at ASPET Journals on September 26, 2021 other bHLH-PAS proteins. HIF-1α is rapidly degraded under normoxic condition by the ubiquitin-proteasome system, whereas the level of Arnt is constant (Huang et al., 1998; Kallio et al., 1999). Hydroxylation of proline-564 and/or 402 residues in the oxygen- dependent degradation domain (ODD) of HIF-1α initiates its ubiquitination and subsequent proteasomal degradation (Masson and Ratcliffe, 2003). Prolyl hydroxylation of HIF-1α is catalyzed by a novel HIF-1α-specific prolyl hydroxylase which requires 2+ O2, 2-oxoglutarate, vitamin C, and Fe (Bruick and McKnight, 2001; Epstein et al., 2001; Ivan et al., 2001; Jaakkola et al., 2001). The tumor suppressor, von Hippel-Lindau (VHL) protein, which functions as an E3 ubiquitin ligase, interacts with the hydroxylated prolines of HIF-1α and brings about the assembly of a complex that activates a ubiquitin-dependent proteasome (Maxwell et al., 1999; Min et al., 2002; Ohh et al., 2000). When cells lack oxygen, proline hydroxylation ceases and HIF-1α protein 4 Molecular Pharmacology Fast Forward. Published on September 9, 2005 as DOI: 10.1124/mol.105.015271 This article has not been copyedited and formatted. The final version may differ from this version. MOL(15271) Regulation of PHD2 activity by MYND-Zn finger accumulates. In mammalian cells, a family of HIF-1α specific prolyl-4 hydroxylases has been identified and given the acronyms PHD1 (HPH3, EGLN2), PHD2 (HPH2, EGLN1), and PHD3 (HPH1, EGLN3) (Huang et al., 2002; Metzen et al., 2005; Taylor, 2001). Although all three PHDs hydroxylate prolines of HIF-1α in vitro, there is evidence that PHD2 has the primary role in vivo (Appelhoff et al., 2004; Berra et al., Downloaded from 2003; Hirsila et al., 2003; Huang et al., 2002). Thus, experiments employing short interfering RNAs revealed that silencing of PHD2 is enough to stabilize and activate molpharm.aspetjournals.org HIF-1α in normoxic cells (Berra et al., 2003). Moreover PHD2 is the most abundant of the three isoforms in most normoxic cells (Appelhoff et al., 2004). These findings suggest that each PHD has its own specific substrate, and that PHD2 is the major form responsible for hydroxylating HIF-1α, and therefore the critical oxygen sensor at ASPET Journals on September 26, 2021 maintaining the low steady state level of HIF-1α in normoxic conditions (Freeman et al., 2003; Huang et al., 2002; Masson et al., 2004; Masson and Ratcliffe, 2003). In addition to hypoxia, Co(II) ion and iron chelators, which inhibit the catalytic activity of PHDs, as well as other agents such as growth factors and the oncogenes Ras, active Src and Akt, have been reported to activate HIF-1α under normoxia (Chan et al., 2002; Karni et al., 2002; Zundel et al., 2000). It is not clear whether these non-hypoxic stimuli repress the catalytic activity of PHD2, to stabilize HIF-1α, or act in some other way. In this study we investigated whether the activity of PHD2 is regulated. By analyzing the catalytic activity of purified PHD2 and truncated mutants, we found that the N-terminal region of PHD2 contains a MYND-type Zn finger domain which inhibits catalytic activity. 5 Molecular Pharmacology Fast Forward. Published on September 9, 2005 as DOI: 10.1124/mol.105.015271 This article has not been copyedited and formatted. The final version may differ from this version. MOL(15271) Regulation of PHD2 activity by MYND-Zn finger Materials and Methods Cells, cDNAs and reagents Human epithelial HeLa cells were cultured in DMEM supplemented with 10% fetal bovine serum (Bio Whittaker), gentamicin (5 µg/ml, Life Technologies Gibco BRL) and Fungizone (0.25 µg/ml, Life Technologies Gibco BRL) Downloaded from in humidified air containing 5% CO2 at 37°C. Cells were made hypoxic by incubation in an anaerobic incubator (Model 1029, Forma Scientific, Inc.) in 5% CO2, 10% H2, and 85% N2 at 37°C or Multi-gas incubator (Model : NU-4950G, NuAire, Inc). We used the molpharm.aspetjournals.org following human cDNAs in expression vectors, transfection assays and in vitro transcription and translation experiments; PHD1 (AJ310544), PHD2 (AJ310543), PHD3 (AJ310545), HIF-1α (U22431), VHL (AF010238). The p(HRE)4-luc reporter plasmid contained 4 copies of the erythropoietin hypoxia-responsive element 5’- at ASPET Journals on September 26, 2021 GATCGCCCTACGTGCTGTCTCA-3’; nucleotides 3449-3470). Anti-HIF-1α was obtained from Transduction Laboratories. We obtained N, N, N’, N’-tetrakis (2- pyridylmethyl) ethylenediamine (TPEN) from Calbiochem, and all remaining chemicals from Sigma Chemical Co. Culture media were purchased from Gibco BRL, fetal bovine serum from Bio Whittaker. Expression of PHDs and HIF-1α Full-length cDNAs for PHD1, 2, and 3 were cloned from a human lymphocyte cDNA library into pcDNA3.1B(+) (Invitrogen).