金屬硫蛋白(MT)在哺乳類細胞中負責調節重金屬離子的含量，將細胞所需的離子維持在適當的濃度，並且螯合對細胞有毒害的金屬離子以減少細胞受損的情形。當細胞受到金屬離子刺激時，金屬感應轉錄因子(MTF-1)會與位於MT基因啟動子的金屬感應序列(MRE)結合，活化MT表現。雖然目前研究對於MTF-1的活化機制尚未十分了解，後修飾作用已被報導為調控其功能的重要因素之一。實驗室先前的研究發現腫瘤抑制因子PTEN與MTF-1會進行交互作用，且發現作用之位置在於MTF-1 acidic domain上，因此我們想了解PTEN對於acidic domain序列中胺基酸的磷酸化狀態是否有影響。我們首先將acidic domain中的serine及threonine進行點突變模擬胺基酸的磷酸化與否，以報導基因活性觀察磷酸化狀態是否影響到MTF-1的轉錄活性，發現單一位置的胺基酸突變並未造成顯著的影響。將該domain中唯一的tyrosine進行點突變後，同樣未顯著影響MTF-1之轉錄活性，亦並未影響MTF-1與PTEN的交互作用。最後我們在大腸桿菌中分別表現PTEN及MTF-1的acidic domain，發現即使該domain未被磷酸化也會與PTEN進行交互作用。

In mammalian cells, metallothionein (MT) functions in regulating the abundance of heavy metal ions, modulating essential metal ions and chelating those hazardous to avoid causing cell damages. Upon stimulation, metal responsive transcription factor-1 (MTF-1) binds to the metal response element (MRE) located on the promoter of MT gene, and induce activation. However, the detailed mechanism of how MTF-1 is regulated is still unknown. Post-translational modification has been reported as one of the key modulations for the function of MTF-1. Previous studies from our lab have confirmed interaction between MTF-1 and the tumor suppressor PTEN (phosphatase and tensin homologue deleted on chromosome 10), the interaction occurred at the acidic domain of MTF-1. Therefore, we attempted to identify the role of PTEN on the phosphorylation state of the acidic domain. First, we performed phospho-mimic experiments on the serines and threonines in the acidic domain with point mutation, and determined transcriptional activity of MTF-1 by reporter gene assay. We found that the mutation at single amino acid is insufficient to cause any effects. We then mutated the only tyrosine in the acidic domain, and obtained the same results: the transcriptional activity of MTF-1 was unaffected by the mutation, and neither was the interaction between MTF-1 and PTEN. Expression of PTEN and the acidic domain individually in E. coli followed by in vitro interaction assay revealed that the acidic domain is capable of interaction with PTEN regardless of its phosphorylation state. The result suggests that PTEN-MTF-1 interaction is independent of the phosphorylation status of the proteins.

Al-Khouri AM, Ma Y, Togo SH, Williams S, Mustelin T (2005) Cooperative phosphorylation of the tumor suppressor phosphatase and tensin homologue (PTEN) by casein kinases and glycogen synthase kinase 3β. J. Biol. Chem. 280: 35195-35202.

Ali IU, Schriml LM, Dean M (1999) Mutational spectra of PTEN/MMAC1 gene: a tumor suppressor with lipid phosphatase activity. J Natl Cancer Inst. 91: 1922-1932.

Auf der Maur A, Belser T, Elgar G, Georgiev O, Schaffner W (1999) Characterization of the transcription factor MTF-1 from the Japanese pufferfish (Fugu rubripes) reveals evolutionary conservation of heavy metal stress response. Biol Chem. 380: 175-185.

Boyle WJ, Smeal T, Defize LH, Angel P, Woodgett JR, Karin M, Hunter T (1991) Activation of protein kinase C decreases phosphorylation of c-Jun at sites that negatively regulate its DNA-binding activity. Cell 64: 573-584.

Bremner I, Beattie JH (1990) Metallothionein and the trace minerals. Annu Rev Nutr 10: 63-83.

Brugnera E, Georgiev O, Radtke F, Heuchel R, Baker E, Sutherland GR, Schaffner W (1994) Cloning, chromosomal mapping and characterization of the human metal-regulatory transcription factor MTF-1. Nucleic Acids Res. 22: 3167-3173.

Chagpar RB, Links PH, Pastor MC, Furber LA, Hawrysh AD, Chamberlain MD, Anderson DH (2010) Direct positive regulation of PTEN by the p85 subunit of phosphatidylinositol 3-kinase. Proc. Natl Acad. Sci. USA 107: 5471-5476.

Chen WY, John JA, Lin CH, Chang CY (2002) Molecular cloning and developmental expression of zinc finger transcription factor MTF-1 gene in zebrafish, Danio rerio. Biochem Biophys Res Commun. 291: 798-805.

Dalton T, Palmiter RD, Andrews GK (1994) Transcriptional induction of the mouse metallothionein-I gene in hydrogen peroxide-treated Hepa cells involves a composite major late transcription factor/antioxidant response element and metal response promoter elements. Nucleic Acids Res. 22: 5016-5023.

Datta J, Majumder S, Kutay H, Motiwala T, Frankel W, Costa R, Cha HC, MacDougald OA, Jacob ST, Ghoshal K (2007) Metallothionein expression is suppressed in primary human hepatocellular carcinomas and is mediated through inactivation of CCAAT/enhancer binding protein alpha by phosphatidylinositol 3-kinase signaling cascade. Cancer Res. 67: 2736-2746.

Denu JM, Stuckey JA, Saper MA, Dixon JE (1996) Form and function in protein dephosphorylation. Cell 87: 361-364.

Drinjakovic J, Jung H, Campbell DS, Strochlic L, Dwivedy A, Holt CE (2010) E3 ligase Nedd4 promotes axon branching by downregulating PTEN. Neuron 65: 341-357.

Eng C (2003) PTEN: one gene, many syndromes. Hum. Mutat. 22: 183-198.

Escrivà M, Peiró S, Herranz N, Villagrasa P, Dave N, Montserrat-Sentís B, Murray SA, Francí C, Gridley T, Virtanen I, García de Herreros A (2008) Repression of PTEN phosphatase by Snail1 transcriptional factor during γ-radiation induced apoptosis. Mol. Cell. Biol. 28: 1528-1540.

Faux MC, Scott JD (1996) More on target with protein phosphorylation: conferring specificity by location. Trends Biochem Sci. 21: 312-315.

Fine B, Hodakoski C, Koujak S, Su T, Saal LH, Maurer M, Hopkins B, Keniry M, Sulis ML, Mense S, Hibshoosh H, Parsons R (2009) Activation of the PI3K pathway in cancer through inhibition of PTEN by exchange factor P-REX2a. Science 325: 1261-1265.

Günes C, Heuchel R, Georgiev O, Müller KH, Lichtlen P, Blüthmann H, Marino S, Aguzzi A, Schaffner W (1998) Embryonic lethality and liver degeneration in mice lacking the metalresponsive transcriptional activator MTF-1. EMBO J. 17: 2846-2854.

Gericke A, Munson M, Ross AH (2006) Regulation of the PTEN phosphatase. Gene. 374: 1-9.

Goldhaber SB (2003) Trace element risk assessment: essentiality vs. toxicity. Regul Toxicol Pharmacol 38: 232-242.

Green CJ, Lichtlen P, Huynh NT, Yanovsky M, Laderoute KR, Schaffner W, Murphy BJ (2001) Placenta growth factor gene expression is induced by hypoxia in fibroblasts: a central role for metal transcription factor-1. Cancer Res. 61: 2696-2703.

Gu T, Zhang Z, Wang J, Guo J, Shen WH, Yin Y (2011) CREB is a novel nuclear target of PTEN phosphatase. Cancer Res. 71: 2821-2825.

Guerrerio AL, Berg JM (2004) Metal ion affinities of the zinc finger domains of the metal responsive element-binding transcription factor-1 (MTF1). Biochemistry 43: 5437-5444.

Guo L, Lichten LA, Ryu MS, Liuzzi JP, Wang F, Cousins RJ (2010) STAT5-glucocorticoid receptor interaction and MTF-1 regulate the expression of ZnT2 (Slc30a2) in pancreatic acinar cells. Proc. Natl. Acad. Sci. U. S. A. 107: 2818-2823.

Hamer DH (1986) Metallothionein. Ann. Rev. Biochem. 55: 913-951.

Hollander MC, Blumenthal GM, Dennis PA (2011) PTEN loss in the continuum of common cancers, rare syndromes and mouse models. Nature Rev. Cancer 11: 289-301.

Jiang H, Fu K, Andrews GK (2004) Gene- and cell-type-specific effects of signal transduction cascades on metal-regulated gene transcription appear to be independent of changes in the phosphorylation of metal-response-element-binding transcription factor-1. Biochem J. 382: 33-41.

Kaler P, Prasad R (2007) Molecular cloning and functional characterization of novel zinc transporter rZip10 (Slc39a10) involved in zinc uptake across rat renal brush-border membrane. Am. J. Physiol. Renal Physiol. 292: 217-229.

Karin M, Ben-Neriah Y (2000) Phosphorylation meets ubiquitination: the control of NF-[kappa]B activity. Annu Rev Immunol. 18: 621-663.

Langmade SJ, Ravindra R, Daniels PJ, Andrews GK (2000) The transcription factor MTF-1 mediates metal regulation of the mouse ZnT1 gene. J. Biol. Chem. 275: 34803-34809.

LaRochelle O, Gagné V, Charron J, Soh JW, Séguin C (2001a) Phosphorylation is involved in the activation of metal-regulatory transcription factor 1 in response to metal ions. J. Biol. Chem. 276: 41879-41888.

LaRochelle O, Stewart G, Moffatt P, Tremblay V, Séguin C (2001b) Characterization of the mouse metal-regulatory-element-binding proteins, metal element protein-1 and metal regulatory transcription factor-1. Biochem. J. 353: 591-601.

Lee JY, Kang MB, Jang SH, Qian T, Kim HJ, Kim CH, Kim Y, Kong G (2009) Id-1 activates Akt-mediated Wnt signaling and p27(Kip1) phosphorylation through PTEN inhibition. Oncogene 28: 824-831.

Li AG, Piluso LG, Cai X, Wei G, Seller WR, Liu X (2006) Mechanistic insights into maintenance of high p53 acetylation by PTEN. Mol Cell. 23: 575-587.

Li J, Yen C, Liaw D, Podsypanina K, Bose S, Wang SI, Puc J, Miliaresis C, Rodgers L, McCombie R, Bigner SH, Giovanella BC, Ittmann M, Tycko B, Hibshoosh H, Wigler MH, Parsons R (1997) PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer. Science 275: 1943-1947.

Li Y, Kimura T, Huyck RW, Laity JH, Andrews GK (2008) Zinc-induced formation of a coactivator complex containing the zinc-sensing transcription factor MTF-1, p300/CBP, and Sp1. Mol. Cell. Biol. 28: 4275-4284.

Li Z, Dong X, Wang Z, Liu W, Deng N, Ding Y, Tang L, Hla T, Zeng R, Li L, Wu D (2005) Regulation of PTEN by Rho small GTPases. Nature Cell Biol. 7: 399-404.

Lin HK, Hu YC, Lee DK, Chang C (2004) Regulation of androgen receptor signaling by PTEN (phosphatase and tensin homolog deleted on chromosome 10) tumor suppressor through distinct mechanisms in prostate cancer cells. Mol Endocrinol. 18: 2409-2423.

Lin MC, Liu YC, Tam MF, Lu YJ, Hsieh YT, Lin LY (2012) PTEN interacts with metal-responsive transcription factor 1 and stimulates its transcriptional activity. Biochem. J. 441: 367-337.

Lindert U, Cramer M, Meuli M, Georgiev O, Schaffner W (2009) Metal-responsive transcription factor 1 (MTF-1) activity is regulated by a nonconventional nuclear localization signal and a metal-responsive transactivation domain. Mol. Cell.Biol. 29: 6283–6293.

Lindert U, Leuzinger L, Steiner K, Georgiev O, Schaffner W (2008) Characterization of metal-responsive transcription factor (MTF-1) from the giant rodent capybara reveals features in common with human as well as with small rodents (mouse, rat). Short communication. Chem Biodivers. 5: 1485-1494.

Liu YC, Lin MC, Chen HC, Tam MF, Lin LY (2011 ) The role of small ubiquitin-like modifier-interacting motif in the assembly and regulation of metal-responsive transcription factor 1. J Biol Chem. 286: 42818-42829.

Lu J, Jeong HW, Kong N, Yang Y, Carroll J, Luo HR, Silberstein LE, Yupoma, Chai L (2009) Stem cell factor SALL4 represses the transcriptions of PTEN and SALL1 through an epigenetic repressor complex. PLoS ONE 4: e5577.

Ma XM, Blenis J (2009) Molecular mechanisms of mTOR-mediated translational control. Nature Rev. Mol. Cell Biol. 10: 307-318.

Maddika S, Kavela S, Rani N, Palicharla VR, Pokorny JL, Sarkaria JN, Chen J (2011) WWP2 is an E3 ubiquitin ligase for PTEN. Nature Cell Biol. 13: 728–733.

Maehama T, Dixon JE (1998) The tumor suppressor, PTEN/MMAC1, dephosphorylates the lipid second messenger, phosphatidylinositol 3,4,5 triphosphate. J. Biol. Chem. 273: 13375-13378.

Manning BD, Cantley LC (2007) AKT/PKB signaling: navigating downstream. Cell 129: 1261-1274.

Margoshes M, Vallee BL (1957) A cadmium protein from equine kidney cortex. Journal of the American Chemical Society 79: 4813-4814.

Mason AZ, Perico N, Moeller R, Thrippleton K, Potter T, Lloyd D (2004) Metal donation and apo-metalloenzyme activation by stable isotopically labeled metallothionein. Mar Environ Res. 58: 371-375.

Mavrakis KJ, Wolfe AL, Oricchio E, Palomero T, Keersmaecker K de, McJunkin K, Zuber J, James T, Khan AA, Leslie CS, Parker JS, Paddison PJ, Tam W, Ferrando A, Wendel HG (2010) Genome-wide RNA-mediated interference screen identifies miR‑19 targets in Notch-induced T‑cell acute lymphoblastic leukaemia. Nature Cell Biol. 12: 372-379.

Mehenni H, Lin-Marq N, Buchet-Poyau K, Reymond A, Collart MA, Picard D, Antonarakis SE (2005) LKB1 interacts with and phosphorylates PTEN: a functional link between two proteins involved in cancer predisposing syndromes. Hum Mol Genet. 14: 2209-2219.

Meng F, Henson R, Wehbe-Janek H, Ghoshal K, Jacob ST, Patel T (2007) MicroRNA-21 regulates expression of the PTEN tumor suppressor gene in human hepatocellular cancer. Gastroenterology 133: 647-658

Méplan C, Richard MJ, Hainaut P (2000) Metalloregulation of the tumor suppressor protein p53: zinc mediates the renaturation of p53 after exposure to metal chelators in vitro and in intact cells. Oncogene. 19: 5227-5236.

Murphy BJ, Kimura T, Sato BG, Shi Y, Andrews GK (2008) Metallothionein induction by hypoxia involves cooperative interactions between metal-responsive transcription factor-1 and hypoxia-inducible transcription factor-1alpha, . Mol. Cancer Res. 6: 483-490.

Myers MP, Stolarov JP, Eng C, Li J, Wang SI, Wigler MH, Parsons R, Tonks NK (1997) PTEN, the tumor suppressor from human chromosome 10q23, is a dual-specificity phosphatase. Proc. Natl Acad. Sci. USA 94: 9052-9057.

Okumura K, Mendoza M, Bachoo RM, DePinho RA, Cavenee WK, Furnari FB (2006) PCAF modulates PTEN activity. J. Biol. Chem. 281: 26562-26568

Okumura K, Zhao M, Depinho RA, Furnari FB, Cavenee WK (2005) Cellular transformation by the MSP58 oncogene is inhibited by its physical interaction with the PTEN tumor suppressor. Proc. Natl Acad. Sci. USA 102: 2703-2706.

Patel L, Pass I, Coxon P, Downes CP, Smith SA, Macphee CH (2001) Tumor suppressor and antiinflammatory actions of PPARγ agonists are mediated via upregulation of PTEN. Curr. Biol. 11: 764-768.

Radtke F, Georgiev O, Müller HP, Brugnera E, Schaffner W (1995) Functional domains of the heavy metal-responsive transcription regulator MTF-1. Nucleic Acids Res. 23: 2277-2286.

Raftopoulou M, Etienne-Manneville S, Self A, Nicholls S, Hall A (2004) Regulation of cell migration by the C2 domain of the tumor suppressor PTEN. Science 303: 1179-1181.

Salmena L, Carracedo A, Pandolfi PP (2008) Tenets of PTEN tumor suppression. Cell 133: 403-414.

Saydam N, Adams TK, Steiner F, Schaffner W, Freedman JH (2002) Regulation of metallothionein transcription by the metal-responsive transcription factor MTF-1: identification of signal transduction cascades that control metal-inducible transcription. J. Biol. Chem. 277: 20438-20445.

Saydam N, Georgiev O, Nakano MY, Greber UF, Schaffner W (2001) Nucleo-cytoplasmic trafficking of metal-regulatory transcription factor 1 is regulated by diverse stress signals. J. Biol. Chem. 276: 25487–25495.

Seo J, Lee KJ (2004) Post-translational Modifications and Their Biological Functions: Proteomic Analysis and Systematic Approaches. Journal of Biochemistry and Molecular Biology 37: 35-44.

Shen WH, Balajee AS, Wang J, Wu H, Eng C, Pandolfi PP, Yin Y (2007) Essential role for nuclear PTEN in maintaining chromosomal integrity. Cell 128: 157-170.

Song MS, Carracedo A, Salmena L, Song SJ, Egia A, Malumbres M, Pandolfi PP (2011) Nuclear PTEN regulates the APC-CDH1 tumor-suppressive complex in a phosphataseindependent manner. Cell 144: 187-199.

Song MS, Salmena L, Pandolfi PP (2012) The functions and regulation of the PTEN tumour suppressor. Nat Rev Mol Cell Biol. 13: 283-296.

Stambolic V, MacPherson D, Sas D, Lin Y, Snow B, Jang Y, Benchimol S, Mak TW (2001) Regulation of PTEN transcription by p53. Mol. Cell 8: 317-325.

Takahashi Y, Morales FC, Kreimann EL, Georgescu MM (2006) PTEN tumor suppressor associates with NHERF proteins to attenuate PDGF receptor signaling. EMBO J. 25: 910–920.

Tamguney T, Stokoe D (2007) New insights into PTEN. J Cell Sci. 120: 4071-4079.

Tamura M, Gu J, Matsumoto K, Aota S, Parsons R, Yamada KM (1998) Inhibition of cell migration, spreading, and focal adhesions by tumor suppressor PTEN. Science (280): 1614-1617.

Van Themsche C, Leblanc V, Parent S, Asselin E (2009) X-linked inhibitor of apoptosis protein (XIAP) regulates PTEN ubiquitination, content, and compartmentalization. J. Biol. Chem. 284: 20462-20466.

TK Adams, N Saydam, F Steiner, W Schaffner, JH Freedman (2002) Activation of gene expression by metal-responsive signal transduction pathways. Environ Health Perspect 5: 813-817.

Trotman LC, Wang X, Alimonti A, Chen Z, Teruya-Feldstein J, Yang H, Pavletich NP, Carver BS, Cordon-Cardo C, Erdjument-Bromage H, Tempst P, Chi SG, Kim HJ, Misteli T, Jiang X, Pandolfi PP (2007) Ubiquitination regulates PTEN nuclear import and tumor suppression. Cell 128: 141-156.

van Diepen MT, Parsons M, Downes CP, Leslie NR, Hindges R, Eickholt BJ (2009) MyosinV controls PTEN function and neuronal cell size. Nature Cell Biol. 11: 1191–1196

Vazquez F, Ramaswamy S, Nakamura N, Sellers WR (2000) Phosphorylation of the PTEN tail regulates protein stability and function. Mol. Cell. Biol. 20: 5010-5018.

Virolle T, Adamson ED, Baron V, Birle D, Mercola D, Mustelin T, de Belle I (2001) The Egr-1 transcription factor directly activates PTEN during irradiation-induced signalling. Nature Cell Biol. 3: 1124-1128.

Wang X, Jiang X (2008) Post-translational regulation of PTEN. Oncogene. 27: 5454-5463.

Wang Y, Wimmer U, Lichtlen P, Inderbitzin D, Stieger B, Meier PJ, Hunziker L, Stallmach T, Forrer R, Rülicke T, Georgiev O, Schaffner W (2004) Metal-responsive transcription factor-1 (MTF-1) is essential for embryonic liver development and heavy metal detoxification in the adult liver. FASEB J. 18: 1071-1079.

Wu X, Hepner K, Castelino-Prabhu S, Do D, Kaye MB, Yuan XJ, Wood J, Ross C, Sawyers CL, Whang YE (2000) Evidence for regulation of the PTEN tumor suppressor by a membrane-localized multi-PDZ domain containing scaffold protein MAGI-2. Proc. Natl Acad. Sci. USA 97: 4233–4238.

Yim EK, Peng G, Dai H, Hu R, Li K, Lu Y, Mills GB, Meric-Bernstam F, Hennessy BT, Craven RJ, Lin SY (2009) Rak functions as a tumor suppressor by regulating PTEN protein stability and function. Cancer Cell 15: 304-314.

Zhang B, Egli D, Georgiev O, Schaffner W (2001) The Drosophila homolog of mammalian zinc finger factor MTF-1 activates transcription in response to heavy metals. Mol Cell Biol. 21: 4505-4514.

Zoncu R, Efeyan A, Sabatini DM (2011) mTOR: from growth signal integration to cancer, diabetes and ageing. Nature Rev. Mol. Cell Biol. 12: 21-35.

曾維昌 (2008). "可怕的重金屬." from http://info.fy.org.tw/59/P09.pdf.