TSG101為一多功能蛋白，參與細胞內許多生物作用的進行，包括調節轉錄作用、蛋白運輸、細胞生長及分化。許多文獻指出，細胞內TSG101蛋白steady-state量必須維持衡定，過多或過少都可能導致腫瘤的形成，然而，細胞內調控TSG101蛋白功能的訊息傳遞路徑尚不清楚。TSG101蛋白上有許多激酶的磷酸化位點，其中包含了2個GSK-3b的磷酸化位點，Ser172 (S172-P-Y-P-S176 ) 以及Ser202 (S202-Q-Y-P-S206)。實驗室前人以in vitro kinase assay得知GSK-3b可以磷酸化TSG101，本研究主旨為探討GSK-3b的訊息傳遞路徑對TSG101蛋白質之穩定性、轉譯後修飾及其在細胞中之分佈的影響。研究結果發現TSG101蛋白平時以接上一個單泛素之形式存在穩定於細胞內，且GSK-3b的活性會影響細胞內單泛素化TSG101蛋白表現量：在HeLa與HEp-2細胞中，透過血清挨餓或是轉染持續活化態GSK-3b之表達質體，可以提高TSG101蛋白的含量，且呈現劑量相關性之變化，反之，透過GSK-3b抑制劑，LiCl與TDZD8的處理，會導致TSG101的表現量下降，此現象可因共同處理proteasome抑制劑MG132而消失；在表現不同量之持續活化型GSK-3bmutS9A蛋白的細胞中，可發現野生型HA-TSG101、HA-TSG101mut S172176A蛋白量都隨著GSK-3b活性的增加而提升，但Ser202磷酸化位點突變的HA-TSG101mutS202206A蛋白量則是隨著GSK-3b活性的增加而減少；透過免疫共沉澱分析發現野生型與突變型HA-TSG101蛋白皆可與GFP-GSK-3b蛋白結合形成複合體，但是Ser202磷酸化位點突變之HA-TSG101蛋白的結合能力會減弱。綜合以上結果，我們推測TSG101蛋白會以接上一個單泛素的形式與GSK-3b形成複合體，且GSK-3b藉由磷酸化TSG101蛋白上Ser202的磷酸化位點來增加此單泛素化TSG101蛋白在細胞中的steady-state量，或許可藉此提升TSG101與GSK-3b複合體的穩定度，而GSK-3b訊息是否會影響TSG101被泛素化修飾的情形以及其在細胞中的蛋白定位，值得深入討論。

The TSG101 protein has been implicated in multiple biological functions including the regulation of gene transcription, vesicular trafficking, cellular growth and differentiation. Previous reports indicated the steady-state level of TSG101 must be maintained in a narrow range. Either deprivation or overexpression of TSG101 protein could result in neoplastic transformation. However, cellular signals that control TSG101 functions are not clear. TSG101 protein contains many kinase phosphorylation sites including two GSK-3β phosphorylation sites, S-172 (S172-P-Y-P-S176 ) and S202 (S202-Q-Y-P-S206). Our previous in vitro kinase assay result indicated TSG101 could be phosphorylated by GSK-3β. In the present study, we demonstrated that 47 kDa TSG101 is monoubiquitination of 42 kDa TSG101. The GSK-3β inhibitors, LiCl and TDZD8 could decrease TSG101 level in both HeLa and HEp-2 cells. On the contrary, activation of GSK-3β by serum starvation or by transfection of a plasmid encodes for constitutive active GSK-3β led to the increase of TSG101 level in a dose-dependent manner. The effect of LiCl and TDZD8 could be blocked by MG132, implying the involvement of proteaosome mediated mechanism. Expression of constitutive active GSK-3bmutS9A led to a dose-dependent increases of wildtype and HA-TSG101mutS172176A, but decrease of HA-TSG101mutS202206A protein. In addition, either wildtype or mutant HA-TSG101 could complex with GFP-GSK-3b. The mutation of S202 GSK-3b phosphorylation site of TSG101 compromised its ability to for complex with GSK-3b. In summary, our data support that GSK-3b is an important cellular signaling in regulation of monoubiquitinated TSG101 steady-state level. Whether it also affects the subcellular localization of TSG101 awaits further investigation.

中文摘要 1
英文摘要 3
前言 5
研究目的 21
材料與方法 22
結果 40
討論 52
未來展望 60
參考文獻 62
圖表 71

Al-Assar O, Crouch DH (2005). Retraction: Inactivation of MAP kinase signalling in Myc transformed cells and rescue by LiCl inhibition of GSK3. Mol Cancer 4: 17.

Alt JR, Cleveland JL, Hannink M, Diehl JA (2000). Phosphorylation-dependent regulation of cyclin D1 nuclear export and cyclin D1-dependent cellular transformation. Genes Dev 14: 3102-14.

Amit I, Yakir L, Katz M, Zwang Y, Marmor MD, Citri A et al (2004). Tal, a Tsg101-specific E3 ubiquitin ligase, regulates receptor endocytosis and retrovirus budding. Genes Dev 18: 1737-52.

Andersen SS, Ashford AJ, Tournebize R, Gavet O, Sobel A, Hyman AA et al (1997). Mitotic chromatin regulates phosphorylation of Stathmin/Op18. Nature 389: 640-3.

Baffa R, Negrini M, Mandes B, Rugge M, Ranzani GN, Hirohashi S et al (1996). Loss of heterozygosity for chromosome 11 in adenocarcinoma of the stomach. Cancer Res 56: 268-72.

Bhat RV, Shanley J, Correll MP, Fieles WE, Keith RA, Scott CW et al (2000). Regulation and localization of tyrosine216 phosphorylation of glycogen synthase kinase-3beta in cellular and animal models of neuronal degeneration. Proc Natl Acad Sci U S A 97: 11074-9.

Byrne JA, Simms LA, Little MH, Algar EM, Smith PJ (1993). Three non-overlapping regions of chromosome arm 11p allele loss identified in infantile tumors of adrenal and liver. Genes Chromosomes Cancer 8: 104-11.

Carstens MJ, Krempler A, Triplett AA, Van Lohuizen M, Wagner KU (2004). Cell cycle arrest and cell death are controlled by p53-dependent and p53-independent mechanisms in Tsg101-deficient cells. J Biol Chem 279: 35984-94.

Cheng TH, Cohen SN (2007). Human MDM2 isoforms translated differentially on constitutive versus p53-regulated transcripts have distinct functions in the p53/MDM2 and TSG101/MDM2 feedback control loops. Mol Cell Biol 27: 111-9.

Cohen P, Frame S (2001). The renaissance of GSK3. Nat Rev Mol Cell Biol 2: 769-76.

Cross DA, Alessi DR, Cohen P, Andjelkovich M, Hemmings BA (1995). Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature 378: 785-9.

Czech C, Tremp G, Pradier L (2000). Presenilins and Alzheimer's disease: biological functions and pathogenic mechanisms. Prog Neurobiol 60: 363-84.

DePaoli-Roach AA (1984). Synergistic phosphorylation and activation of ATP-Mg-dependent phosphoprotein phosphatase by F A/GSK-3 and casein kinase II (PC0.7). J Biol Chem 259: 12144-52.

Diehl JA, Cheng M, Roussel MF, Sherr CJ (1998). Glycogen synthase kinase-3beta regulates cyclin D1 proteolysis and subcellular localization. Genes Dev 12: 3499-511.

Embi N, Rylatt DB, Cohen P (1980). Glycogen synthase kinase-3 from rabbit skeletal muscle. Separation from cyclic-AMP-dependent protein kinase and phosphorylase kinase. Eur J Biochem 107: 519-27.

Farr GH, 3rd, Ferkey DM, Yost C, Pierce SB, Weaver C, Kimelman D (2000). Interaction among GSK-3, GBP, axin, and APC in Xenopus axis specification. J Cell Biol 148: 691-702.

Feng GH, Lih CJ, Cohen SN (2000). TSG101 protein steady-state level is regulated posttranslationally by an evolutionarily conserved COOH-terminal sequence. Cancer Res 60: 1736-41.

Finley D, Chau V (1991). Ubiquitination. Annu Rev Cell Biol 7: 25-69.

Fiol CJ, Haseman JH, Wang YH, Roach PJ, Roeske RW, Kowalczuk M et al (1988). Phosphoserine as a recognition determinant for glycogen synthase kinase-3: phosphorylation of a synthetic peptide based on the G-component of protein phosphatase-1. Arch Biochem Biophys 267: 797-802.

Frame S, Cohen P (2001). GSK3 takes centre stage more than 20 years after its discovery. Biochem J 359: 1-16.

Gantier R, Gilbert D, Dumanchin C, Campion D, Davoust D, Toma F et al (2000). The pathogenic L392V mutation of presenilin 1 decreases the affinity to glycogen synthase kinase-3 beta. Neurosci Lett 283: 217-20.

Garrus JE, von Schwedler UK, Pornillos OW, Morham SG, Zavitz KH, Wang HE et al (2001). Tsg101 and the vacuolar protein sorting pathway are essential for HIV-1 budding. Cell 107: 55-65.

Grimes CA, Jope RS (2001). The multifaceted roles of glycogen synthase kinase 3beta in cellular signaling. Prog Neurobiol 65: 391-426.

Haq S, Choukroun G, Kang ZB, Ranu H, Matsui T, Rosenzweig A et al (2000). Glycogen synthase kinase-3beta is a negative regulator of cardiomyocyte hypertrophy. J Cell Biol 151: 117-30.

Hartigan JA, Johnson GV (1999). Transient increases in intracellular calcium result in prolonged site-selective increases in Tau phosphorylation through a glycogen synthase kinase 3beta-dependent pathway. J Biol Chem 274: 21395-401.

Hicke L (2001). Protein regulation by monoubiquitin. Nat Rev Mol Cell Biol 2: 195-201.

Ikeda S, Kishida S, Yamamoto H, Murai H, Koyama S, Kikuchi A (1998). Axin, a negative regulator of the Wnt signaling pathway, forms a complex with GSK-3beta and beta-catenin and promotes GSK-3beta-dependent phosphorylation of beta-catenin. EMBO J 17: 1371-84.

Jope RS, Johnson GV (2004). The glamour and gloom of glycogen synthase kinase-3. Trends Biochem Sci 29: 95-102.

Katzmann DJ, Babst M, Emr SD (2001). Ubiquitin-dependent sorting into the multivesicular body pathway requires the function of a conserved endosomal protein sorting complex, ESCRT-I. Cell 106: 145-55.

Kim BY, Olzmann JA, Barsh GS, Chin LS, Li L (2007). Spongiform neurodegeneration-associated E3 ligase Mahogunin ubiquitylates TSG101 and regulates endosomal trafficking. Mol Biol Cell 18: 1129-42.

Kishida S, Yamamoto H, Ikeda S, Kishida M, Sakamoto I, Koyama S et al (1998). Axin, a negative regulator of the wnt signaling pathway, directly interacts with adenomatous polyposis coli and regulates the stabilization of beta-catenin. J Biol Chem 273: 10823-6.

Koonin EV, Abagyan RA (1997). TSG101 may be the prototype of a class of dominant negative ubiquitin regulators. Nat Genet 16: 330-1.

Krylova O, Messenger MJ, Salinas PC (2000). Dishevelled-1 regulates microtubule stability: a new function mediated by glycogen synthase kinase-3beta. J Cell Biol 151: 83-94.

Lee MP, Feinberg AP (1997). Aberrant splicing but not mutations of TSG101 in human breast cancer. Cancer Res 57: 3131-4.

Li L, Cohen SN (1996). Tsg101: a novel tumor susceptibility gene isolated by controlled homozygous functional knockout of allelic loci in mammalian cells. Cell 85: 319-29.

Li L, Li X, Francke U, Cohen SN (1997). The TSG101 tumor susceptibility gene is located in chromosome 11 band p15 and is mutated in human breast cancer. Cell 88: 143-54.

Li L, Liao J, Ruland J, Mak TW, Cohen SN (2001). A TSG101/MDM2 regulatory loop modulates MDM2 degradation and MDM2/p53 feedback control. Proc Natl Acad Sci U S A 98: 1619-24.

Li L, Yuan H, Weaver CD, Mao J, Farr GH, 3rd, Sussman DJ et al (1999). Axin and Frat1 interact with dvl and GSK, bridging Dvl to GSK in Wnt-mediated regulation of LEF-1. EMBO J 18: 4233-40.

Lin PM, Liu TC, Chang JG, Chen TP, Lin SF (1998). Aberrant TSG101 transcripts in acute myeloid leukaemia. Br J Haematol 102: 753-8.

Liu RT, Huang CC, You HL, Chou FF, Hu CC, Chao FP et al (2002). Overexpression of tumor susceptibility gene TSG101 in human papillary thyroid carcinomas. Oncogene 21: 4830-7.

Martin-Serrano J, Zang T, Bieniasz PD (2001). HIV-1 and Ebola virus encode small peptide motifs that recruit Tsg101 to sites of particle assembly to facilitate egress. Nat Med 7: 1313-9.

Maucuer A, Camonis JH, Sobel A (1995). Stathmin interaction with a putative kinase and coiled-coil-forming protein domains. Proc Natl Acad Sci U S A 92: 3100-4.

Moyret-Lalle C, Duriez C, Van Kerckhove J, Gilbert C, Wang Q, Puisieux A (2001). p53 induction prevents accumulation of aberrant transcripts in cancer cells. Cancer Res 61: 486-8.

Oh H, Mammucari C, Nenci A, Cabodi S, Cohen SN, Dotto GP (2002). Negative regulation of cell growth and differentiation by TSG101 through association with p21(Cip1/WAF1). Proc Natl Acad Sci U S A 99: 5430-5.

Ohta T, Fukuda M (2004). Ubiquitin and breast cancer. Oncogene 23: 2079-88.

Orford K, Crockett C, Jensen JP, Weissman AM, Byers SW (1997). Serine phosphorylation-regulated ubiquitination and degradation of beta-catenin. J Biol Chem 272: 24735-8.

Ou CY, Pi H, Chien CT (2003). Control of protein degradation by E3 ubiquitin ligases in Drosophila eye development. Trends Genet 19: 382-9.

Palombella VJ, Rando OJ, Goldberg AL, Maniatis T (1994). The ubiquitin-proteasome pathway is required for processing the NF-kappa B1 precursor protein and the activation of NF-kappa B. Cell 78: 773-85.

Piper RC, Luzio JP (2001). Late endosomes: sorting and partitioning in multivesicular bodies. Traffic 2: 612-21.

Plyte SE, Hughes K, Nikolakaki E, Pulverer BJ, Woodgett JR (1992). Glycogen synthase kinase-3: functions in oncogenesis and development. Biochim Biophys Acta 1114: 147-62.

Ponting CP, Cai YD, Bork P (1997). The breast cancer gene product TSG101: a regulator of ubiquitination? J Mol Med 75: 467-9.

Pornillos O, Alam SL, Davis DR, Sundquist WI (2002). Structure of the Tsg101 UEV domain in complex with the PTAP motif of the HIV-1 p6 protein. Nat Struct Biol 9: 812-7.

Pornillos O, Alam SL, Rich RL, Myszka DG, Davis DR, Sundquist WI (2002). Structure and functional interactions of the Tsg101 UEV domain. EMBO J 21: 2397-406.

Rice AM, Sartorelli AC (2001). Inhibition of 20 S and 26 S proteasome activity by lithium chloride: impact on the differentiation of leukemia cells by all-trans retinoic acid. J Biol Chem 276: 42722-7.

Richards PI, Benson MA, Steiner A (2003). In situ complexation of lithium chloride by amphiprotic cyclophosphazenes. Chem Commun (Camb): 1392-3.

Roach PJ (1990). Control of glycogen synthase by hierarchal protein phosphorylation. FASEB J 4: 2961-8.

Ross SE, Erickson RL, Hemati N, MacDougald OA (1999). Glycogen synthase kinase 3 is an insulin-regulated C/EBPalpha kinase. Mol Cell Biol 19: 8433-41.

Rubinfeld B, Albert I, Porfiri E, Fiol C, Munemitsu S, Polakis P (1996). Binding of GSK3beta to the APC-beta-catenin complex and regulation of complex assembly. Science 272: 1023-6.

Ruland J, Sirard C, Elia A, MacPherson D, Wakeham A, Li L et al (2001). p53 accumulation, defective cell proliferation, and early embryonic lethality in mice lacking tsg101. Proc Natl Acad Sci U S A 98: 1859-64.

Rylatt DB, Aitken A, Bilham T, Condon GD, Embi N, Cohen P (1980). Glycogen synthase from rabbit skeletal muscle. Amino acid sequence at the sites phosphorylated by glycogen synthase kinase-3, and extension of the N-terminal sequence containing the site phosphorylated by phosphorylase kinase. Eur J Biochem 107: 529-37.

Sanchez C, Diaz-Nido J, Avila J (2000). Phosphorylation of microtubule-associated protein 2 (MAP2) and its relevance for the regulation of the neuronal cytoskeleton function. Prog Neurobiol 61: 133-68.

Steiner P, Barnes DM, Harris WH, Weinberg RA (1997). Absence of rearrangements in the tumour susceptibility gene TSG101 in human breast cancer. Nat Genet 16: 332-3.

Sun W, Qureshi HY, Cafferty PW, Sobue K, Agarwal-Mawal A, Neufield KD et al (2002). Glycogen synthase kinase-3beta is complexed with tau protein in brain microtubules. J Biol Chem 277: 11933-40.

Sun Z, Pan J, Bubley G, Balk SP (1997). Frequent abnormalities of TSG101 transcripts in human prostate cancer. Oncogene 15: 3121-5.

Sun Z, Pan J, Hope WX, Cohen SN, Balk SP (1999). Tumor susceptibility gene 101 protein represses androgen receptor transactivation and interacts with p300. Cancer 86: 689-96.

ter Haar E, Coll JT, Austen DA, Hsiao HM, Swenson L, Jain J (2001). Structure of GSK3beta reveals a primed phosphorylation mechanism. Nat Struct Biol 8: 593-6.

Trink B, Pai SI, Spunt SL, Raman V, Cairns P, Jen J et al (1998). Absence of TSG101 transcript abnormalities in human cancers. Oncogene 16: 2815-8.

Turenne GA, Price BD (2001). Glycogen synthase kinase3 beta phosphorylates serine 33 of p53 and activates p53's transcriptional activity. BMC Cell Biol 2: 12.

Turpin E, Dalle B, de Roquancourt A, Plassa LF, Marty M, Janin A et al (1999). Stress-induced aberrant splicing of TSG101: association to high tumor grade and p53 status in breast cancers. Oncogene 18: 7834-7.

van Weeren PC, de Bruyn KM, de Vries-Smits AM, van Lint J, Burgering BM (1998). Essential role for protein kinase B (PKB) in insulin-induced glycogen synthase kinase 3 inactivation. Characterization of dominant-negative mutant of PKB. J Biol Chem 273: 13150-6.

VanDemark AP, Hofmann RM, Tsui C, Pickart CM, Wolberger C (2001). Molecular insights into polyubiquitin chain assembly: crystal structure of the Mms2/Ubc13 heterodimer. Cell 105: 711-20.

VerPlank L, Bouamr F, LaGrassa TJ, Agresta B, Kikonyogo A, Leis J et al (2001). Tsg101, a homologue of ubiquitin-conjugating (E2) enzymes, binds the L domain in HIV type 1 Pr55(Gag). Proc Natl Acad Sci U S A 98: 7724-9.

Wang NM, Chang JG, Liu TC, Lin SF, Peng CT, Tsai FJ et al (2000). Aberrant transcripts of FHIT, TSG101 and PTEN/MMAC1 genes in normal peripheral mononuclear cells. Int J Oncol 16: 75-80.

Wang QM, Fiol CJ, DePaoli-Roach AA, Roach PJ (1994). Glycogen synthase kinase-3 beta is a dual specificity kinase differentially regulated by tyrosine and serine/threonine phosphorylation. J Biol Chem 269: 14566-74.

Watanabe M, Yanagi Y, Masuhiro Y, Yano T, Yoshikawa H, Yanagisawa J et al (1998). A putative tumor suppressor, TSG101, acts as a transcriptional suppressor through its coiled-coil domain. Biochem Biophys Res Commun 245: 900-5.

Woodgett JR (1990). Molecular cloning and expression of glycogen synthase kinase-3/factor A. EMBO J 9: 2431-8.

Woodgett JR (1991). cDNA cloning and properties of glycogen synthase kinase-3. Methods Enzymol 200: 564-77.

Xavier IJ, Mercier PA, McLoughlin CM, Ali A, Woodgett JR, Ovsenek N (2000). Glycogen synthase kinase 3beta negatively regulates both DNA-binding and transcriptional activities of heat shock factor 1. J Biol Chem 275: 29147-52.

Xie W, Li L, Cohen SN (1998). Cell cycle-dependent subcellular localization of the TSG101 protein and mitotic and nuclear abnormalities associated with TSG101 deficiency. Proc Natl Acad Sci U S A 95: 1595-600.

Yamamoto H, Kishida S, Kishida M, Ikeda S, Takada S, Kikuchi A (1999). Phosphorylation of axin, a Wnt signal negative regulator, by glycogen synthase kinase-3beta regulates its stability. J Biol Chem 274: 10681-4.

Yost C, Farr GH, 3rd, Pierce SB, Ferkey DM, Chen MM, Kimelman D (1998). GBP, an inhibitor of GSK-3, is implicated in Xenopus development and oncogenesis. Cell 93: 1031-41.

Yost C, Torres M, Miller JR, Huang E, Kimelman D, Moon RT (1996). The axis-inducing activity, stability, and subcellular distribution of beta-catenin is regulated in Xenopus embryos by glycogen synthase kinase 3. Genes Dev 10: 1443-54.

You HL, Eng HL, Hsu SF, Chen CM, Ye TC, Liao WT et al (2007). A PKC-Sp1 signaling pathway induces early differentiation of human keratinocytes through upregulation of TSG101. Cell Signal 19: 1201-11.

Zhong Q, Chen Y, Jones D, Lee WH (1998). Perturbation of TSG101 protein affects cell cycle progression. Cancer Res 58: 2699-702.

Zhou BP, Deng J, Xia W, Xu J, Li YM, Gunduz M et al (2004). Dual regulation of Snail by GSK-3beta-mediated phosphorylation in control of epithelial-mesenchymal transition. Nat Cell Biol 6: 931-40.