中文摘要：
TSG101為一多功能蛋白，對大部分的蛋白質而言，特定生物功能的執行或是結構的穩定性，一般都會透過蛋白質磷酸化或其它蛋白修飾作用來加以調節。由胺基酸序列得知TSG101上具有GSK-3β磷酸化位點，經in vitro kinase assay得知GSK-3β確實可磷酸化TSG101。由文獻得知GSK-3β可調節其標的蛋白之穩定性及其細胞功能，本研究主旨為探討TSG101蛋白上之GSK-3β磷酸化位點對TSG101蛋白穩定性之影響。初期研究結果發現：若以GSK-3β抑制劑 LiCl處理COS1細胞，會致使內生性TSG101的表現量隨處理劑量而下降，故猜測GSK-3β對TSG101蛋白的磷酸化作用，對維持其蛋白穩定性有關鍵性的影響；在經轉染活化態GSK-3β表達質體GSK-3β/pEGFP情況下，TSG101的表現又有些變化，發現到有約40 kD左右TSG101的產物，也額外發現到有二個約50至80 kD之間的蛋白質產物，故猜測TSG101經GSK-3β的磷酸化作用後，可能對其蛋白質的修飾作用或降解有所影響；進一步以定點突變法，將TSG101上的GSK-3β磷酸化位點Serine突變成Alanine，再將此突變表達質體送入COS1細胞內表現，結果顯現具GSK-3β磷酸化位點突變的TSG101蛋白表現量比全長野生型的TSG101多，猜測突變的蛋白因無法被GSK-3β磷酸化作用而降解，故較野生型穩定；細胞經由LiCl處理後，野生型TSG101蛋白表現量會因GSK-3β受到抑制而回升，而S172AS176A、S172AS176AS202AS206A突變型也有上升的趨勢，但S202AS206A突變型則不受到LiCl的影響，故猜測GSK-3β可能透過S202磷酸化位點而調控TSG101蛋白之穩定性及其生物活性，值得深入探討。

Abstract：
Tumor susceptibility gene 101, TSG101, is a protein exhibits multiple biological functions. For most protein, its specific function or structure stability can be regulated through protein phosphorylation or modification. The analysis of the amino acid sequence of TSG101 revealed that it has two GSK-3β phosphorylation concensus sequences. Our previous data of in vitro kinase assay have demonstrated that TSG101 can be phosphorylated by GSK-3β, a wellknown protein kinase that regulates the stability and function of it’s target protein. To investigate the effect of GSK-3β phosphorylation on the stability and the function of TSG101 protein, we first exploited the effect of GSK-3βinhibitor, LiCl, on endogenous TSG101 protein in COS1 cells. The results suggested that inhibition of GSK-3β phosphorylation could impact on the stability of TSG101 protein. Upon the transfection of an active form GSK-3β expression plasmid GSK-3β/pEGFP, additional protein products of 40, 50-80 kD were detected, suggesting that GSK-3β phosphorylation might induce modification or degradation of TSG101 protein. GSK-3β phosphorylation site mutant TSG101 protein expression plasmids were constructed using site-directed mutagenesis, and were transfected into COS1 cells to evaluate the effect of GSK-3β on TSG101 level. The results showed that GSK-3β phosphorylation site mutant TSG101 protein is more stable then wild type TSG101 due to the lack of GSK-3β phosphorylation site. The inhibition of GSK-3β activity by LiCl treatment resulted in the increase of wildtype as well as the S172AS176 and S172AS176AS202AS206A mutant TSG101 proteins, whereas the S202AS206A mutant TSG101 protein level was not affected by LiCl treatment. The above data indicated that GSK-3β might regulate the stability and biological activity of TSG101 protein through phosphorylation of serine residue at position 202, which is worthy of further investigation.

中文摘要 1
英文摘要 2
背景介紹 3
材料與方法 9
結果 20
討論 24
參考文獻 28
圖表 35
附錄 51

參考文獻：
Al-Assar O and Crouch DH. (2005). Inactivation of MAP kinase signalling in Myc transformed cells and rescue by LiCl inhibition of GSK3. Mol Cancer, 4:13.

Ali IU, Lidereau R, Theillet C and Callahan R. (1987). Reduction to homozygosity of genes on chromosome 11 in human breast neoplasia. Scinece, 238:185-188.

Babst M, Odorizzi G, Estepa EJ and Emr SD. (2000). Mammalian tumor susceptibility gene 101 (TSG101) and the yeast homologue, Vps23p, both function in late endosomal trafficking. Traffic, 1:248-258.

Baffa R, Negrini M, Mandes B, Rugge M, Ranzani GN, Hirohashi S and Croce CM. (1996). Loss of heterozygosity for chromosome 11 in adenocarcinoma of the stomach. Cancer Res, 56:268-272

Bijur GN and Jope RS. (2000). Opposing action of phosphatidylinositol 3-kinase and glycogen synthase kinase-3β in the regulation of HSF-1 avtivity. J Neurochem, 75:2401-2408.

Bijur GN, DeSarno P and Jope RS. (2000). Glycogen synthase kinase-3β facilitates staurosporine- and heat shock-induced apoptosis. Protection by Lithium. J Biol Chem, 275:7583-7590.

Bournat JC, Brown AM, Soler AP. (2000). Wnt-1 dependent activation of the survival factor NF-κB in PC12 cell. J Neurosci Res, 61:21-32.

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 avtivity. Cell, 64:573-584.

Byrne JA, Simms LA, Little MH, Algar EM and Smith PJ. (1993). Three non-overlapping regions of chromosome arm 11p allale loss identified in infantile tumors of adrenal and liver. Genes Chromosomes Cancer, 8:104-111.

Chang JG, Su TH, Wei HJ, Wang JC, Chen YJ, Chang CP, Jeng CJ. (1999). Analysis of TSG101 tumour susceptibility gene transcripts in cervical and endometrial cancers. Br J Cancer, 79:445-450.

Carney ME, Maxwell GL, Lancaster JM, Gumbs C, Marks J, Berchuck A, Futreal PA. (1998). Aberrant splicing of the TSG101 tumor suppressor gene in human breast and ovarian cacers. J Soc Gynecol Investig, 5:281-285.

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, 279:35984-35994.

Chen YJ, Chen PH, Lin SY, Chang JG. (1999). Analysis of aberrant transcription of TSG101 in hepatocellular carcinomas. Eur J Cancer, 35:302-308.

Chu B, Soncin F, Price BD, Stevenson MA, Calderwood SK. (1996). Sequential phosphorylation by mitogen-activated protein kinase and glycogen synthase kinase 3 represses transcriptional avtivation by heat shock factor-1. J Biol Chem, 271:30847-30857.

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, 378:785-789.

Dale TC. (1998). Signal transduction by the Wnt Family of ligand. Biochem J, 329:209-223.

Datta SR, Brunet A, Greenberg ME. (1999). Cellular survival: a play in three Akt. Genes Deu, 13:2905-2927.

Demirov DG, Ono A, Orenstein JM, Freed EO. (2002). Overexpression of the N-terminal domain of TSG101 inhibits HIV-1 budding by blocking late domain function. Proc Natl Acad Sci USA, 99:955-960.

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, 107:519-527.

Feng GH, Lih CJ, Cohen SN. (2000). TSG101 protein steady-state level is regulated posttranslationally by an evolutionarily conserved COOH-terminal sequence. Cancer Res, 60:1736-1741.

Ferkey DM and Kimelman D. (2000). GSK-3. New thoughts on an old enzyme. Dev. Biol, 225:471-479.

Garrus JE, von Schwedler UK, Pornillos OW, Morham SG, Zavitz KH, Wang HE, Wettstein DA, Stray KM, Cote M, Rich RL, Myszka DG, Sundquist WI. (2001). Tsg101 and the vacuolar protein sorting pathway are essential for HIV-1 budding. Cell, 107:55-65.

Goff A, Ehrlich LS, Cohen SN, Carter CA. (2003). Tsg101 control of human immunodeficiency virus type 1 Gag trafficking and release. J Virol, 77:9173-9182.

Hetman M, Cavanaugh JE, Kimelman D, Xia Z. (2000). Role of glycogen synthase kinase-3β in neuronal apoptosis induced by trophic withdrawal. J Neurosci, 20:2567-2674.

Hosokawa Y, Nagai E, Seto M. (2000). Truncated TSG101 transcripts in human leukemia and lymphoma cell lines. J Cancer Res Clin Oncol, 126:79-84.

John son MC, Spidel JL, Ako-Adjei D, Wills JW, Vogt VM. (2005). The C-terminal half of TSG101 blocks Rous sarcoma virus budding and sequesters Gag into unique nonendosomal structures. J Virol, 79:3775-3786.

Koonin EV, Abagyan RA. (1997). TSG101 may be the prototype of a class of dominant negative ubiquitin regulators. Nat Genet, 16:330-331.

Lee MP and Feinberg AP. (1997). Aberrant splicing but not mutations of TSG101 in human breast cancer. Cancer Res, 57:3131-3134.

Li L, Cohen SN. (1996). Tsg101: a novel tumor susceptibility gene isolated by controlled homozygous functional knockout of allelic loci in mammalian cells. Cell, 85:319-329.

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, 88:143-154.

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 USA , 98:1619-1624.

Lin PM, Liu TC, Chang JG, Chen TP, Lin SF. (1998). Aberrant TSG101 transcripts in acute myeloid leukaemia. Br J Haematol, 102:753-758.

Liu RT, Huang CC, You HL, Chou FF, Hu CC, Chao FP, Chen CM, Chen JT. (2002). Overexpression of tumor susceptibility gene TSG101 in human papillary thyroid carcinomas. Oncogene, 21:4830-4837.

Li X, Bijur GN, Jope RS. (2002). Glycogen synthase kinase-3beta, mood stabilizers, and neuroprotection. Bipolar Disord, 4:137-144.

Lothe RA, Hastie N, Heimdal K, Fossa SD, Stenwig AE, Borresen AL. (1993). Frequent loss of 11p13 and 11p15 loci in male germ cell tumours. Genes Chromosomes Cancer, 7:96-101

Lo YF, Chen TC, Chen SC, Chao CC. (2000). Aberrant expression of TSG101 in Taiwan Chinese breast cancer. Breast Cancer Res Treat, 60:259-266.

Ludwig CU, Raefle G, Dalquen P, Stulz P, Stahel R, Obrecht JP. (1991). Allelic loss on the short arm of chromosome 11 in non-small-cell lung cancer. Int J Cancer, 49:661-665

Martin-Serrano J, Zang T, Bieniasz PD. (2003). Role of ESCRT-I in retroviral budding. J Virol, 77:4794-4804.

Maucuer A, Camonis JH, Sobel A. (1995). Stathmin interaction with a putative kinase and coiled-coil-forming protein domains. Proc Natl Acad Sci USA, 92:3100-3104.

Mclver B, Grebe SK, Wang L, Hay ID, Yokomizo A, Liu W, Goellner JR, Grant CS, Smith DI, Eberhardt NL. (2000). FHIT and TSG101 in thyroid tumours: aberrant transcripts reflect rare abnormal RNA processing events of uncertain pathogenetic or clinical significance. Clin Endocrinol (Oxf), 52:749-577.

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, 61:486-488.

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 p21Cip/WAF1. Proc Natl Acad Sci USA, 99:5430-5.

Oh Y, Proctor ML, Fan YH, Su LK, Hong WK, Fong KM, Sekido YS, Gazdar AF, Minna JD, Mao L. (1998). TSG101 is not mutated in lung cancer but a shortened transcript is frequently expressed in small cell lung cancer. Oncogene, 17:1141-1148.

Pap M and Cooper GM. (1998). Role of glycogen synthase kinase-3 in the phosphatidylinositol 3-kinase/Akt cell survival pathway. J Biol chem, 273:19929-19932.

Ponting CP, Cai YD, Bork P. (1997). The breast cancer gene product TSG101: a regulator of ubiquitination? J Mol Med, 75:467-469.

Pornillos O, Alam SL, Rich RL, Myszka DG, David DR, Sundquist WI. (2002). Structure and function interactions of the TSG101 UEV domain. EMBO J, 21:2397-2406.

Reeve AE, Sih SA, Raizis AM and Feinberg AP. (1989). Loss of allelic heterozygosity at a second locus on chromosome 11 in sporadic Wilm’s tumor cells. Mol Cell Biol, 9:1799-1803.

Rubinfeld B, Albert I, Porfiri R, Fiol C, Munemitsu S, Polakis P. (1996). Binding of GSK-3β to the APC-β-catenin complex and regulation of complex assembly. Science, 272:1023-1026.

Ruland J, Sirard C, Elia A, MacPherson D, Wakeham A, Li L, de la Pompa JL, Cohen SN, Mak TW. (2001). p53 accumulation, defective cell proliferation, and early embryonic lethality in mice lacking tsg101. Proc Natl Acad Sci U S A, 98:1859-1864.

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, 107:529-537.

Segura-Morales C, Pescia C, Chatellard-Causse C, Sadoul R, Bertrand E, Basyuk E. (2005). Tsg101 and Alix interact with MLV Gag and cooperate with Nedd4 ubiquitin-ligases during budding. J Biol Chem, 280:27004-27012.

Shaw ME and Knowles MA. (1995). Deletion mapping of chromosome 11 in carcinoma of the bladder. Genes Chromosomes Cancer, 13:1-8

Steiner P, Barnes DM, Harris WH, Weinberg RA. (1997). Absence of rearrangements in the tumour susceptibility gene TSG101 in human breast cancer. Nat Genet, 16:332-333.

Turenne GA and Price BD. (2001). Glycogen synthase kinase 3 beta phosphorylates serine 33 of p53 and activates p53’s transcriptional activity. BMC Cell Biol, 2:12

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

Weitzel JN, Patel J, Smith DM, Goodman A, Safaii H, Ball HG.. (1994). Molecular genetic changes associated with ovarian cancer. Gynecol Oncol, 55:245-252.

Willeke F, Ridder R, Bork P, Klaes R, Mechtersheimer G, Schwarzbach M, ZimmerD, Kloor M, Lehnert T, Herfarth C, von Kenbel Doeberitz M. (1998). Identical variant TSG101 transcripts in soft tissue sarcomas and various non-neoplastic tissues. Mol Carcinog, 23:195-200.

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 USA, 95:1595-1600.

Yost C, Torres M, Miller JR, Huang E, Kimelman D, Moon RT. (1996). The axis-inducing activity, stability, and subcellular distribution of β-catenin is regulated in Xenopus embryos by glycogen synthase kinase 3. Gene Deu, 10:1443-1454.

Zhong Q, Chen Y, Jones D, Lee WH. (1998). Perturbation of TSG101 protein affects cell cycle progression. Cancer Res, 58:2699-2702.

Zhou BP, Deng J, Xia W, Xu J, Li YM, Gunduz M, Hung MC. (2004). Dual regulation of Snail by GSK-3beta-mediated phosphorylation in control of epithelial-mesenchymal transition. Nat Cell Biol, 6:931-940.

蔡弘原. (2003). TSG101蛋白表現及其磷酸化之分析. 國立中山大學碩士論文.