臺灣博碩士論文加值系統

許多的細胞外刺激皆會引起 MAPK 途徑的活化，這些刺激可能為紫外光、放射線、熱休克或促發炎細胞激素等。其中 MAPK 的活化主要藉上游激酶磷酸化 MAPK 活化環中的 TXY 區域。MAPK 蛋白被認為在調控細胞凋亡上扮演重要的角色；它也被認為和敗血症及關節炎造成的原因有密切的關係。而蛋白質轉譯後的相撲化修飾作用 (SUMOylation) 也負責調控許多細胞功能，諸如訊息傳遞、蛋白質的標靶運送、穩定蛋白質、轉錄作用的活化或調控細胞凋亡等。先前本實驗室研究結果得知當幽門螺旋桿菌感染時，SUMO-1的 mRNA 及蛋白質會表現較多，MAPK 途徑有被活化，細胞凋亡現象也有上升的情形。因此我們推測幽門螺旋桿菌感染相關的細胞凋亡現象可能與相撲化修飾作用及 MAPK 途徑有關係。
本實驗研究結果顯示當胃癌上皮細胞大量表現 RFP-SUMOs 或GFP-MAPK 時，MAPK 的磷酸化及細胞凋亡有上升的趨勢；特別在幽門螺旋桿菌感染時更加顯著。而此現象可被 MAPK 抑制劑所抑制。當 AGS 細胞大量表現GFP-MAPK 時，MAPK蛋白質磷酸化及細胞凋亡也有上升的趨勢，因此我們推測 MAPK 蛋白為 SUMOs 的修飾蛋白。

Diverse extracellular stimuli, including ultraviolet light, irradiation, heat shock, proinflammatory cytokines, trigger activation of MAPK pathway through phosphorylation on a TGY motif within the kinase activation loop. Protein MAPK appears to play a major role in apoptosis. It has been causally implicated in sepsis and arthritis. The translational small ubiquitin related modifier (SUMO) modification of proteins has been shown to play multiple functional roles in several cellular processes, including signal transduction, protein targeting, stabilization, transcriptional activation and apoptosis. Our previous study demonstrated that the expression levels of SUMO-1 rnRNA and proteins were enhanced in Helicobacter pylori infected human gastric epithelial cells. The activation of MAPK pathway and cellular apoptosis of AGS cell lines were increased during Helicobacter pylori infection. It was hypothesized that Helicobacter pylori functioning as a biological stress that induced MAPK mediated apoptosis which may be regulated by sumoylation.
Results showed that MAPK phosphorylation and cellular apoptosis were enhanced in RFP-SUMOs or GFP-MAPK expressing cells, especially during Helicobacter pylori infection. It was inhibited by pretreatment of MAPK inhibitor. The enhanced phosphorylation and apoptosis were observed during GFP-MAPK overexpression. It’s suggested that MAPK is a target protein for SUMOs.

中文摘要……………………………………………………………… 1
英文摘要……………………………………………………………… 2
縮寫表………………………………………………………………… 3
壹、緒論……………………………………………………………… 6
一、幽門螺旋桿菌 (Helicobacter pylori)…………………………… 6
1.1細胞毒素相關基因致病性基因島嶼
(Cytotoxin associated gene pathogenicity island, Cag-PAI)… 7
1.2 細胞毒素相關基因A (Cytotoxin associated gene A, CagA). 9
1.3 液泡式細胞毒素A (Vacuolating cytotoxin A, VacA)……… 11
1.4 幽門螺旋桿菌與細胞凋亡的關係………………………… 12
二、絲裂原活化蛋白激酶途徑 (MAPK pathway)………………… 14
2.1 p38 MAPK.………………………………………………… 15
2.2 p38 MAPK活化途徑及其功能…………………………… 16
2.3 p38α 結構與功能…………………………………………… 19
2.4 p38 MAPK於細胞內之位置與其功能…………………… 20
2.5 p38 MAPK與細胞凋亡的關係…………………………… 21
三、相撲蛋白 (Small ubiquitin-related modifiers, SUMOs)……….. 22
3.1 相撲化 (SUMOylation) …………………………………… 23
3.2 相撲蛋白在細胞中的功能………………………………….25
3.2.1 調控核體 (nuclear bodies) 的組成………………………25
3.2.2 調控蛋白質在細胞中的位置……………………………..26
3.2.3 調控蛋白質的活性及穩定性…………………………….. 28
3.3 相撲蛋白與細胞凋亡的關係………………………………. 30
貳、研究目的………………………………………………………… 31
參、實驗材料及方法………………………………………………… 32
一、 DNA Cloning…………………………………………………… 32
二、 E. Coli transformation及重組基因之確認……………………. 36
三、由點導引的突變產生法 (Site-directed mutagenesis) …………. 39
四、細胞培養…….………………………………………………… 41
五、基因轉殖……………………………………………………… 45
六、幽門螺旋桿菌感染 (Hp infection) …………………………… 47
七、蛋白質電泳與西方墨點法 (Western blots, WB) ……………… 49
八、螢光顯微鏡分析 (Fluorescence microscope analysis) ………… 55
九、免疫細胞化學法 (Immunocytochemistry, ICC) ……………… 57
十、細胞週期測試 (Cell cycle assay) .……………………………… 60
十一、蛋白質表現、純化及保存…………………………………… 62
十二、試管內相撲化反應 (In vitro sumoylation assay) …………… 67
肆、結果……………………………………………………………… 69
一、過度表現SUMOs時可放大p38的活化……………………… 69
二、 SUMOs可與內生性p-p38形成核體結構…………………… 72
三、野生型p38-wt及其突變質體的建構…………………………… 77
四、p38-79*與p38-P*不具放大細胞凋亡之功能…………………… 80
五、p38-79*可被磷酸化的能力明顯的下降………………………… 83
六、SB203580 抑制含 GFP-p38-wt 細胞凋亡之功能…………… 86
七、p38-wt，p38-79* 及 p38-P* 與 SUMO形成核體…………… 89
八、p38 聚集於 SUMOs-PML 核體上…………………………… 93
九、p38的試管中相撲化反應 (in vitro sumoylation) ……………… 96
伍、討論……………………………………………………………… 101
陸、未來工作………………………………………………………… 109
柒、參考文獻………………………………………………………… 110
捌、附錄……………………………………………………………… 127
一、SUMOs誘發p38 MAPK磷酸化的原始資料…………………… 127
二、GFP-p38-wt、GFP-p38-79*、GFP-p38-P*影響細胞凋亡的原始
資料……………………………………………………………… 128
三、GFP-p38-79*對於其磷酸化修飾功能之影響的原始資料……… 129
四、SB203580影響GFP-p38誘導之細胞凋亡的原始資料……… 130
圖目錄
圖一、p38 的活化在含 RFP-SUMOs 的細胞中明顯的增加……… 70
圖二 (A, B)、H. pylori 感染時內生性的p-p38在細胞中表現的情形 73
圖二 (C, D)、H. pylori 感染時，內生性的p-p38與不活化的
RFP-SUMOΔs 在細胞中作用的情形………………………… 75
圖二 (E, F)、H. pylori 感染時，內生性的p-p38與活化的RFP-SUMOs在細胞中作用的情形…………………………… 76
圖三 (A, B)、野生型GFP-p38及其突變質體的建構……………… 78
圖三 (C, D)、野生型GFP-p38及其突變質體蛋白的表現………… 79
圖四、GFP-p38-P* 與 GFP-p38-79* 無法放大細胞凋亡之功能… 81
圖五、突變型的GFP-p38-79* 可被磷酸化的能力明顯的下降…… 84
圖六、SB203580 抑制含 GFP-p38-wt細胞凋亡之功能…………… 87
圖七、GFP-p38 與 RFP-SUMOs 在細胞中的交互作用…………… 90
圖八、GFP-p38-wt和其突變蛋白與 RFP-SUMOs-PML共存於核體 94
圖九、p38 的試管中相撲化反應 (A) …….………………………… 98
圖九、p38 的試管中相撲化反應 (B) …….………………………… 99
αΔ

[1] Algeciras-Schimnich A, Shen L, Barnhart BC, Murmann AE, Burkhardt JK, Peter ME. (2002) Molecular ordering of the initial signaling events of CD95. Mol Cell Biol. 22(1):207-20.

[2] Allison CC, Kufer TA, Kremmer E, Kaparakis M, Ferrero RL. (2009) Helicobacter pylori induce MAPK phosphorylation and AP-1 activation via a NOD1-dependent mechanism. J Immunol. 183(12):8099-109.

[3] Ashktorab H, Ahmed A, Littleton G, Wang XW, Allen CR, Tackey R, Walters C, Smoot DT. (2003) p53 and p14 increase sensitivity of gastric cells to H. pylori-induced apoptosis. Dig Dis Sci. 48(7):1284-91.

[4] Avruch J. (1998) Insulin signal transduction through protein kinase cascades. Mol Cell Biochem. 182(1-2):31-48.

[5] Azuma T, Yamazaki S, Yamakawa A, Ohtani M, Muramatsu A, Suto H, Ito Y, Dojo M, Yamazaki Y, Kuriyama M, Keida Y, Higashi H, Hatakeyama M. (2004) Association between diversity in the Src homology 2 domain--containing tyrosine phosphatase binding site of Helicobacter pylori CagA protein and gastric atrophy and cancer. J Infect Dis. 189(5):820-7.

[6] Backert S and Selbach M. (2008) Role of type IV secretion in Helicobacter pylori pathogenesis. Cell Microbiol. 10(8):1573-81.

[7] Barger PM, Browning AC, Garner AN, Kelly DP. (2001) p38 mitogen-activated protein kinase activates peroxisome proliferator-activated receptor alpha: a potential role in the cardiac metabolic stress response. J Biol Chem. 276(48):44495-501.

[8] Bayer P, Arndt A, Metzger S, Mahajan R, Melchior F, Jaenicke R, Becker J. (1998) Structure determination of the small ubiquitin-related modifier SUMO-1. J Mol Biol. 280(2):275-86.

[9] Ben-Levy R, Hooper S, Wilson R, Paterson HF, Marshall CJ. (1998) Nuclear export of the stress-activated protein kinase p38 mediated by its substrate MAPKAP kinase-2. Curr Biol. 8(19):1049-57.

[10] Bland DA, Suarez G, Beswick EJ, Sierra JC, Reyes VE. (2006) H. pylori receptor MHC class II contributes to the dynamic gastric epithelial apoptotic response. World J Gastroenterol. 12(33):5306-10.

[11] Blaser MJ and Atherton JC. (2004) Helicobacter pylori persistence: biology and disease. J Clin Invest. 113(3):321-33

[12] Boncristiano M, Paccani SR, Barone S, Ulivieri C, Patrussi L, Ilver D, Amedei A, D''Elios MM, Telford JL, Baldari CT. (2003) The Helicobacter pylori vacuolating toxin inhibits T cell activation by two independent mechanisms. J Exp Med. 198(12):1887-97.

[13] Borden KL. (2002) Pondering the promyelocytic leukemia protein (PML) puzzle: possible functions for PML nuclear bodies. Mol Cell Biol. 22(15):5259-69.

[14] Bossis G and Melchior F. (2006) SUMO: regulating the regulator. Cell Div. 1:13.

[15] Bourzac KM and Guillemin K. (2005) Helicobacter pylori-host cell interactions mediated by type IV secretion. Cell Microbiol. 7(7):911-9.

[16] Brand P, Plochmann S, Valk E, Zahn S, Saloga J, Knop J, Becker D. (2002) Activation and translocation of p38 mitogen-activated protein kinase after stimulation of monocytes with contact sensitizers. J Invest Dermatol. 119(1):99-106.

[17] Braun H, Koop R, Ertmer A, Nacht S, Suske G. (2001) Transcription factor Sp3 is regulated by acetylation. Nucleic Acids Res. 29(24):4994-5000.

[18] Bulavin DV, Higashimoto Y, Popoff IJ, Gaarde WA, Basrur V, Potapova O, Appella E, Fornace AJ Jr. (2001) Initiation of a G2/M checkpoint after ultraviolet radiation requires p38 kinase. Nature. 411(6833):102-7.

[19] Cascales E and Christie PJ. (2003) The versatile bacterial type IV secretion systems. Nat Rev Microbiol. 1(2):137-49.

[20] Censini S, Lange C, Xiang Z, Crabtree JE, Ghiara P, Borodovsky M, Rappuoli R, Covacci A. (1996) cag, a pathogenicity island of Helicobacter pylori, encodes type I-specific and disease-associated virulence factors. Proc Natl Acad Sci USA. 93(25):14648-53

[21] Chen A, Wang PY, Yang YC, Huang YH, Yeh JJ, Chou YH, Cheng JT, Hong YR, Li SS. (2006) SUMO regulates the cytoplasmonuclear transport of its target protein Daxx. J Cell Biochem. 98(4):895-911.

[22] Chang CI, Xu BE, Akella R, Cobb MH, Goldsmith EJ. (2002) Crystal structures of MAP kinase p38 complexed to the docking sites on its nuclear substrate MEF2A and activator MKK3b. Mol Cell. 9(6):1241-9.

[23] Chen YR, Wang X, Templeton D, Davis RJ, Tan TH. (1996) The role of c-Jun N-terminal kinase (JNK) in apoptosis induced by ultraviolet C and gamma radiation. Duration of JNK activation may determine cell death and proliferation. J Biol Chem. 271(50):31929-36.

[24] Chen Z, Gibson TB, Robinson F, Silvestro L, Pearson G, Xu B, Wright A, Vanderbilt C, Cobb MH. (2001) MAP kinases. Chem Rev. 101(8):2449-76.

[25] Churin Y, Al-Ghoul L, Kepp O, Meyer TF, Birchmeier W, Naumann M. (2003) Helicobacter pylori CagA protein targets the c-Met receptor and enhances the motogenic response. J Cell Biol. 161(2):249-55.

[26] Cobb MH, Goldsmith EJ. (1995) How MAP kinases are regulated. J Biol Chem. 270(25):14843-6.

[27] Court NW, dos Remedios CG, Cordell J, Bogoyevitch MA. (2002) Cardiac expression and subcellular localization of the p38 mitogen-activated protein kinase member, stress-activated protein kinase-3 (SAPK3). J Mol Cell Cardiol. 34(4):413-26.

[28] Cover TL, Hanson PI, Heuser JE. (1997) Acid-induced dissociation of VacA, the Helicobacter pylori vacuolating cytotoxin, reveals its pattern of assembly. J Cell Biol. 138(4):759-69.

[29] Crabtree JE, Covacci A, Farmery SM, Xiang Z, Tompkins DS, Perry S, Lindley IJ, Rappuoli R. (1995) Helicobacter pylori induced interleukin-8 expression in gastric epithelial cells is associated with CagA positive phenotype. J Clin Pathol. 48(1):41-5.

[30] Cuadrado A, Lafarga V, Cheung PC, Dolado I, Llanos S, Cohen P, Nebreda AR. (2007) A new p38 MAP kinase-regulated transcriptional coactivator that stimulates p53-dependent apoptosis. EMBO J. 26(8):2115-26.

[31] Curtin JF and Cotter TG. (2003) Live and let die: regulatory mechanisms in Fas-mediated apoptosis. Cell Signal. 15(11):983-92.

[32] Czajkowsky DM, Iwamoto H, Cover TL, Shao Z. (1999) The vacuolating toxin from Helicobacter pylori forms hexameric pores in lipid bilayers at low pH. Proc Natl Acad Sci U S A. 96(5):2001-6.

[33] Dean JL, Brook M, Clark AR, Saklatvala J. (1999) p38 mitogen-activated protein kinase regulates cyclooxygenase-2 mRNA stability and transcription in lipopolysaccharide-treated human monocytes. J Biol Chem. 274(1):264-9.

[34] Dérijard B, Raingeaud J, Barrett T, Wu IH, Han J, Ulevitch RJ, Davis RJ. (1995) Independent human MAP-kinase signal transduction pathways defined by MEK and MKK isoforms. Science. 267(5198):682-5.

[35] Desterro JM, Rodriguez MS, Hay RT. (1998) SUMO-1 modification of IkappaBalpha inhibits NF-kappaB activation. Mol Cell. 2(2):233-9.

[36] Ding SZ, Smith MF Jr, Goldberg JB. (2008) Helicobacter pylori and mitogen-activated protein kinases regulate the cell cycle, proliferation and apoptosis in gastric epithelial cells. J Gastroenterol Hepatol. 23(7 Pt 2):e67-78.

[37] Ferrigno P, Posas F, Koepp D, Saito H, Silver PA. (1998) Regulated nucleo/cytoplasmic exchange of HOG1 MAPK requires the importin beta homologs NMD5 and XPO1. EMBO J. 17(19):5606-14.

[38] Fischer W, Puls J, Buhrdorf R, Gebert B, Odenbreit S, Haas R. (2001) Systematic mutagenesis of the Helicobacter pylori cag pathogenicity island: essential genes for CagA translocation in host cells and induction of interleukin-8. Mol Microbiol. 42(5):1337-48.

[39] Freshney NW, Rawlinson L, Guesdon F, Jones E, Cowley S, Hsuan J, Saklatvala J. (1994) Interleukin-1 activates a novel protein kinase cascade that results in the phosphorylation of Hsp27. Cell 78:1039–1049.

[40] Fu C, Ahmed K, Ding H, Ding X, Lan J, Yang Z, Miao Y, Zhu Y, Shi Y, Zhu J, Huang H, Yao X. (2005) Stabilization of PML nuclear localization by conjugation and oligomerization of SUMO-3. Oncogene. 24(35):5401-13.

[41] Galmiche A, Rassow J, Doye A, Cagnol S, Chambard JC, Contamin S, de Thillot V, Just I, Ricci V, Solcia E, Van Obberghen E, Boquet P. (2000) The N-terminal 34 kDa fragment of Helicobacter pylori vacuolating cytotoxin targets mitochondria and induces cytochrome c release. EMBO J. 19(23):6361-70.

[42] Geiss-Friedlander R, Melchior F. (2007) Concepts in sumoylation: a decade on. Nat Rev Mol Cell Biol. 8(12):947-56.

[43] Gorog DA, Jabr RI, Tanno M, Sarafraz N, Clark JE, Fisher SG, Cao XB, Bellahcene M, Dighe K, Kabir AM, Quinlan RA, Kato K, Gaestel M, Marber MS, Heads RJ. (2009) MAPKAPK-2 modulates p38-MAPK localization and small heat shock protein phosphorylation but does not mediate the injury associated with p38-MAPK activation during myocardial ischemia. Cell Stress Chaperones. 14(5):477-89.

[44] Gostissa M, Hengstermann A, Fogal V, Sandy P, Schwarz SE, Scheffner M, Del Sal G. (1999) Activation of p53 by conjugation to the ubiquitin-like protein SUMO-1. EMBO J. 18(22):6462-71.

[45] Gresko E, Möller A, Roscic A, Schmitz ML. (2005) Covalent modification of human homeodomain interacting protein kinase 2 by SUMO-1 at lysine 25 affects its stability. Biochem Biophys Res Commun. 329(4):1293-9.

[46] Guo D, Li M, Zhang Y, Yang P, Eckenrode S, Hopkins D, Zheng W, Purohit S, Podolsky RH, Muir A, Wang J, Dong Z, Brusko T, Atkinson M, Pozzilli P, Zeidler A, Raffel LJ, Jacob CO, Park Y, Serrano-Rios M, Larrad MT, Zhang Z, Garchon HJ, Bach JF, Rotter JI, She JX, Wang CY. (2004) A functional variant of SUMO4, a new I kappa B alpha modifier, is associated with type 1 diabetes. Nat Genet. 36(8):837-41.

[47] Han J, Jiang Y, Li Z, Kravchenko VV, Ulevitch RJ. (1997) Activation of the transcription factor MEF2C by the MAP kinase p38 in inflammation. Nature. 386(6622):296-9.

[48] Han J, Lee JD, Bibbs L, Ulevitch RJ. (1994) A MAP kinase targeted by endotoxin and hyperosmolarity in mammalian cells. Science. 265(5173):808-11.

[49] Hanks SK and Hunter T. (1995) Protein kinases 6. The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification. FASEB J. 9(8):576-96.

[50] Hatakeyama M. (2004) Oncogenic mechanisms of the Helicobacter pylori CagA protein. Nat Rev Cancer. 4(9):688-94.

[51] He LZ, Merghoub T, Pandolfi PP. (1999) In vivo analysis of the molecular pathogenesis of acute promyelocytic leukemia in the mouse and its therapeutic implications. Oncogene. 18(38):5278-92.

[52] Higashi H, Tsutsumi R, Muto S, Sugiyama T, Azuma T, Asaka M, Hatakeyama M. (2002) SHP-2 tyrosine phosphatase as an intracellular target of Helicobacter pylori CagA protein. Science. 295(5555):683-6.

[53] Higashi H, Nakaya A, Tsutsumi R, Yokoyama K, Fujii Y, Ishikawa S, Higuchi M, Takahashi A, Kurashima Y, Teishikata Y, Tanaka S, Azuma T, [54] Hatakeyama M. (2004) Helicobacter pylori CagA induces Ras-independent morphogenetic response through SHP-2 recruitment and activation. J Biol Chem. 279(17):17205-16.

[55] Hofmann TG and Will H. (2003) Body language: the function of PML nuclear bodies in apoptosis regulation. Cell Death Differ. 10(12):1290-9.

[56] Huang TT, Wuerzberger-Davis SM, Wu ZH, Miyamoto S. (2003) Sequential modification of NEMO/IKKgamma by SUMO-1 and ubiquitin mediates NF-kappaB activation by genotoxic stress. Cell. 115(5):565-76.

[57] Johnson ES. (2004) Protein modification by SUMO. Annu Rev Biochem. 73:355-82.

[58] Johnson ES and Blobel G. (1997) Ubc9p is the conjugating enzyme for the ubiquitin-like protein Smt3p. J Biol Chem. 272(43):26799-802.

[59] Johnson ES, Schwienhorst I, Dohmen RJ, Blobel G. (1997) The ubiquitin-like protein Smt3p is activated for conjugation to other proteins by an Aos1p/Uba2p heterodimer. EMBO J. 16(18):5509-19.

[60] Johnson NL, Gardner AM, Diener KM, Lange-Carter CA, Gleavy J, Jarpe MB, Minden A, Karin M, Zon LI, Johnson GL. (1996) Signal transduction pathways regulated by mitogen-activated/extracellular response kinase kinase kinase induce cell death. J Biol Chem. 271(6):3229-37.

[61] Johnson SE, Shah N, Bajer AA, LeBien TW. (2008) IL-7 activates the phosphatidylinositol 3-kinase/AKT pathway in normal human thymocytes but not normal human B cell precursors. J Immunol. 180(12):8109-17.

[62] Kaukinen P, Vaheri A, Plyusnin A. (2003) Non-covalent interaction between nucleocapsid protein of Tula hantavirus and small ubiquitin-related modifier-1, SUMO-1. Virus Res. 92(1):37-45.

[63] Knipscheer P, Flotho A, Klug H, Olsen JV, van Dijk WJ, Fish A, Johnson ES, Mann M, Sixma TK, Pichler A. (2008) Ubc9 sumoylation regulates SUMO target discrimination. Mol Cell. 31(3):371-82.

[64] Ko LJ and Prives C. (1996) p53: puzzle and paradigm. Genes Dev. 10(9):1054-72.

[65] Kuck D, Kolmerer B, Iking-Konert C, Krammer PH, Stremmel W, Rudi J. (2001) Vacuolating cytotoxin of Helicobacter pylori induces apoptosis in the human gastric epithelial cell line AGS. Infect Immun. 69(8):5080-7.

[66] Kumar S, McDonnell PC, Gum RJ, Hand AT, Lee JC, Young PR. (1997) Novel homologues of CSBP/p38 MAP kinase: activation, substrate specificity and sensitivity to inhibition by pyridinyl imidazoles. Biochem Biophys Res Commun. 235(3):533-8.

[67] Kyriakis JM and Avruch J. (2001) Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation. Physiol Rev. 81(2):807-69.

[68] Lanzavecchia S, Bellon PL, Lupetti P, Dallai R, Rappuoli R, Telford JL. (1998) Three-dimensional reconstruction of metal replicas of the Helicobacter pylori vacuolating cytotoxin. J Struct Biol. 121(1):9-18.

[69] Lee JC, Laydon JT, McDonnell PC, Gallagher TF, Kumar S, Green D, McNulty D, Blumenthal MJ, Heys JR, (1994) A protein kinase involved in the regulation of inflammatory cytokine biosynthesis. Nature. 372(6508):739-46.

[70] Li Q, Zhang N, Zhang D, Wang Y, Lin T, Wang Y, Zhou H, Ye Z, Zhang F, Lin SC, Han J. (2008) Determinants that control the distinct subcellular localization of p38alpha-PRAK and p38beta-PRAK complexes. J Biol Chem. 283(16):11014-23.

[71] Li X, Zhang R, Luo D, Park SJ, Wang Q, Kim Y, Min W. (2005) Tumor necrosis factor alpha-induced desumoylation and cytoplasmic translocation of homeodomain-interacting protein kinase 1 are critical for apoptosis signal-regulating kinase 1-JNK/p38 activation. J Biol Chem. 280(15):15061-70.

[72] Lin DY, Huang YS, Jeng JC, Kuo HY, Chang CC, Chao TT, Ho CC, Chen YC, Lin TP, Fang HI, Hung CC, Suen CS, Hwang MJ, Chang KS, Maul GG, Shih HM. (2006) Role of SUMO-interacting motif in Daxx SUMO modification, subnuclear localization, and repression of sumoylated transcription factors. Mol Cell. 24(3):341-54.

[73] Liou ML and Liou HC. (1999) The ubiquitin-homology protein, DAP-1, associates with tumor necrosis factor receptor (p60) death domain and induces apoptosis. J Biol Chem. 274(15):10145-53.

[74] Lu G, Kang YJ, Han J, Herschman HR, Stefani E, Wang Y. (2006) TAB-1 modulates intracellular localization of p38 MAP kinase and downstream signaling. J Biol Chem. 281(9):6087-95.

[75] Lupetti P, Heuser JE, Manetti R, Massari P, Lanzavecchia S, Bellon PL, Dallai R, Rappuoli R, Telford JL. (1996) Oligomeric and subunit structure of the Helicobacter pylori vacuolating cytotoxin. J Cell Biol. 133(4):801-7.

[76] Mabb AM, Wuerzberger-Davis SM, Miyamoto S. (2006) PIASy mediates NEMO sumoylation and NF-kappaB activation in response to genotoxic stress. Nat Cell Biol. 8(9):986-93.

[77] MacFarlane M, Cohen GM, Dickens M. (2000) JNK (c-Jun N-terminal kinase) and p38 activation in receptor-mediated and chemically-induced apoptosis of T-cells: differential requirements for caspase activation. Biochem J. 348 Pt 1:93-101.

[78] Maeda A, Lee BH, Yoshimatsu K, Saijo M, Kurane I, Arikawa J, Morikawa S. (2003) The intracellular association of the nucleocapsid protein (NP) of hantaan virus (HTNV) with small ubiquitin-like modifier-1 (SUMO-1) conjugating enzyme 9 (Ubc9). Virology. 305(2):288-97.

[79] Mahajan R, Delphin C, Guan T, Gerace L, Melchior F. (1997) A small ubiquitin-related polypeptide involved in targeting RanGAP1 to nuclear pore complex protein RanBP2. Cell. 88(1):97-107.

[80] Mahajan R, Gerace L, Melchior F. (1998) Molecular characterization of the SUMO-1 modification of RanGAP1 and its role in nuclear envelope association. J Cell Biol. 140(2):259-70.

[81] Maltzman W and Czyzyk L. (1984) UV irradiation stimulates levels of p53 cellular tumor antigen in nontransformed mouse cells. Mol Cell Biol. 4(9):1689-94.

[82] Maniatis T. (1999) A ubiquitin ligase complex essential for the NF-kappaB, Wnt/Wingless, and Hedgehog signaling pathways. Genes Dev. 13(5):505-10.

[83] Manke IA, Nguyen A, Lim D, Stewart MQ, Elia AE, Yaffe MB. (2005) MAPKAP kinase-2 is a cell cycle checkpoint kinase that regulates the G2/M transition and S phase progression in response to UV irradiation. Mol Cell. 17(1):37-48.

[84] Markström E, Svensson ECh, Shao R, Svanberg B, Billig H. (2002) Survival factors regulating ovarian apoptosis -- dependence on follicle differentiation. Reproduction. 123(1):23-30.

[85] Matunis MJ, Coutavas E, Blobel G. (1996) A novel ubiquitin-like modification modulates the partitioning of the Ran-GTPase-activating protein RanGAP1 between the cytosol and the nuclear pore complex. J Cell Biol. 135(6 Pt 1):1457-70.

[86] Matunis MJ, Wu J, Blobel G. (1998) SUMO-1 modification and its role in targeting the Ran GTPase-activating protein, RanGAP1, to the nuclear pore complex. J Cell Biol. 140(3):499-509.

[87] Mbalaviele G, Anderson G, Jones A, De Ciechi P, Settle S, Mnich S, Thiede M, Abu-Amer Y, Portanova J, Monahan J. (2006) Inhibition of p38 mitogen-activated protein kinase prevents inflammatory bone destruction. J Pharmacol Exp Ther. 317(3):1044-53.

[88] Melchior F. (2000) SUMO--nonclassical ubiquitin. Annu Rev Cell Dev Biol. 16:591-626.

[89] Melchior F, Schergaut M, Pichler A. (2003) SUMO: ligases, isopeptidases and nuclear pores. Trends Biochem Sci. 28(11):612-8.

[90] Molinari M, Salio M, Galli C, Norais N, Rappuoli R, Lanzavecchia A, Montecucco C. (1998) Selective inhibition of Ii-dependent antigen presentation by Helicobacter pylori toxin VacA. J Exp Med. 187(1):135-40.

[91] Monack DM, Mueller A, Falkow S. (2004) Persistent bacterial infections: the interface of the pathogen and the host immune system. Nat Rev Microbiol. 2(9):747-65.

[92] Montecucco C and Rappuoli R. (2001) Living dangerously: how Helicobacter pylori survives in the human stomach. Nat Rev Mol Cell Biol. 2(6):457-66.

[93] Moss SF, Sordillo EM, Abdalla AM, Makarov V, Hanzely Z, Perez-Perez GI, Blaser MJ, Holt PR. (2001) Increased gastric epithelial cell apoptosis associated with colonization with cagA + Helicobacter pylori strains. Cancer Res. 61(4):1406-11.

[94] Müller S, Matunis MJ, Dejean A. (1998) Conjugation with the ubiquitin-related modifier SUMO-1 regulates the partitioning of PML within the nucleus. EMBO J. 17(1):61-70.

[95] Muratani M, Gerlich D, Janicki SM, Gebhard M, Eils R, Spector DL. (2002) Metabolic-energy-dependent movement of PML bodies within the mammalian cell nucleus. Nat Cell Biol. 4(2):106-10.

[96] Nagata S. (1997) Apoptosis by death factor. Cell. 88(3):355-65.

[97] Negorev D, Maul GG. (2001) Cellular proteins localized at and interacting within ND10/PML nuclear bodies/PODs suggest functions of a nuclear depot. Oncogene. 20(49):7234-42.

[98] New L, Jiang Y, Han J. (2003) Regulation of PRAK subcellular location by p38 MAP kinases. Mol Biol Cell. 14(6):2603-16.

[99] Nguyen VQ, Caprioli RM, Cover TL. (2001) Carboxy-terminal proteolytic processing of Helicobacter pylori vacuolating toxin. Infect Immun. 69(1):543-6.

[100] Okura T, Gong L, Kamitani T, Wada T, Okura I, Wei CF, Chang HM, Yeh ET. (1996) Protection against Fas/APO-1- and tumor necrosis factor-mediated cell death by a novel protein, sentrin. J Immunol. 157(10):4277-81.

[101] Philipsen S and Suske G. (1999) A tale of three fingers: the family of mammalian Sp/XKLF transcription factors. Nucleic Acids Res. 27(15):2991-3000.

[102] Potthoff A, Ledig S, Martin J, Jandl O, Cornberg M, Obst B, Beil W, Manns MP, Wagner S. (2002) Significance of the caspase family in Helicobacter pylori induced gastric epithelial apoptosis. Helicobacter. 7(6):367-77.

[103] Raingeaud J, Gupta S, Rogers JS, Dickens M, Han J, Ulevitch RJ, Davis RJ. (1995) Pro-inflammatory cytokines and environmental stress cause p38 mitogen-activated protein kinase activation by dual phosphorylation on tyrosine and threonine. J Biol Chem. 270(13):7420-6.

[104] Raingeaud J, Whitmarsh AJ, Barrett T, Dérijard B, Davis RJ. (1996) MKK3- and MKK6-regulated gene expression is mediated by the p38 mitogen-activated protein kinase signal transduction pathway. Mol Cell Biol. 16(3):1247-55.

[105] Ridley SH, Sarsfield SJ, Lee JC, Bigg HF, Cawston TE, Taylor DJ, DeWitt DL, Saklatvala J. (1997) Actions of IL-1 are selectively controlled by p38 mitogen-activated protein kinase: regulation of prostaglandin H synthase-2, metalloproteinases, and IL-6 at different levels. J Immunol. 158(7):3165-73.

[106] Rodriguez MS, Desterro JM, Lain S, Midgley CA, Lane DP, Hay RT. (1999) SUMO-1 modification activates the transcriptional response of p53. EMBO J. 18(22):6455-61.

[107] Rohde M, Püls J, Buhrdorf R, Fischer W, Haas R. (2003) A novel sheathed surface organelle of the Helicobacter pylori cag type IV secretion system. Mol Microbiol. 49(1):219-34.

[108] Ross S, Best JL, Zon LI, Gill G. (2002) SUMO-1 modification represses Sp3 transcriptional activation and modulates its subnuclear localization. Mol Cell. 10(4):831-42.

[109] Rothenbacher D and Brenner H. (2003) Burden of Helicobacter pylori and H. pylori-related diseases in developed countries: recent developments and future implications. Microbes Infect. 5(8):693-703.

[110] Rouse J, Cohen P, Trigon S, Morange M, Alonso-Llamazares A, Zamanillo D, Hunt T, Nebreda AR. (1994). A novel kinase cascade triggered by stress and heat shock that stimulates MAPKAP kinase-2 and phosphorylation of the small heat shock proteins. Cell 78:1027–1037.

[111] Rudi J, Kuck D, Strand S, von Herbay A, Mariani SM, Krammer PH, Galle PR, Stremmel W. (1998) Involvement of the CD95 (APO-1/Fas) receptor and ligand system in Helicobacter pylori-induced gastric epithelial apoptosis. J Clin Invest. 102(8):1506-14.

[112] Saeki K, Kobayashi N, Inazawa Y, Zhang H, Nishitoh H, Ichijo H, Saeki K, Isemura M, Yuo A. (2002) Oxidation-triggered c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein (MAP) kinase pathways for apoptosis in human leukaemic cells stimulated by epigallocatechin-3-gallate (EGCG): a distinct pathway from those of chemically induced and receptor-mediated apoptosis. Biochem J. 368(Pt 3):705-20.

[113] Saitoh H and Hinchey J. (2000) Functional heterogeneity of small ubiquitin-related protein modifiers SUMO-1 versus SUMO-2/3. J Biol Chem. 275(9):6252-8.

[114] Schindler JF, Monahan JB, Smith WG. (2007) p38 pathway kinases as anti-inflammatory drug targets. J Dent Res. 86(9):800-11.

[115] Schindler JF, Godbey A, Hood WF, Bolten SL, Broadus RM, Kasten TP, Cassely AJ, Hirsch JL, Merwood MA, Nagy MA, Fok KF, Saabye MJ, Morgan HM, Compton RP, Mourey RJ, Wittwer AJ, Monahan JB. (2002) Examination of the kinetic mechanism of mitogen-activated protein kinase activated protein kinase-2. Biochim Biophys Acta. 1598(1-2):88-97.

[116] Seeler JS and Dejean A. (2003) Nuclear and unclear functions of SUMO. Nat Rev Mol Cell Biol. 4(9):690-9.

[117] Selbach M, Moese S, Hauck CR, Meyer TF, Backert S. (2002) Src is the kinase of the Helicobacter pylori CagA protein in vitro and in vivo. J Biol Chem. 277(9):6775-8.

[118] Seternes OM, Johansen B, Hegge B, Johannessen M, Keyse SM, Moens U. (2002) Both binding and activation of p38 mitogen-activated protein kinase (MAPK) play essential roles in regulation of the nucleocytoplasmic distribution of MAPK-activated protein kinase 5 by cellular stress. Mol Cell Biol. 22(20):6931-45.

[119] Shao R, Rung E, Weijdegård B, Billig H. (2006) Induction of apoptosis increases SUMO-1 protein expression and conjugation in mouse periovulatory granulosa cells in vitro. Mol Reprod Dev. 73(1):50-60.

[120] Shen TH, Lin HK, Scaglioni PP, Yung TM, Pandolfi PP. (2006) The mechanisms of PML-nuclear body formation. Mol Cell. 24(3):331-9.

[121] Shimada K, Nakamura M, Ishida E, Kishi M, Konishi N. (2003) Roles of p38- and c-jun NH2-terminal kinase-mediated pathways in 2-methoxyestradiol-induced p53 induction and apoptosis. Carcinogenesis. 24(6):1067-75.

[122] Sternsdorf T, Jensen K, Will H. (1997) Evidence for covalent modification of the nuclear dot-associated proteins PML and Sp100 by PIC1/SUMO-1. J Cell Biol. 139(7):1621-34.

[123] Svensson EC, Markström E, Andersson M, Billig H. (2000) Progesterone receptor-mediated inhibition of apoptosis in granulosa cells isolated from rats treated with human chorionic gonadotropin. Biol Reprod. 63(5):1457-64.

[124] Tanaka J, Suzuki T, Mimuro H, Sasakawa C. (2003) Structural definition on the surface of Helicobacter pylori type IV secretion apparatus. Cell Microbiol. 5(6):395-404.

[125] Tatham MH, Jaffray E, Vaughan OA, Desterro JM, Botting CH, Naismith JH, Hay RT. (2001) Polymeric chains of SUMO-2 and SUMO-3 are conjugated to protein substrates by SAE1/SAE2 and Ubc9. J Biol Chem. 276(38):35368-74.

[126] Tobiume K, Saitoh M, Ichijo H. (2002) Activation of apoptosis signal-regulating kinase 1 by the stress-induced activating phosphorylation of pre-formed oligomer. J Cell Physiol. 191(1):95-104.

[127] Toledo F and Wahl GM. (2006) Regulating the p53 pathway: in vitro hypotheses, in vivo veritas. Nat Rev Cancer. 6(12):909-23.

[128] Tombola F, Carlesso C, Szabò I, de Bernard M, Reyrat JM, Telford JL, Rappuoli R, Montecucco C, Papini E, Zoratti M. (1999) Helicobacter pylori vacuolating toxin forms anion-selective channels in planar lipid bilayers: possible implications for the mechanism of cellular vacuolation. Biophys J. 76(3):1401-9.

[129] Tong L, Pav S, White DM, Rogers S, Crane KM, Cywin CL, Brown ML, Pargellis CA. (1997) A highly specific inhibitor of human p38 MAP kinase binds in the ATP pocket. Nat Struct Biol. 4(4):311-6.

[130] Tourian L Jr, Zhao H, Srikant CB. (2004) p38alpha, but not p38beta, inhibits the phosphorylation and presence of c-FLIPS in DISC to potentiate Fas-mediated caspase-8 activation and type I apoptotic signaling. J Cell Sci. 117(Pt 26):6459-71.

[131] Underwood DC, Osborn RR, Bochnowicz S, Webb EF, Rieman DJ, Lee JC, Romanic AM, Adams JL, Hay DW, Griswold DE. (2000) SB 239063, a p38 MAPK inhibitor, reduces neutrophilia, inflammatory cytokines, MMP-9, and fibrosis in lung. Am J Physiol Lung Cell Mol Physiol. 279(5):L895-902.

[132] Vertegaal AC, Andersen JS, Ogg SC, Hay RT, Mann M, Lamond AI. (2006) Distinct and overlapping sets of SUMO-1 and SUMO-2 target proteins revealed by quantitative proteomics. Mol Cell Proteomics. 5(12):2298-310.

[133] Wang Z, Canagarajah BJ, Boehm JC, Kassisà S, Cobb MH, Young PR, Abdel-Meguid S, Adams JL, Goldsmith EJ. (1998) Structural basis of inhibitor selectivity in MAP kinases. Structure. 6(9):1117-28.

[134] Wang XZ and Ron D. (1996) Stress-induced phosphorylation and activation of the transcription factor CHOP (GADD153) by p38 MAP Kinase. Science. 272(5266):1347-9.

[135] Watson IR and Irwin MS. (2006) Ubiquitin and ubiquitin-like modifications of the p53 family. Neoplasia. 8(8):655-66.

[136] White A, Pargellis CA, Studts JM, Werneburg BG, Farmer BT 2nd. (2007) Molecular basis of MAPK-activated protein kinase 2:p38 assembly. Proc Natl Acad Sci U S A. 104(15):6353-8.

[137] Wiesmeijer K, Molenaar C, Bekeer IM, Tanke HJ, Dirks RW. (2002) Mobile foci of Sp100 do not contain PML: PML bodies are immobile but PML and Sp100 proteins are not. J Struct Biol. 140(1-3):180-8.

[138] Wilson KP, Fitzgibbon MJ, Caron PR, Griffith JP, Chen W, McCaffrey PG, Chambers SP, Su MS. (1996) Crystal structure of p38 mitogen-activated protein kinase. J Biol Chem. 271(44):27696-700.

[139] Wood CD, Thornton TM, Sabio G, Davis RA, Rincon M. (2009) Nuclear localization of p38 MAPK in response to DNA damage. Int J Biol Sci. 5(5):428-37.

[140] Wrobleski ST and Doweyko AM. (2005) Structural comparison of p38 inhibitor-protein complexes: a review of recent p38 inhibitors having unique binding interactions. Curr Top Med Chem. 5(10):1005-16.

[141] Xia Z, Dickens M, Raingeaud J, Davis RJ, Greenberg ME. (1995) Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science. 270(5240):1326-31.

[142] Yu L, Hébert MC, Zhang YE. (2002) TGF-beta receptor-activated p38 MAP kinase mediates Smad-independent TGF-beta responses. EMBO J. 21(14):3749-59.

[143] Zeidan A, Javadov S, Chakrabarti S, Karmazyn M. (2008) Leptin-induced cardiomyocyte hypertrophy involves selective caveolae and RhoA/ROCK-dependent p38 MAPK translocation to nuclei. Cardiovasc Res. 77(1):64-72.

[144] Zhong S, Müller S, Ronchetti S, Freemont PS, Dejean A, Pandolfi PP. (2000) Role of SUMO-1-modified PML in nuclear body formation. Blood. 95(9):2748-52.