CPAP蛋白在過去的研究中被認為是細胞中心體蛋白 (centrosomal protein)，位於細胞內中心體的位置，對其功能扮演重要的角色，例如細胞內中心體處微小管的生成 (microtubule nucleation) 及在細胞分裂時紡綞絲 (mitotic spindle) 的形成；此外，CPAP蛋白也參與了細胞微小管不穩定性 (microtubule instability) 的調控。除了在細胞中心體的功能外，CPAP蛋白亦具有轉錄輔助活化因子 (transcriptional co-activator) 的活性，在Prolatin或TNFalpha的刺激下，會與STAT5或NF-kappaB相互作用而進入細胞核中，進而增加STAT5或NFkappaB的轉錄活性 (transcriptional activity)。在本論文中，我們利用siRNA knockdown的技術，使A549細胞中內生性CPAP蛋白的表現量減少，結果發現在TNFalpha誘導下，受NF-kappaB活化的下游標的基因的表現均受到抑制。在reporter assay中觀察到，CPAP蛋白可增加NF-kappaB的轉錄活性。進一步利用immunoprecipitation assay，發現CPAP蛋白可能是藉由網羅更多的IKKbeta到CPAP/p65 complexes中，使得IkappaBalpha及p65/RelA磷酸化增加，進而加速IkappaBalpha的降解與NF-kappaB的活化，最後促進標的基因的表現。此外，immunofluorescence assay的結果顯示，CPAP蛋白在TNFalpha誘導下，會移動到細胞核中。由ChIP assay的結果，發現CPAP蛋白可幫助更多p65/RelA結合到COX2啟動子上，而CPAP可能也會藉由與p65/RelA的相互作用而結合上去。由以上結果顯示，CPAP蛋白在A549細胞中的確參與了透過TNFalpha誘導NF-kappaB活化下游基因的訊息傳遞路徑。此外，in vivo及in vitro sumoylation assay證實CPAP蛋白會受到SUMO蛋白的修飾。至於受SUMO修飾的CPAP是否有參與在TNFalpha誘導NF-kappaB活化的訊息傳遞路徑中，則是我們目前正在研究的課題。

CPAP is originally identified as a centrosomal protein that plays important roles on centrosome function, such as: centrosomal microtubule nucleation and mitotic spindle formation. Besides, CPAP can translocate into nucleus, where to act as a transcriptional co-activator, and through the interaction with STAT5 or NF-kappaB to enhance their transcriptional activity upon prolatin or TNFalpha simulation. In this study, when treated A549 cells with TNFalpha, the NF-kappaB was activated and then transactivated the target genes. By CPAP RNA interference assay, the expression of NF-kappaB-target genes was attenuated in the CPAP knockdown cells under TNFalpha stimulus. Moreover, CPAP contribute to the NF-kappaB-driven transcriptional activation in reporter assay. Co-immunoprecipitation analysis demonstrated that CPAP may activate NF-kappaB signaling pathway through recruiting more IKKbeta to the inactivated NF-kappaB complex to enhance the degradation of IkappaBalpha and to phosphorylate more NF-kappaB/p65. Besides, the nuclear translocation of CPAP was induced by TNFalpha treatment. Chromatin immunoprecipitation assay indicated that the binding of p65 to COX2 promoter was increased by ectopic expressed GFP-CPAP. These results suggested that CPAP may involve in the signaling pathway of TNFalpha-induced NF-kappaB mediated activation. Furthermore, the polypeptide sequence of CPAP contains several potential SUMO-acceptor sites. In vitro and in vivo sumoylation assay demonstrated that CPAP polypeptide can be sumoylated. The role of sumoylated CPAP in the TNFalpha-induced NF-kappaB avtivation pathway is currently under investigation.

1. Ghosh S, May MJ, Kopp EB: NF-kappa B and Rel proteins: evolutionarily conserved mediators of immune responses. Annu Rev Immunol 1998, 16:225-260.
2. Hayden MS, Ghosh S: Signaling to NF-kappaB. Genes Dev 2004, 18(18):2195-2224.
3. Gilmore TD: Introduction to NF-kappaB: players, pathways, perspectives. Oncogene 2006, 25(51):6680-6684.
4. Baldwin AS, Jr.: The NF-kappa B and I kappa B proteins: new discoveries and insights. Annu Rev Immunol 1996, 14:649-683.
5. Bonizzi G, Karin M: The two NF-kappaB activation pathways and their role in innate and adaptive immunity. Trends Immunol 2004, 25(6):280-288.
6. Basseres DS, Baldwin AS: Nuclear factor-kappaB and inhibitor of kappaB kinase pathways in oncogenic initiation and progression. Oncogene 2006, 25(51):6817-6830.
7. Perkins ND: Integrating cell-signalling pathways with NF-kappaB and IKK function. Nat Rev Mol Cell Biol 2007, 8(1):49-62.
8. Scheidereit C: IkappaB kinase complexes: gateways to NF-kappaB activation and transcription. Oncogene 2006, 25(51):6685-6705.
9. Chen LF, Greene WC: Shaping the nuclear action of NF-kappaB. Nat Rev Mol Cell Biol 2004, 5(5):392-401.
10. Hanahan D, Weinberg RA: The hallmarks of cancer. Cell 2000, 100(1):57-70.
11. Karin M, Cao Y, Greten FR, Li ZW: NF-kappaB in cancer: from innocent bystander to major culprit. Nat Rev Cancer 2002, 2(4):301-310.
12. Karin M: Nuclear factor-kappaB in cancer development and progression. Nature 2006, 441(7092):431-436.
13. Karin M, Greten FR: NF-kappaB: linking inflammation and immunity to cancer development and progression. Nat Rev Immunol 2005, 5(10):749-759.
14. Li Q, Withoff S, Verma IM: Inflammation-associated cancer: NF-kappaB is the lynchpin. Trends Immunol 2005, 26(6):318-325.
15. Koch AE, Polverini PJ, Kunkel SL, Harlow LA, DiPietro LA, Elner VM, Elner SG, Strieter RM: Interleukin-8 as a macrophage-derived mediator of angiogenesis. Science 1992, 258(5089):1798-1801.
16. Guttridge DC, Albanese C, Reuther JY, Pestell RG, Baldwin AS, Jr.: NF-kappaB controls cell growth and differentiation through transcriptional regulation of cyclin D1. Mol Cell Biol 1999, 19(8):5785-5799.
17. Hinz M, Krappmann D, Eichten A, Heder A, Scheidereit C, Strauss M: NF-kappaB function in growth control: regulation of cyclin D1 expression and G0/G1-to-S-phase transition. Mol Cell Biol 1999, 19(4):2690-2698.
18. Karin M, Lin A: NF-kappaB at the crossroads of life and death. Nat Immunol 2002, 3(3):221-227.
19. Baldwin AS: Control of oncogenesis and cancer therapy resistance by the transcription factor NF-kappaB. J Clin Invest 2001, 107(3):241-246.
20. Yamamoto Y, Gaynor RB: Therapeutic potential of inhibition of the NF-kappaB pathway in the treatment of inflammation and cancer. J Clin Invest 2001, 107(2):135-142.
21. Gilmore TD, Herscovitch M: Inhibitors of NF-kappaB signaling: 785 and counting. Oncogene 2006, 25(51):6887-6899.
22. Karin M, Yamamoto Y, Wang QM: The IKK NF-kappa B system: a treasure trove for drug development. Nat Rev Drug Discov 2004, 3(1):17-26.
23. Nakanishi C, Toi M: Nuclear factor-kappaB inhibitors as sensitizers to anticancer drugs. Nat Rev Cancer 2005, 5(4):297-309.
24. Pikarsky E, Ben-Neriah Y: NF-kappaB inhibition: a double-edged sword in cancer? Eur J Cancer 2006, 42(6):779-784.
25. Inoue J, Gohda J, Akiyama T, Semba K: NF-kappaB activation in development and progression of cancer. Cancer Sci 2007, 98(3):268-274.
26. Verger A, Perdomo J, Crossley M: Modification with SUMO. A role in transcriptional regulation. EMBO Rep 2003, 4(2):137-142.
27. Gill G: SUMO and ubiquitin in the nucleus: different functions, similar mechanisms? Genes Dev 2004, 18(17):2046-2059.
28. Walsh G, Jefferis R: Post-translational modifications in the context of therapeutic proteins. Nat Biotechnol 2006, 24(10):1241-1252.
29. Martin S, Wilkinson KA, Nishimune A, Henley JM: Emerging extranuclear roles of protein SUMOylation in neuronal function and dysfunction. Nat Rev Neurosci 2007, 8(12):948-959.
30. Johnson ES: Protein modification by SUMO. Annu Rev Biochem 2004, 73:355-382.
31. Guo B, Yang SH, Witty J, Sharrocks AD: Signalling pathways and the regulation of SUMO modification. Biochem Soc Trans 2007, 35(Pt 6):1414-1418.
32. Seeler JS, Dejean A: Nuclear and unclear functions of SUMO. Nat Rev Mol Cell Biol 2003, 4(9):690-699.
33. Desterro JM, Rodriguez MS, Hay RT: SUMO-1 modification of IkappaBalpha inhibits NF-kappaB activation. Mol Cell 1998, 2(2):233-239.
34. Matunis MJ, Wu J, Blobel G: SUMO-1 modification and its role in targeting the Ran GTPase-activating protein, RanGAP1, to the nuclear pore complex. J Cell Biol 1998, 140(3):499-509.
35. Seeler JS, Dejean A: SUMO: of branched proteins and nuclear bodies. Oncogene 2001, 20(49):7243-7249.
36. Hoege C, Pfander B, Moldovan GL, Pyrowolakis G, Jentsch S: RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO. Nature 2002, 419(6903):135-141.
37. Pichler A, Melchior F: Ubiquitin-related modifier SUMO1 and nucleocytoplasmic transport. Traffic 2002, 3(6):381-387.
38. Schmitz ML, Stelzer G, Altmann H, Meisterernst M, Baeuerle PA: Interaction of the COOH-terminal transactivation domain of p65 NF-kappa B with TATA-binding protein, transcription factor IIB, and coactivators. J Biol Chem 1995, 270(13):7219-7226.
39. Gerritsen ME, Williams AJ, Neish AS, Moore S, Shi Y, Collins T: CREB-binding protein/p300 are transcriptional coactivators of p65. Proc Natl Acad Sci U S A 1997, 94(7):2927-2932.
40. Zhong H, Voll RE, Ghosh S: Phosphorylation of NF-kappa B p65 by PKA stimulates transcriptional activity by promoting a novel bivalent interaction with the coactivator CBP/p300. Mol Cell 1998, 1(5):661-671.
41. Sheppard KA, Rose DW, Haque ZK, Kurokawa R, McInerney E, Westin S, Thanos D, Rosenfeld MG, Glass CK, Collins T: Transcriptional activation by NF-kappaB requires multiple coactivators. Mol Cell Biol 1999, 19(9):6367-6378.
42. Werbajh S, Nojek I, Lanz R, Costas MA: RAC-3 is a NF-kappa B coactivator. FEBS Lett 2000, 485(2-3):195-199.
43. Koyanagi M, Hijikata M, Watashi K, Masui O, Shimotohno K: Centrosomal P4.1-associated protein is a new member of transcriptional coactivators for nuclear factor-kappaB. J Biol Chem 2005, 280(13):12430-12437.
44. Hung LY, Tang CJ, Tang TK: Protein 4.1 R-135 interacts with a novel centrosomal protein (CPAP) which is associated with the gamma-tubulin complex. Mol Cell Biol 2000, 20(20):7813-7825.
45. Hung LY, Chen HL, Chang CW, Li BR, Tang TK: Identification of a novel microtubule-destabilizing motif in CPAP that binds to tubulin heterodimers and inhibits microtubule assembly. Mol Biol Cell 2004, 15(6):2697-2706.
46. Hsu WB, Hung LY, Tang CJ, Su CL, Chang Y, Tang TK: Functional characterization of the microtubule-binding and -destabilizing domains of CPAP and d-SAS-4. Exp Cell Res 2008.
47. Peng B, Sutherland KD, Sum EY, Olayioye M, Wittlin S, Tang TK, Lindeman GJ, Visvader JE: CPAP is a novel stat5-interacting cofactor that augments stat5-mediated transcriptional activity. Mol Endocrinol 2002, 16(9):2019-2033.
48. Lin DY, Huang YS, Jeng JC, Kuo HY, Chang CC, Chao TT, Ho CC, Chen YC, Lin TP, Fang HI et al: Role of SUMO-interacting motif in Daxx SUMO modification, subnuclear localization, and repression of sumoylated transcription factors. Mol Cell 2006, 24(3):341-354.
49. Chao TT, Chang CC, Shih HM: SUMO modification modulates the transrepression activity of PLZF. Biochem Biophys Res Commun 2007, 358(2):475-482.
50. Hu MC, Hung MC: Role of IkappaB kinase in tumorigenesis. Future Oncol 2005, 1(1):67-78.
51. Tam WF, Sen R: IkappaB family members function by different mechanisms. J Biol Chem 2001, 276(11):7701-7704.
52. Tam WF, Wang W, Sen R: Cell-specific association and shuttling of IkappaBalpha provides a mechanism for nuclear NF-kappaB in B lymphocytes. Mol Cell Biol 2001, 21(14):4837-4846.
53. Perkins ND: Post-translational modifications regulating the activity and function of the nuclear factor kappa B pathway. Oncogene 2006, 25(51):6717-6730.
54. Hoberg JE, Popko AE, Ramsey CS, Mayo MW: IkappaB kinase alpha-mediated derepression of SMRT potentiates acetylation of RelA/p65 by p300. Mol Cell Biol 2006, 26(2):457-471.
55. Okazaki T, Sakon S, Sasazuki T, Sakurai H, Doi T, Yagita H, Okumura K, Nakano H: Phosphorylation of serine 276 is essential for p65 NF-kappaB subunit-dependent cellular responses. Biochem Biophys Res Commun 2003, 300(4):807-812.
56. Sasaki CY, Barberi TJ, Ghosh P, Longo DL: Phosphorylation of RelA/p65 on serine 536 defines an I{kappa}B{alpha}-independent NF-{kappa}B pathway. J Biol Chem 2005, 280(41):34538-34547.
57. Naar AM, Lemon BD, Tjian R: Transcriptional coactivator complexes. Annu Rev Biochem 2001, 70:475-501.
58. McKenna NJ, O'Malley BW: Minireview: nuclear receptor coactivators--an update. Endocrinology 2002, 143(7):2461-2465.
59. Xu W: Nuclear receptor coactivators: the key to unlock chromatin. Biochem Cell Biol 2005, 83(4):418-428.
60. Chan HM, La Thangue NB: p300/CBP proteins: HATs for transcriptional bridges and scaffolds. J Cell Sci 2001, 114(Pt 13):2363-2373.
61. Shiama N: The p300/CBP family: integrating signals with transcription factors and chromatin. Trends Cell Biol 1997, 7(6):230-236.
62. Goodman RH, Smolik S: CBP/p300 in cell growth, transformation, and development. Genes Dev 2000, 14(13):1553-1577.
63. Xu L, Glass CK, Rosenfeld MG: Coactivator and corepressor complexes in nuclear receptor function. Curr Opin Genet Dev 1999, 9(2):140-147.
64. Lee DY, Hayes JJ, Pruss D, Wolffe AP: A positive role for histone acetylation in transcription factor access to nucleosomal DNA. Cell 1993, 72(1):73-84.
65. Vettese-Dadey M, Grant PA, Hebbes TR, Crane- Robinson C, Allis CD, Workman JL: Acetylation of histone H4 plays a primary role in enhancing transcription factor binding to nucleosomal DNA in vitro. EMBO J 1996, 15(10):2508-2518.
66. Tse C, Fletcher TM, Hansen JC: Enhanced transcription factor access to arrays of histone H3/H4 tetramer.DNA complexes in vitro: implications for replication and transcription. Proc Natl Acad Sci U S A 1998, 95(21):12169-12173.
67. Tse C, Sera T, Wolffe AP, Hansen JC: Disruption of higher-order folding by core histone acetylation dramatically enhances transcription of nucleosomal arrays by RNA polymerase III. Mol Cell Biol 1998, 18(8):4629-4638.
68. Ura K, Kurumizaka H, Dimitrov S, Almouzni G, Wolffe AP: Histone acetylation: influence on transcription, nucleosome mobility and positioning, and linker histone-dependent transcriptional repression. EMBO J 1997, 16(8):2096-2107.
69. Nightingale KP, Wellinger RE, Sogo JM, Becker PB: Histone acetylation facilitates RNA polymerase II transcription of the Drosophila hsp26 gene in chromatin. EMBO J 1998, 17(10):2865-2876.
70. Sterner DE, Berger SL: Acetylation of histones and transcription-related factors. Microbiol Mol Biol Rev 2000, 64(2):435-459.
71. Chen H, Tini M, Evans RM: HATs on and beyond chromatin. Curr Opin Cell Biol 2001, 13(2):218-224.
72. Chen L, Fischle W, Verdin E, Greene WC: Duration of nuclear NF-kappaB action regulated by reversible acetylation. Science 2001, 293(5535):1653-1657.
73. Chen LF, Mu Y, Greene WC: Acetylation of RelA at discrete sites regulates distinct nuclear functions of NF-kappaB. EMBO J 2002, 21(23):6539-6548.
74. Arenzana-Seisdedos F, Thompson J, Rodriguez MS, Bachelerie F, Thomas D, Hay RT: Inducible nuclear expression of newly synthesized I kappa B alpha negatively regulates DNA-binding and transcriptional activities of NF-kappa B. Mol Cell Biol 1995, 15(5):2689-2696.
75. Arenzana-Seisdedos F, Turpin P, Rodriguez M, Thomas D, Hay RT, Virelizier JL, Dargemont C: Nuclear localization of I kappa B alpha promotes active transport of NF-kappa B from the nucleus to the cytoplasm. J Cell Sci 1997, 110 ( Pt 3):369-378.
76. Furia B, Deng L, Wu K, Baylor S, Kehn K, Li H, Donnelly R, Coleman T, Kashanchi F: Enhancement of nuclear factor-kappa B acetylation by coactivator p300 and HIV-1 Tat proteins. J Biol Chem 2002, 277(7):4973-4980.
77. Deng WG, Zhu Y, Wu KK: Up-regulation of p300 binding and p50 acetylation in tumor necrosis factor-alpha-induced cyclooxygenase-2 promoter activation. J Biol Chem 2003, 278(7):4770-4777.
78. Kerscher O: SUMO junction-what's your function? New insights through SUMO-interacting motifs. EMBO Rep 2007, 8(6):550-555.
79. Prajapati S, Tu Z, Yamamoto Y, Gaynor RB: IKKalpha regulates the mitotic phase of the cell cycle by modulating Aurora A phosphorylation. Cell Cycle 2006, 5(20):2371-2380.
80. Briassouli P, Chan F, Savage K, Reis-Filho JS, Linardopoulos S: Aurora-A regulation of nuclear factor-kappaB signaling by phosphorylation of IkappaBalpha. Cancer Res 2007, 67(4):1689-1695.
81. Linardopoulos S: Aurora-A kinase regulates NF-kappaB activity: lessons from combination studies. J BUON 2007, 12 Suppl 1:S67-70.
82. Sun C, Chan F, Briassouli P, Linardopoulos S: Aurora kinase inhibition downregulates NF-kappaB and sensitises tumour cells to chemotherapeutic agents. Biochem Biophys Res Commun 2007, 352(1):220-225.