Eps8是receptor tyrosine kinase (例如EGFR) 和non-receptor tyrosine kinase (例如Src) 的受質。已知其具有兩種isoforms，分別是p97Eps8和p68Eps8。先前的研究已經證明p97Eps8參與在EGF所誘導的細胞分裂與腫瘤的生成中。在我們實驗室則發現p97Eps8可以促進C3H10T1/2纖維母細胞轉型。在Ha-Ras transformed cells中，我們發現Eps8的表現量增加，而且Eps8會參與在Ha-Ras促進細胞分裂和轉型的過程中。為了探討Eps8是如何受到Ha-Ras所調控，我們選擇了可以用IPTG誘導Ha-Ras表達的7-4細胞做實驗。在IPTG成功誘導Ha-Ras表現後，Eps8的mRNA和蛋白質的表現量都會增加。為了確認其調控機制是否是藉由影響啟動區活性而提高基因轉錄的能力。我們設計一系列的primer，將可能的啟動區序列分離出來，接在pXP-1這個帶有luciferase gene的載體上。得到-1489 ~ +109 (pXE 1489)， -985 ~ +109 (pXE 985)， -474 ~ +109 (pXE 474)，-313 ~ +109 (pXE 313)，-154 ~ +109 (pXE 154)，-52 ~ +109 (pXE 52)。 Dual-light luciferase assay的結果顯示在7-4細胞中，這幾個不同片段長度的啟動區，其luciferase activity都會隨著IPTG的處理而稍微增加。另ㄧ方面，Ha-Ras可藉由多種的signal pathway調控下游基因的表現。當使用LY294002 (PI3K inhibitor) 處理細胞時，我們發現藥物可抑制IPTG所造成的Eps8表現量增加，PD98059 (MEK inhibitor)及PP2 (Src inhibitor)有部份抑制的作用。但是LY294002卻無法抑制pXE 313及pXE 154的luciferase activity增加。顯示Ha-Ras所活化的PI3K pathway或許並不作用在這個區域之中。細胞外送dominant negative p85確認了PI3K在Ha-Ras調控Eps8表現上確實扮演重要的角色。在actinomycin D處理下發現Ha-Ras會促進Eps8 mRNA的分解，降低它的半衰期。當使用Cycloheximide時，也顯示Ha-Ras會加快Eps8 protein分解的速度。總而言之，在本篇研究中發現Ha-Ras主要是藉由PI3 kinase pathway造成Eps8表現量的增加；另ㄧ方面也會促進Eps8的mRNA及蛋白質的分解速率。

Eps8 is a common substrate of both receptor tyrosine kinase (ie, EGFR) and non-receptor tyrosine kinase (ie, Src). P97Eps8 has been demonstrated to participate in EGF-induced mitogenesis and tumorigenesis. Our previous studies indicated that over-expression of p97Eps8 induced foci formation and tumorigenesis in murine C3H10T1/2 fibroblasts. We also found that Eps8 is over-expressed in Ha-Ras transformed cells and participates in Ha-Ras-mediated cell proliferation and transformation. In order to study how Ha-Ras mediated eps8 gene expression, we use a Ha-Ras-inducible cells line, 7-4, as the studying materials. We found that inducible Ha-Ras could increase the expression of both mRNA and protein of Eps8. Next, we want to know how Ha-Ras could increase the transcription of eps8. At first, the promoter region of human eps8 was cloned into pXP-1 luciferase expression vectors. Then, we generated several deleted constructs of it, including -1489 ~ +109 (pXE 1489), -985 ~ +109 (pXE 985), -474 ~ +109 (pXE 474), -313 ~ +109 (pXE 313), -154 ~ +109 (pXE 154), -52 ~ +109 (pXE 52), and characterized their promoter activity in 7-4 cell in the presence or absence of Ha-Ras. Our study indicated that the induction of Ha-Ras by IPTG could slightly increase the luciferase activity of these constructs. Western blotting and RT-PCR indicated that PI3K inhibitor, LY294002, could inhibit Ha-Ras-mediated eps8 expression. Similar results were observed by dominant negative PI3K transfection. However, LY294002 did not abolish the luciferase activity of pXE 313 and pXE 154. This indicated the PI3K responsible element(s) is not present in this region. Finally, we observed Ha-Ras could increase the turnover rate of both eps8 mRNA and its protein in 7-4 cell.

1. Albertson, D.G. (2006). Gene amplification in cancer. Trends Genet 22, 447-455.
2. Avantaggiato, V., Torino, A., Wong, W.T., Di Fiore, P.P., and Simeone, A. (1995). Expression of the receptor tyrosine kinase substrate genes eps8 and eps15 during mouse development. Oncogene 11, 1191-1198.
3. Azambuja, E., Durbecq, V., Rosa, D.D., Colozza, M., Larsimont, D., Piccart-Gebhart, M., and Cardoso, F. (2008). HER-2 overexpression/amplification and its interaction with taxane-based therapy in breast cancer. Ann Oncol 19, 223-232.
4. Biesova, Z., Piccoli, C., and Wong, W.T. (1997). Isolation and characterization of e3B1, an eps8 binding protein that regulates cell growth. Oncogene 14, 233-241.
5. Bos, J.L. (1989). ras oncogenes in human cancer: a review. Cancer Res 49, 4682-4689.
6. Campbell, P.M., and Der, C.J. (2004). Oncogenic Ras and its role in tumor cell invasion and metastasis. Semin Cancer Biol 14, 105-114.
7. Castagnino, P., Biesova, Z., Wong, W.T., Fazioli, F., Gill, G.N., and Di Fiore, P.P. (1995). Direct binding of eps8 to the juxtamembrane domain of EGFR is phosphotyrosine- and SH2-independent. Oncogene 10, 723-729.
8. Fazioli, F., Bottaro, D.P., Minichiello, L., Auricchio, A., Wong, W.T., Segatto, O., and Di Fiore, P.P. (1992). Identification and biochemical characterization of novel putative substrates for the epidermal growth factor receptor kinase. J Biol Chem 267, 5155-5161.
9. Fazioli, F., Minichiello, L., Matoska, V., Castagnino, P., Miki, T., Wong, W.T., and Di Fiore, P.P. (1993). Eps8, a substrate for the epidermal growth factor receptor kinase, enhances EGF-dependent mitogenic signals. EMBO J 12, 3799-3808.
10. Feinstein, E. (2005). Ral-GTPases: good chances for a long-lasting fame. Oncogene 24, 326-328.
11. Ferbeyre, G., de Stanchina, E., Lin, A.W., Querido, E., McCurrach, M.E., Hannon, G.J., and Lowe, S.W. (2002). Oncogenic ras and p53 cooperate to induce cellular senescence. Mol Cell Biol 22, 3497-3508.
12. Forough, R., Weylie, B., Collins, C., Parker, J.L., Zhu, J., Barhoumi, R., and Watson, D.K. (2006). Transcription factor Ets-1 regulates fibroblast growth factor-1-mediated angiogenesis in vivo: role of Ets-1 in the regulation of the PI3K/AKT/MMP-1 pathway. J Vasc Res 43, 327-337.
13. Innocenti, M., Frittoli, E., Ponzanelli, I., Falck, J.R., Brachmann, S.M., Di Fiore, P.P., and Scita, G. (2003). Phosphoinositide 3-kinase activates Rac by entering in a complex with Eps8, Abi1, and Sos-1. J Cell Biol 160, 17-23.
14. Inobe, M., Katsube, K., Miyagoe, Y., Nabeshima, Y., and Takeda, S. (1999). Identification of EPS8 as a Dvl1-associated molecule. Biochem Biophys Res Commun 266, 216-221.
15. Kirkegaard, K., Taylor, M.P., and Jackson, W.T. (2004). Cellular autophagy: surrender, avoidance and subversion by microorganisms. Nat Rev Microbiol 2, 301-314.
16. Kishan, K.V., Scita, G., Wong, W.T., Di Fiore, P.P., and Newcomer, M.E. (1997). The SH3 domain of Eps8 exists as a novel intertwined dimer. Nat Struct Biol 4, 739-743.
17. Koera, K., Nakamura, K., Nakao, K., Miyoshi, J., Toyoshima, K., Hatta, T., Otani, H., Aiba, A., and Katsuki, M. (1997). K-ras is essential for the development of the mouse embryo. Oncogene 15, 1151-1159.
18. Lambert, J.M., Lambert, Q.T., Reuther, G.W., Malliri, A., Siderovski, D.P., Sondek, J., Collard, J.G., and Der, C.J. (2002). Tiam1 mediates Ras activation of Rac by a PI(3)K-independent mechanism. Nat Cell Biol 4, 621-625.
19. Lanzetti, L., Rybin, V., Malabarba, M.G., Christoforidis, S., Scita, G., Zerial, M., and Di Fiore, P.P. (2000). The Eps8 protein coordinates EGF receptor signalling through Rac and trafficking through Rab5. Nature 408, 374-377.
20. Leu, T.H., Yeh, H.H., Huang, C.C., Chuang, Y.C., Su, S.L., and Maa, M.C. (2004). Participation of p97Eps8 in Src-mediated transformation. J Biol Chem 279, 9875-9881.
21. Lim, K.H., Baines, A.T., Fiordalisi, J.J., Shipitsin, M., Feig, L.A., Cox, A.D., Der, C.J., and Counter, C.M. (2005). Activation of RalA is critical for Ras-induced tumorigenesis of human cells. Cancer Cell 7, 533-545.
22. Maa, M.C., Hsieh, C.Y., and Leu, T.H. (2001). Overexpression of p97Eps8 leads to cellular transformation: implication of pleckstrin homology domain in p97Eps8-mediated ERK activation. Oncogene 20, 106-112.
23. Maa, M.C., Lai, J.R., Lin, R.W., and Leu, T.H. (1999). Enhancement of tyrosyl phosphorylation and protein expression of eps8 by v-Src. Biochim Biophys Acta 1450, 341-351.
24. Maa, M.C., Lee, J.C., Chen, Y.J., Lee, Y.C., Wang, S.T., Huang, C.C., Chow, N.H., and Leu, T.H. (2007). Eps8 facilitates cellular growth and motility of colon cancer cells by increasing the expression and activity of focal adhesion kinase. J Biol Chem 282, 19399-19409.
25. Malaney, S., and Daly, R.J. (2001). The ras signaling pathway in mammary tumorigenesis and metastasis. J Mammary Gland Biol Neoplasia 6, 101-113.
26. Marinov, M., Fischer, B., and Arcaro, A. (2007). Targeting mTOR signaling in lung cancer. Crit Rev Oncol Hematol 63, 172-182.
27. Matoskova, B., Wong, W.T., Nomura, N., Robbins, K.C., and Di Fiore, P.P. (1996). RN-tre specifically binds to the SH3 domain of eps8 with high affinity and confers growth advantage to NIH3T3 upon carboxy-terminal truncation. Oncogene 12, 2679-2688.
28. Matoskova, B., Wong, W.T., Salcini, A.E., Pelicci, P.G., and Di Fiore, P.P. (1995). Constitutive phosphorylation of eps8 in tumor cell lines: relevance to malignant transformation. Mol Cell Biol 15, 3805-3812.
29. Mertens, A.E., Roovers, R.C., and Collard, J.G. (2003). Regulation of Tiam1-Rac signalling. FEBS Lett 546, 11-16.
30. Miyakis, S., Sourvinos, G., and Spandidos, D.A. (1998). Differential expression and mutation of the ras family genes in human breast cancer. Biochem Biophys Res Commun 251, 609-612.
31. Nalepa, G., Rolfe, M., and Harper, J.W. (2006). Drug discovery in the ubiquitin-proteasome system. Nat Rev Drug Discov 5, 596-613.
32. Offenhauser, N., Borgonovo, A., Disanza, A., Romano, P., Ponzanelli, I., Iannolo, G., Di Fiore, P.P., and Scita, G. (2004). The eps8 family of proteins links growth factor stimulation to actin reorganization generating functional redundancy in the Ras/Rac pathway. Mol Biol Cell 15, 91-98.
33. Omerovic, J., Laude, A.J., and Prior, I.A. (2007). Ras proteins: paradigms for compartmentalised and isoform-specific signalling. Cell Mol Life Sci 64, 2575-2589.
34. Park, M.J., Kwak, H.J., Lee, H.C., Yoo, D.H., Park, I.C., Kim, M.S., Lee, S.H., Rhee, C.H., and Hong, S.I. (2007). Nerve growth factor induces endothelial cell invasion and cord formation by promoting matrix metalloproteinase-2 expression through the phosphatidylinositol 3-kinase/Akt signaling pathway and AP-2 transcription factor. J Biol Chem 282, 30485-30496.
35. Rajalingam, K., Schreck, R., Rapp, U.R., and Albert, S. (2007). Ras oncogenes and their downstream targets. Biochim Biophys Acta 1773, 1177-1195.
36. Rapp, U.R., Troppmair, J., Beck, T., and Birrer, M.J. (1994). Transformation by Raf and other oncogenes renders cells differentially sensitive to growth inhibition by a dominant negative c-jun mutant. Oncogene 9, 3493-3498.
37. Rodriguez-Viciana, P., Warne, P.H., Dhand, R., Vanhaesebroeck, B., Gout, I., Fry, M.J., Waterfield, M.D., and Downward, J. (1994). Phosphatidylinositol-3-OH kinase as a direct target of Ras. Nature 370, 527-532.
38. Ross, J. (1996). Control of messenger RNA stability in higher eukaryotes. Trends Genet 12, 171-175.
39. Schubbert, S., Shannon, K., and Bollag, G. (2007). Hyperactive Ras in developmental disorders and cancer. Nat Rev Cancer 7, 295-308.
40. Scita, G., Nordstrom, J., Carbone, R., Tenca, P., Giardina, G., Gutkind, S., Bjarnegard, M., Betsholtz, C., and Di Fiore, P.P. (1999). EPS8 and E3B1 transduce signals from Ras to Rac. Nature 401, 290-293.
41. Scita, G., Tenca, P., Areces, L.B., Tocchetti, A., Frittoli, E., Giardina, G., Ponzanelli, I., Sini, P., Innocenti, M., and Di Fiore, P.P. (2001). An effector region in Eps8 is responsible for the activation of the Rac-specific GEF activity of Sos-1 and for the proper localization of the Rac-based actin-polymerizing machine. J Cell Biol 154, 1031-1044.
42. Shaw, R.J., and Cantley, L.C. (2006). Ras, PI(3)K and mTOR signalling controls tumour cell growth. Nature 441, 424-430.
43. Song, C., Hu, C.D., Masago, M., Kariyai, K., Yamawaki-Kataoka, Y., Shibatohge, M., Wu, D., Satoh, T., and Kataoka, T. (2001). Regulation of a novel human phospholipase C, PLCepsilon, through membrane targeting by Ras. J Biol Chem 276, 2752-2757.
44. Tartaglia, M., and Gelb, B.D. (2005). Noonan syndrome and related disorders: genetics and pathogenesis. Annu Rev Genomics Hum Genet 6, 45-68.
45. Tseng, Y.S., Tzeng, C.C., Chiu, A.W., Lin, C.H., Won, S.J., Wu, I.C., and Liu, H.S. (2003). Ha-ras overexpression mediated cell apoptosis in the presence of 5-fluorouracil. Exp Cell Res 288, 403-414.
46. Wong, W.T., Carlomagno, F., Druck, T., Barletta, C., Croce, C.M., Huebner, K., Kraus, M.H., and Di Fiore, P.P. (1994). Evolutionary conservation of the EPS8 gene and its mapping to human chromosome 12q23-q24. Oncogene 9, 3057-3061.
47. Yan, J., Roy, S., Apolloni, A., Lane, A., and Hancock, J.F. (1998). Ras isoforms vary in their ability to activate Raf-1 and phosphoinositide 3-kinase. J Biol Chem 273, 24052-24056.