臺灣博碩士論文加值系統

巨噬細胞在人體的防禦系統，包括先天性免疫及適應性免疫反應中扮演重要的角色。當人體受到病原菌感染時，會引起發炎反應，同時會活化巨噬細胞分泌活性氮以及釋放許多細胞激素，並且造成細胞型態改變，促進細胞移動以及細菌的吞噬作用。目前研究指出，在內毒素刺激下，主要是活化Toll-like receptor 4而提升巨噬細胞吞噬細菌的能力來幫助細菌的清除。然而，目前對於其作用機轉則不清楚。在本篇文章中，我們發現LPS誘導巨噬細胞內Src蛋白質的表現量增加是TLR4和MyD88-dependent，當細胞大量表現src siRNA時，則會降低內毒素所誘導的吞噬作用，然而在送入siRNA-resistant Src之後，吞噬作用則有回復的情形。有趣的是，我們發現Eps8，為Src的受質之一，在內毒素刺激下，也會促進其蛋白質的表達，將細胞內Eps8 knockdown或大量表達PH-truncated Eps8 (i.e. 261-p97Eps8) 時，細胞的吞噬能力會有顯著的下降。此外，我們也發現Eps8和TLR4會形成複合物而共同存在於細胞質以及phagosome中。當細胞表現eps8 siRNA 或261-p97Eps8時，則會破壞Eps8-TLR4複合物的形成而抑制內毒素所誘導TLR4和MyD88的結合，並且伴隨著抑制細胞內Src, FAK以及p38 MAPK的活性。最後，我們發現抑制Eps8 蛋白質的表現會抑制巨噬細胞清除細菌的能力。 因此，綜合以上結果，我們認為在內毒素的刺激下，Eps8會藉由增加TLR4和MyD88的結合而促進巨噬細胞進行吞噬作用。

Macrophage-mediated phagocytosis and subsequent degradation of pathogens by macrophages play a pivotal role in host innate immune responses to microbial infection. Recent studies have shown that Toll-like receptors (TLRs) play an important role in promoting the clearance of bacteria by up-regulating the phagocytic activity of macrophages under LPS stimulation. However, the underlying mechanism is not well defined. In the present study, we demonstrated the induction of Src in LPS-treated macrophages was TLR4- and MyD88-dependent and attenuation of Src by src-specific siRNA reduced LPS-promoted phagocytosis. And reintroduction of siRNA-resistant Src could restore the defect. Interestingly, we observed Eps8 was also up-regulated under LPS stimulation in a Src-dependent manner and Eps8 knockdown or overexpression of PH-truncated Eps8 (i.e. 261-p97Eps8) decreased LPS-mediated macrophage phagocytosis. In addition, confocal microscopy indicated Eps8 and TLR4 were colocolized in the cytosol and at the phagosome in RAW264.7 cells. Consistently, both Eps8 and TLR4 immunoprecipitated together from the lysates of RAW264.7 cells with or without LPS stimulation. This Eps8-TLR4 complex formation was required for LPS-induced TLR4-MyD88 interaction and the following activation of Src, FAK, and p38 MAPK. Importantly, Eps8 knockdown reduced bacterium killing ability of macrophages. Thus, Eps8 is a key protein regulating LPS-stimulated TLR4-MyD88 interaction and the following macrophage phagocytosis.

中文摘要……………………………………………………………Ⅰ
ABSTRACT……………………………………………………………Ⅱ
致謝…………………………………………………………………Ⅲ
CONTENTS……………………………………………………………Ⅴ
FIGURE CONTENTS……………………………………………………Ⅶ
ABBREVIATIONS……………………………………………………..Ⅸ
Ⅰ. Introduction…………………………………………………………1
A. Overviews of infection and the mammalian immune system………………………………1
B. The innate immunity…………………………………………………………………2
a. Toll-like receptors (TLRs)……………………………………………………………………2
1. Structure of TLRs and their ligands………………………………………………………………….2
2. TLRs signaling pathways…………………………………………………………………3
b. RIG-I-like receptors (RLRs)……………………………………………………………………4
1. Structures and ligands of the RLRs family…………………………………………………………4
2. RLRs signaling pathway…………………………………………………………4
c. NOD-like receptors (NLRs)……………………………………………………………5
1. Structures and ligands of the NLRs family………………………………………………………5
2. The NOD1 and NOD2 signaling pathways……………………………………………………5
C. Phagocytosis………………………………………………5
a. The steps of phagocytosis in phagocytes……………………………………………………5
b. The role of TLRs in phagocytosis……………………………………………………6
D. Eps8 (EGF receptor pathway substrate No. 8)……………………………………………………7
a. Structural features of Eps8…………………………………………………………………7
b. The role of Eps8 in tumorigenesis……………………………………………………8
Ⅱ. Specific aims……………………………………………………………10
Ⅲ. Materials and Experimental procedures……………………………11
1. Reagents and antibodies……………………………………………………11
2. Bacterial
strain…………………………………………………………11
3. Animals……………………………………………………………11
4. Cell culture and collection of PEMs or BMDMs…………………………………………..…………11
5. Generation of macrophages expressing nonspecific siRNA, src siRNA, fak siRNA, luciferase siRNA, eps8 siRNA, myd88 siRNA, or 261-p97Eps8………………………….………12
6. Lysate preparation and immunoblot analysis……………………………………………….………….13
7. Phagocytosis…………………………………………………13
8. Confocal microscopy……………………………………………………………13
9. Bacterial killing assay…………………………………………………………14
10. Statistical analysis………………………………………………………………14
Ⅳ. Results…………………………………………………………15
1. LPS-induced Src is TLR4-dependent and plays important roles in regulating macrophage phagocytosis…………………………………………………………15
2. Concomitant up-regulation of p97Eps8 and Src in LPS-stimulated macrophages……….16
3. Attenuation of Eps8 reduces LPS-stimulated Src Pi-416, FAK Pi-Y861, actin polymerization, and phagocytosis…………………………………………………………16
4. FAK is required for LPS-induced Eps8 and macrophage phagocytosis…………….……..17
5. Eps8 is required for LPS-induced TLR4-MyD88 complex formation in macrophages…………………………………………………………18
6. PH-truncated 261-p97Eps8 inhibits LPS-induced TLR4-MyD88 complex formation, Src Pi-Y416, FAK Pi-Y861, and phagocytosis in macrophages……………………………….18
7. Eps8 sustains TLR4-mediated activation of p38 MAPK………………………………….………19
8. Complex formation between Eps8 and FAK in RAW cells………………………………………19
9. Eps8 does not regulate LPS-induced PYK2 Pi-Y402 in macrophages……………………..20
10. Attenuation of Eps8, Src, or FAK impairs intracellular killing of E. coli in macrophages……………………………………………………………20
Ⅴ. Conclusion…………………………………………………………...22
Ⅵ. Discussion………………………………………………………………23
Ⅶ. References………………………………………………………………30
Ⅷ. Figures…………………………………………………………………..40
Ⅸ. Appendix………………………………………………………………...65

Abou-Kheir, W., Isaac, B., Yamaguchi, H., and Cox, D. (2008). Membrane targeting of WAVE2 is not sufficient for WAVE2-dependent actin polymerization: a role for IRSp53 in mediating the interaction between Rac and WAVE2. J Cell Sci 121, 379-390.

Aderem, A., and Underhill, D.M. (1999). Mechanisms of phagocytosis in macrophages. Annu Rev Immunol 17, 593-623.

Akira, S., Uematsu, S., and Takeuchi, O. (2006). Pathogen recognition and innate immunity. Cell 124, 783-801.

Allen, L.H., and Aderem, A. (1995). A role for MARCKS, the alpha isozyme of protein kinase C and myosin I in zymosan phagocytosis by macrophages. J Exp Med 182, 829-840.

Anand, R.J., Kohler, J.W., Cavallo, J.A., Li, J., Dubowski, T., and Hackam, D.J. (2007). Toll-like receptor 4 plays a role in macrophage phagocytosis during peritoneal sepsis. J Pediatr Surg 42, 927-932; discussion 933.

Antonieta Cote-Velez, M.J., Ortega, E., and Ortega, A. (2001). Involvement of pp125FAK and p60SRC in the signaling through Fc gamma RII-Fc gamma RIII in murine macrophages. Immunol Lett 78, 189-194.

Beutler, B. (2002). Toll-like receptors: how they work and what they do. Curr Opin Hematol 9, 2-10.

Beutler, B. (2004). Inferences, questions and possibilities in Toll-like receptor signalling. Nature 430, 257-263.

Beutler, B., Eidenschenk, C., Crozat, K., Imler, J.L., Takeuchi, O., Hoffmann, J.A., and Akira, S. (2007). Genetic analysis of resistance to viral infection. Nat Rev Immunol 7, 753-766.

Beutler, B., Jiang, Z., Georgel, P., Crozat, K., Croker, B., Rutschmann, S., Du, X., and Hoebe, K. (2006). Genetic analysis of host resistance: Toll-like receptor signaling and immunity at large. Annu Rev Immunol 24, 353-389.

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.

Blander, J.M., and Medzhitov, R. (2004). Regulation of phagosome maturation by signals from toll-like receptors. Science 304, 1014-1018.

Brown, E.J. (1995). Phagocytosis. Bioessays 17, 109-117.

Burg, N.D., and Pillinger, M.H. (2001). The neutrophil: function and regulation in innate and humoral immunity. Clin Immunol 99, 7-17.

Chamaillard, M., Hashimoto, M., Horie, Y., Masumoto, J., Qiu, S., Saab, L., Ogura, Y., Kawasaki, A., Fukase, K., Kusumoto, S., et al. (2003). An essential role for NOD1 in host recognition of bacterial peptidoglycan containing diaminopimelic acid. Nat Immunol 4, 702-707.

Check, J., Byrd, C.L., Menio, J., Rippe, R.A., Hines, I.N., and Wheeler, M.D. (2010). Src kinase participates in LPS-induced activation of NADPH oxidase. Mol Immunol 47, 756-762.

Chen, Y.J., Shen, M.R., Maa, M.C., and Leu, T.H. (2008). Eps8 decreases chemosensitivity and affects survival of cervical cancer patients. Mol Cancer Ther 7, 1376-1385.

Croce, A., Cassata, G., Disanza, A., Gagliani, M.C., Tacchetti, C., Malabarba, M.G., Carlier, M.F., Scita, G., Baumeister, R., and Di Fiore, P.P. (2004). A novel actin barbed-end-capping activity in EPS-8 regulates apical morphogenesis in intestinal cells of Caenorhabditis elegans. Nat Cell Biol 6, 1173-1179.

Cui, S., Eisenacher, K., Kirchhofer, A., Brzozka, K., Lammens, A., Lammens, K., Fujita, T., Conzelmann, K.K., Krug, A., and Hopfner, K.P. (2008). The C-terminal regulatory domain is the RNA 5'-triphosphate sensor of RIG-I. Mol Cell 29, 169-179.

Defilippi, P., Di Stefano, P., and Cabodi, S. (2006). p130Cas: a versatile scaffold in signaling networks. Trends Cell Biol 16, 257-263.
DeLeo, F.R., Allen, L.A., Apicella, M., and Nauseef, W.M. (1999). NADPH oxidase activation and assembly during phagocytosis. J Immunol 163, 6732-6740.

Disanza, A., Carlier, M.F., Stradal, T.E., Didry, D., Frittoli, E., Confalonieri, S., Croce, A., Wehland, J., Di Fiore, P.P., and Scita, G. (2004). Eps8 controls actin-based motility by capping the barbed ends of actin filaments. Nat Cell Biol 6, 1180-1188.

Disanza, A., Steffen, A., Hertzog, M., Frittoli, E., Rottner, K., and Scita, G. (2005). Actin polymerization machinery: the finish line of signaling networks, the starting point of cellular movement. Cell Mol Life Sci 62, 955-970.

Doyle, S.E., O'Connell, R.M., Miranda, G.A., Vaidya, S.A., Chow, E.K., Liu, P.T., Suzuki, S., Suzuki, N., Modlin, R.L., Yeh, W.C., et al. (2004). Toll-like receptors induce a phagocytic gene program through p38. J Exp Med 199, 81-90.

Duong, L.T., Nakamura, I., Lakkakorpi, P.T., Lipfert, L., Bett, A.J., and Rodan, G.A. (2001). Inhibition of osteoclast function by adenovirus expressing antisense protein-tyrosine kinase 2. J Biol Chem 276, 7484-7492.

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.

Finnemann, S.C. (2003). Focal adhesion kinase signaling promotes phagocytosis of integrin-bound photoreceptors. EMBO J 22, 4143-4154.

Frittoli, E., Matteoli, G., Palamidessi, A., Mazzini, E., Maddaluno, L., Disanza, A., Yang, C., Svitkina, T., Rescigno, M., and Scita, G. (2011). The signaling adaptor Eps8 is an essential actin capping protein for dendritic cell migration. Immunity 35, 388-399.

Funato, Y., Terabayashi, T., Suenaga, N., Seiki, M., Takenawa, T., and Miki, H. (2004). IRSp53/Eps8 complex is important for positive regulation of Rac and cancer cell motility/invasiveness. Cancer Res 64, 5237-5244.

Gillespie, J.P., Kanost, M.R., and Trenczek, T. (1997). Biological mediators of insect immunity. Annu Rev Entomol 42, 611-643.

Girardin, S.E., Boneca, I.G., Carneiro, L.A., Antignac, A., Jehanno, M., Viala, J., Tedin, K., Taha, M.K., Labigne, A., Zahringer, U., et al. (2003a). Nod1 detects a unique muropeptide from gram-negative bacterial peptidoglycan. Science 300, 1584-1587.

Girardin, S.E., Boneca, I.G., Viala, J., Chamaillard, M., Labigne, A., Thomas, G., Philpott, D.J., and Sansonetti, P.J. (2003b). Nod2 is a general sensor of peptidoglycan through muramyl dipeptide (MDP) detection. J Biol Chem 278, 8869-8872.

Greenberg, S., and Grinstein, S. (2002). Phagocytosis and innate immunity. Curr Opin Immunol 14, 136-145.

Guha, M., and Mackman, N. (2001). LPS induction of gene expression in human monocytes. Cell Signal 13, 85-94.

Hansson, G.K., Libby, P., Schonbeck, U., and Yan, Z.Q. (2002). Innate and adaptive immunity in the pathogenesis of atherosclerosis. Circ Res 91, 281-291.

Hasegawa, M., Fujimoto, Y., Lucas, P.C., Nakano, H., Fukase, K., Nunez, G., and Inohara, N. (2008). A critical role of RICK/RIP2 polyubiquitination in Nod-induced NF-kappaB activation. EMBO J 27, 373-383.

Hoffmann, J.A. (2003). The immune response of Drosophila. Nature 426, 33-38.

Hsu, Y.M., Zhang, Y., You, Y., Wang, D., Li, H., Duramad, O., Qin, X.F., Dong, C., and Lin, X. (2007). The adaptor protein CARD9 is required for innate immune responses to intracellular pathogens. Nat Immunol 8, 198-205.

Inohara, Chamaillard, McDonald, C., and Nunez, G. (2005). NOD-LRR proteins: role in host-microbial interactions and inflammatory disease. Annu Rev Biochem 74, 355-383.

Inohara, N., Ogura, Y., Fontalba, A., Gutierrez, O., Pons, F., Crespo, J., Fukase, K., Inamura, S., Kusumoto, S., Hashimoto, M., et al. (2003). Host recognition of bacterial muramyl dipeptide mediated through NOD2. Implications for Crohn's disease. J Biol Chem 278, 5509-5512.

Janssens, S., Burns, K., Tschopp, J., and Beyaert, R. (2002). Regulation of interleukin-1- and lipopolysaccharide-induced NF-kappaB activation by alternative splicing of MyD88. Curr Biol 12, 467-471.

Jones, G.E., Zicha, D., Dunn, G.A., Blundell, M., and Thrasher, A. (2002). Restoration of podosomes and chemotaxis in Wiskott-Aldrich syndrome macrophages following induced expression of WASp. Int J Biochem Cell Biol 34, 806-815.

Karlsson, T., Songyang, Z., Landgren, E., Lavergne, C., Di Fiore, P.P., Anafi, M., Pawson, T., Cantley, L.C., Claesson-Welsh, L., and Welsh, M. (1995). Molecular interactions of the Src homology 2 domain protein Shb with phosphotyrosine residues, tyrosine kinase receptors and Src homology 3 domain proteins. Oncogene 10, 1475-1483.

Kasorn, A., Alcaide, P., Jia, Y., Subramanian, K.K., Sarraj, B., Li, Y., Loison, F., Hattori, H., Silberstein, L.E., Luscinskas, W.F., et al. (2009). Focal adhesion kinase regulates pathogen-killing capability and life span of neutrophils via mediating both adhesion-dependent and -independent cellular signals. J Immunol 183, 1032-1043.

Kawai, T., and Akira, S. (2009). The roles of TLRs, RLRs and NLRs in pathogen recognition. Int Immunol 21, 317-337.

Kawai, T., Sato, S., Ishii, K.J., Coban, C., Hemmi, H., Yamamoto, M., Terai, K., Matsuda, M., Inoue, J., Uematsu, S., et al. (2004). Interferon-alpha induction through Toll-like receptors involves a direct interaction of IRF7 with MyD88 and TRAF6. Nat Immunol 5, 1061-1068.

Kheir, W.A., Gevrey, J.C., Yamaguchi, H., Isaac, B., and Cox, D. (2005). A WAVE2-Abi1 complex mediates CSF-1-induced F-actin-rich membrane protrusions and migration in macrophages. J Cell Sci 118, 5369-5379.

Kong, L., and Ge, B.X. (2008). MyD88-independent activation of a novel actin-Cdc42/Rac pathway is required for Toll-like receptor-stimulated phagocytosis. Cell Res 18, 745-755.

Lakkakorpi, P.T., Bett, A.J., Lipfert, L., Rodan, G.A., and Duong le, T. (2003). PYK2 autophosphorylation, but not kinase activity, is necessary for adhesion-induced association with c-Src, osteoclast spreading, and bone resorption. J Biol Chem 278, 11502-11512.

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.

Leu, T.H., Charoenfuprasert, S., Yen, C.K., Fan, C.W., and Maa, M.C. (2006). Lipopolysaccharide-induced c-Src expression plays a role in nitric oxide and TNFalpha secretion in macrophages. Mol Immunol 43, 308-316.

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.

Liu, P.S., Jong, T.H., Maa, M.C., and Leu, T.H. (2010). The interplay between Eps8 and IRSp53 contributes to Src-mediated transformation. Oncogene 29, 3977-3989.

Maa, M.C., Chang, M.Y., Chen, Y.J., Lin, C.H., Yu, C.J., Yang, Y.L., Li, J., Chen, P.R., Tang, C.H., Lei, H.Y., et al. (2008). Requirement of inducible nitric-oxide synthase in lipopolysaccharide-mediated Src induction and macrophage migration. J Biol Chem 283, 31408-31416.

Maa, M.C., Chang, M.Y., Li, J., Li, Y.Y., Hsieh, M.Y., Yang, C.J., Chen, Y.J., Li, Y., Chen, H.C., Cheng, W.E., et al. (2011). The iNOS/Src/FAK axis is critical in Toll-like receptor-mediated cell motility in macrophages. Biochim Biophys Acta 1813, 136-147.

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.

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.

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.

Maa, M.C., and Leu, T.H. (1998). Vanadate-dependent FAK activation is accomplished by the sustained FAK Tyr-576/577 phosphorylation. Biochem Biophys Res Commun 251, 344-349.

Massol, P., Montcourrier, P., Guillemot, J.C., and Chavrier, P. (1998). Fc receptor-mediated phagocytosis requires CDC42 and Rac1. EMBO J 17, 6219-6229.

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.

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.

May, R.C., Caron, E., Hall, A., and Machesky, L.M. (2000). Involvement of the Arp2/3 complex in phagocytosis mediated by FcgammaR or CR3. Nat Cell Biol 2, 246-248.

Medzhitov, R. (2007). Recognition of microorganisms and activation of the immune response. Nature 449, 819-826.

Medzhitov, R., and Janeway, C.A., Jr. (1997). Innate immunity: impact on the adaptive immune response. Curr Opin Immunol 9, 4-9.

Metheniti, A., Paraskevopoulou, N., Lambropoulou, M., and Marmaras, V.J. (2001). Involvement of FAK/Src complex in the processes of Escherichia coli phagocytosis by insect hemocytes. FEBS Lett 496, 55-59.

Michallet, M.C., Meylan, E., Ermolaeva, M.A., Vazquez, J., Rebsamen, M., Curran, J., Poeck, H., Bscheider, M., Hartmann, G., Konig, M., et al. (2008). TRADD protein is an essential component of the RIG-like helicase antiviral pathway. Immunity 28, 651-661.

Morrison, D.C., and Ryan, J.L. (1979). Bacterial endotoxins and host immune responses. Adv Immunol 28, 293-450.

O'Neill, L. (2000). The Toll/interleukin-1 receptor domain: a molecular switch for inflammation and host defence. Biochem Soc Trans 28, 557-563.

Owen, K.A., Thomas, K.S., and Bouton, A.H. (2007). The differential expression of Yersinia pseudotuberculosis adhesins determines the requirement for FAK and/or Pyk2 during bacterial phagocytosis by macrophages. Cell Microbiol 9, 596-609.

Park, J.H., Kim, Y.G., Shaw, M., Kanneganti, T.D., Fujimoto, Y., Fukase, K., Inohara, N., and Nunez, G. (2007). Nod1/RICK and TLR signaling regulate chemokine and antimicrobial innate immune responses in mesothelial cells. J Immunol 179, 514-521.

Poltorak, A., He, X., Smirnova, I., Liu, M.Y., Van Huffel, C., Du, X., Birdwell, D., Alejos, E., Silva, M., Galanos, C., et al. (1998). Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 282, 2085-2088.

Ponting, C.P. (1996). Novel domains in NADPH oxidase subunits, sorting nexins, and PtdIns 3-kinases: binding partners of SH3 domains? Protein Sci 5, 2353-2357.

Schnare, M., Barton, G.M., Holt, A.C., Takeda, K., Akira, S., and Medzhitov, R. (2001). Toll-like receptors control activation of adaptive immune responses. Nat Immunol 2, 947-950.

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.

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.

Takahasi, K., Yoneyama, M., Nishihori, T., Hirai, R., Kumeta, H., Narita, R., Gale, M., Jr., Inagaki, F., and Fujita, T. (2008). Nonself RNA-sensing mechanism of RIG-I helicase and activation of antiviral immune responses. Mol Cell 29, 428-440.

Takenawa, T., and Suetsugu, S. (2007). The WASP-WAVE protein network: connecting the membrane to the cytoskeleton. Nat Rev Mol Cell Biol 8, 37-48.

Takeda, K., Kaisho, T., and Akira, S. (2003). Toll-like receptors. Annu Rev Immunol 21, 335-376.

Takeuchi, O., and Akira, S. (2010). Pattern recognition receptors and inflammation. Cell 140, 805-820.

Uematsu, S., Sato, S., Yamamoto, M., Hirotani, T., Kato, H., Takeshita, F., Matsuda, M., Coban, C., Ishii, K.J., Kawai, T., et al. (2005). Interleukin-1 receptor-associated kinase-1 plays an essential role for Toll-like receptor (TLR)7- and TLR9-mediated interferon-{alpha} induction. J Exp Med 201, 915-923.

Ulevitch, R.J., and Tobias, P.S. (1999). Recognition of gram-negative bacteria and endotoxin by the innate immune system. Curr Opin Immunol 11, 19-22.

Underhill, D.M., and Gantner, B. (2004). Integration of Toll-like receptor and phagocytic signaling for tailored immunity. Microbes Infect 6, 1368-1373.

Underhill, D.M., and Ozinsky, A. (2002). Phagocytosis of microbes: complexity in action. Annu Rev Immunol 20, 825-852.

Watters, T.M., Kenny, E.F., and O'Neill, L.A. (2007). Structure, function and regulation of the Toll/IL-1 receptor adaptor proteins. Immunol Cell Biol 85, 411-419.

Welsch, T., Endlich, K., Giese, T., Buchler, M.W., and Schmidt, J. (2007). Eps8 is increased in pancreatic cancer and required for dynamic actin-based cell protrusions and intercellular cytoskeletal organization. Cancer Lett 255, 205-218.

Wiedemann, A., Linder, S., Grassl, G., Albert, M., Autenrieth, I., and Aepfelbacher, M. (2001). Yersinia enterocolitica invasin triggers phagocytosis via beta1 integrins, CDC42Hs and WASp in macrophages. Cell Microbiol 3, 693-702.

Xi, C.X., Xiong, F., Zhou, Z., Mei, L., and Xiong, W.C. (2010). PYK2 interacts with MyD88 and regulates MyD88-mediated NF-kappaB activation in macrophages. J Leukoc Biol 87, 415-423.

Xu, M., Shorts-Cary, L., Knox, A.J., Kleinsmidt-DeMasters, B., Lillehei, K., and Wierman, M.E. (2009). Epidermal growth factor receptor pathway substrate 8 is overexpressed in human pituitary tumors: role in proliferation and survival. Endocrinology 150, 2064-2071.

Yap, L.F., Jenei, V., Robinson, C.M., Moutasim, K., Benn, T.M., Threadgold, S.P., Lopes, V., Wei, W., Thomas, G.J., and Paterson, I.C. (2009). Upregulation of Eps8 in oral squamous cell carcinoma promotes cell migration and invasion through integrin-dependent Rac1 activation. Oncogene 28, 2524-2534.

Yoneyama, M., and Fujita, T. (2008). Structural mechanism of RNA recognition by the RIG-I-like receptors. Immunity 29, 178-181