Focal adhesion kinase (FAK)是一個細胞內的酪胺酸磷酸酶，它活化後會產生一連串細胞的生理反應，包括細胞的吸附、遷移、入侵、增生和存活。在數種癌症中，常會見到FAK 的過量表現，因此FAK 有潛力作為一個抗癌的藥物標的。
FAK 上酪胺酸的磷酸化對訊息傳遞的調控扮演重要的角色，其中一個在FAT domain 上第925 個胺基酸酪胺酸(Tyrosine)，位於螺旋1 上，存在有Grb2-SH2 domain 的結合序列pYXNX，當Y925 磷酸化後，會與Grb2-SH2 結合，接著活化Ras-MAPK 的訊息傳遞路徑，且Y925 的磷酸化與促進腫瘤形成的血管增生有關。
多數的SH2 domain 會和延伸的胜肽結合，但Grb2-SH2 內Trp 的側鏈會防止它和延伸的胜肽卻和形成β-turn 構形的胜肽結合，然而FAT 上的Y925 卻位於α 螺旋。本研究想了解是否FAT 上的螺旋1 在與Grb2-SH2 結合後產生構形的變化為何？如此便能更清楚此兩個重要分子的結合模式及調控情形，未來或能設計抑制劑阻斷此一結合。
本篇利用圓二色光譜及核磁共振的方法來研究Grb2-SH2 及FAT衍生的胜肽pY925 其結合時構型的變化。圓二色光譜的結果顯示pY925 胜肽加入TFE 之後，會有螺旋的二級結構出現，而再加入Grb2-SH2 後，pY925 胜肽構型會有明顯的二級結構變化，且二維及三維核磁共振的結果顯示pY925 胜肽與Grb2-SH2 結合後化學位移劇烈變化的胺基酸與pY925/Grb2-SH2 的複合物晶體所解出的胜肽結合模式(β-turn)的變化一致，因此推論pY925 在與Grb2-SH2 結合時，FAT 會有構型的變化。

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that plays a central role in signal transduction pathways that are initiated at sites of integrin-mediated cell adhesions and by growth factor receptors. FAK functions in the control of cell survival, proliferation, migration and invasion, and overexpression of FAK is found in many forms of cancer. Therefore FAK might be a potential target for anticancer drug development.
Tyrosine phosphorylation of FAK plays a significant role in regulating FAK-mediated signaling. One of the FAK phosphorylation sites is at tyrosine Y925 in human which located in the FAT domain.Y925 exists in a consensus Grb2-SH2 domain binding site pYXNX , and has been shown to bind to Grb2-SH2 domain when Y925 is phosphorylated. The FAT-Grb2-SH2 interaction activates the Ras/MAPK signaling pathway, and this linkage is essential in promoting angiogenesis.
Grb2 SH2 prefers a typical β-turn conformation for targeted motif binding because of steric hindrance caused by a bulky side chain of Trp 121, however, Y925 is located at helix1 of the four helix bundle in the FAT domain. The goal of this study is to determine the necessary of conformational change of FAK derived phosphopeptide bound to Grb2-SH2.
Thus, NMR (nuclear magnetic resonance) and CD (circular dichroism) spectroscopy and X-ray diffraction method were utilized to elucidate the peptide adaptation. The FAK phosphopeptide [NDKV(pY)ENVTG] in TFE solvent reveals the helical property as that presented in FAT domain. Interestingly, the CD spectra of FAK peptide bound to Grb2-SH2 in different molar ratio show beta-structure and helical conformation exchange. In addition, the structure of Grb2-SH2 complexed with FAK peptide was determined by heteronuclear multidimensional NMR and X-ray diffraction. Those data indicate the chemical shift-perturbed region on the domain is not linear but bent. Our NMR data, along with CD experiments suggest that phosphorylation of Y925 and subsequent Grb2 binding require FAT domain to undergo conformational change.

1. Arold, S.T., Hoellerer, M.K., and Noble, M.E.M. (2002). The Structural Basis of Localization and Signaling by the Focal Adhesion Targeting Domain. Structure 10, 319-327.
2. Bradshaw, J.M., and Waksman, G. (2002). Molecular recognition by SH2 domains. Anvances in Protein Chemistry 61, 161-210.
3. Buday, L., and Downward, J. (1993). Epidermal growth factor regulates p21ras through the formation of a complex of receptor, Grb2 adapter protein, and Sos nucleotide exchange factor. Cell 73, 611-620.
4. Chardin, P., Cussac, D., Maignan, S.b., and Ducruix, A. (1995). The Grb2 adaptor. FEBS Letlers 369 47-51.
5. Cussac, D., Frech, M., and Chardin, P. (1994). Binding of the Grb2 SH2 domain to phosphotyrosine motifs does not change the affinity of its SH3 domains for Sos proline-rich motifs. The EMBO Journal 13 4011 - 4021.
6. Daly, R.J., Binder, M.D., and Sutherland, R.L. (1994). Overexpression of the Grb2 gene in human breast cancer cell lines. Oncogene 9, 2723-2727.
7. Dyson, H.J., and Wright, P.E. (1998). Equilibrium NMR studies of unfolded and partially folded proteins. Nature Structural Biology 5, 499 - 503.
8. Eck, M.J., Atwell, S.K., Shoelson, S.E., and Harrison, S.C. (1993). Structure of the regulatory domains of the Src-family tyrosine kinase Lck. Nature 362, 764-769.
9. Foster, M.P., Wuttke, D.S., Clemens, K.R., Jahnke, W., Radhakrishnan, I., Tennant, L., Reymond, M., Chung, J., and Wright, P.E. (1998). Chemical shift as a probe of molecular interfaces: NMR studies of DNA binding by the three amino-terminal zinc finger domains from transcription factor IIIA. Journal of Biomolecular NMR 12, 51-71.
10. Gale, N.W., Kaplan, S., Lowenstein, E.J., Schlessinger, J., and Bar-Sagi, D. (1993). Grb2 mediates the EGF-dependent activation of guanine nucleotide exchange on Ras. Natyre 363, 88-92.
11. Garrett, D.S., Seok, Y.-J., Peterkofsky, A., Clore, G.M., and M.Gronenborn, A. (1997). Identification by NMR of the Binding Surface for the Histidine-Containing Phosphocarrier Protein HPr on the N-Terminal Domain of Enzyme I of the Escherichia coli Phosphotransferase System. Biochemistry 4393-4398.
12. Greenfield, N.J. (2006). Using circular dichroism spectra to estimate protein secondary structure. Nature protocols 1 2876-2890.

13. Grzesiek, S., Bax, A., Clore, G.M., Gronenborn, A.M., Hu, J.S., Kaufman, J., Palmer, I., Stahl, S.J., and Wingfield, P.T. (1996). The solution structure of HIV-1 Nef reveals an unexpected fold and permits delineation of the binding surface for the SH3 domain of Hck tyrosine protein kinase. Natural Structural Biology 3, 340-345.
14. Hayashi, I., Vuori, K., and C.Liddington, R. (2002). The focal adhesion targeting (FAT) region of focal adhesion kinase is a four-helix bundle that binds paxillin. Nature Structural Biology 9, 101-106.
15. Hong, D.-P., Hoshino, M., Kuboi, R., and Goto, Y. (1999). Clustering of Fluorine-Substituted Alcohols as a Factor Responsible for Their Marked Effects on Proteins and Peptides. Journal of American Chemical Society 121, 8427-8433.
16. Hubbard, S.R. (1997). Crystal structure of the activated insulin receptor tyrosine kinase in complex with peptide substrate and ATP analog. The EMBO Journal 16 5573–5581.
Kelly, S.M., Jess, T.J., and Price, N.C. (2005). How to study proteins by circular dichroism. Biochimica et Biophysica Acta 1751, 119-139.
17. Kuriyan, J., and Cowburn, D. (1997). Modular peptide recognition domains in eukaryotic signaling. Annu Rev Biophys Biomol Struct 26, 259-288.
18. Li, N., Batzer, A., Daly, R., Yajnik, V., Skolnik, E., Chardin, P., Bar-Sagi, D., Margolis, B., and Schlessinger, J. (1993). Guanine-nucleotide-releasing factor hSos1 binds to Grb2 and links receptor tyrosine kinases to Ras signalling. Nature 363, 85-88.
19. Liang, L., Tajmir-Riahi, H.A., and Subirade, M. (2008). Interaction of β-Lactoglobulin with Resveratrol and its Biological Implications. Biomacromolecules 9, 50-56.
20. Lietha, D., Cai, X., Ceccarelli, D.F.J., Li, Y., Schaller, M.D., and Eck, M.J. (2007). Structural Basis for the Autoinhibition of Focal Adhesion Kinase. Cell 129, 1177-1187.
21. Liu, G., Guibao, C.D., and Zheng, J. (2002). Structural Insight into the Mechanisms of Targeting and Signaling of Focal Adhesion Kinase. Molecular and Cellular Biology 2751-2760.
22. Lowensteina, E.J., Dalya, R.J., Batzera, A.G., Lia, W., Margolisa, B., Lammersb, R., Ullrichc, A., Skolnika, E.Y., Bar-Sagib, D., and Schlessingera, J. (1992). The SH2 and SH3 domain-containing protein GRB2 links receptor tyrosine kinases to ras signaling. Cell 70, 431-442.
23. Machida, K., and Mayer, B.J. (2005). The SH2 domain: versatile signaling module and pharmaceutical target. Biochimica et Biophysica Acta 1747, 1 - 25.
24. Magis, A.T., M., K., Bailey, Kurenova, E.V., Prada, J.A.H.n., Canceb, W.G., and Ostrova, D.A. (2008). Crystallization of the focal adhesion kinase targeting (FAT) domain in a primitive orthorhombic space group. Acta Cryst (2008) F64, 564–566 F64, 564–566.

25. Marley, J., Lu, M., and Bracken, C. (2001). A method for efficient isotopic labeling of recombinant proteins. Journal of Biomolecular NMR 20, 71–75.
26. Mitra, S., Mikolon, D., Molina, J., Hsia, D., Hanson, D., Chi, A., Lim, S.-T., Bernard-Trifilo, J., Ilic, D., Stupack, D., et al. (2006). Intrinsic FAK activity and Y925 phosphorylation facilitatean angiogenic switch in tumors. Oncogene 25, 5969.
27. Myers, J.K., Pace, C.N., and Scholtz, J.M. (1998). Trifluoroethanol effects on helix propensity and electrostatic interactions in the helical peptide from ribonuclease TI. Prorein Science 7383-7388.
28. Nimwegen, M.J.v., and Water, B.v.d. (2007). Focal adhesion kinase: A potential target in cancer therapy. biochemical pharmacology 73, 597-609.
29. Nioche, P., Liu, W.-Q., Broutin, I., Charbonnier, F., Latreille, M.-T.r., Vidal, M., Roques, B., Garbay, C., and Ducruix, A. (2002). Crystal Structures of the SH2 Domain of Grb2: Highlight on the Binding of a New High-affinity Inhibitor. Journal of Molecular Biology 315, 1167-1177.
30. Ogura, K., Shiga, T., Yokochi, M., Yuzawa, S., Burke, T.R., Jr., and Inagaki, F. (2008). Solution structure of the Grb2 SH2 domain complexed with a high-affinity inhibitor. Journal of Biomolecular NMR 42, 197-207.
31. Ogura, K., Tsuchiya, S., Terasawa, H., Yuzawa, S., Hatanaka, H., Mandiyan, V., Schlessinger, J., and Inagaki, F. (1999). Solution Structure of the SH2 Domain of Grb2 Complexed with the Shc-derived Phosphotyrosinecontaining Peptide. Journal of Molecular Biology 289, 439-445.
32. Ogura, K., Tsuchiyaa, S., Terasawaa, H., Yuzawaa, S., Hatanakaa, H., Mandiyand, V., Schlessingerd, J., and Inagakia, F. (1997). Conformation of an Shc-derived phosphotyrosine-containing peptide complexed with the Grb2 SH2 domain. Journal of Biomolecular NMR 10 273-278.
33. Povey, J.F., Smales, C.M., Hassard, S.J., and Howard, M.J. (2007). Comparison of the eVects of 2,2,2-triXuoroethanol on peptide and protein structure and function. Journal of Structural Biology 157, 329-338.
34. Pronk, G.J., AMM, d.V.-S., Buday, L., Downward, J., Maassen, J.A., R.H.Medema, and Bos, J.L. (1994). Involvement of Shc in insulin- and epidermal growth factor-induced activation of p21ras. Molecular and Cellular Biology 14, 1575-1581.
35. Prutzman, K.C., Gao, G., King, M.L., Iyer, V.V., Mueller, G.A., Schaller, M.D., and Campbell, S.L. (2004). The Focal Adhesion Targeting Domain of Focal Adhesion Kinase Contains a Hinge Region that Modulates Tyrosine 926 Phosphorylation. Structure 12, 881-891.

36. Rahuel, J., Gay, B., Erdmann, D., Strauss, A., GarcõÂa-EcheverrõÂa, C., Furet, P., Caravatti, G., Fretz, H., and Schoepfer, J.G.t., M. G. (1996). Structural basis for specificity of GRB2-SH2 revealed by a novel ligand binding mode. Nature Structal Biology 3, 586-589.
37. Rozakis-Adcock, M., McGlade, J., Mbamalu, G., Pelicci, G., R.Daly, W.Li, A.Batzer, S.Thomas, J.Brugge, Pelicci, P.G., et al. (1992). Association of the Shc and Grb2/Sem5 SH2-containing proteins is implicated in activation of the Ras pathway by tyrosine kinases. Nature 360, 689.
38. Schiering, N., Casale, E., Caccia, P., Giordano, P., and Battistini, C. (2000). Dimer Formation through Domain Swapping in the Crystal Structure of the Grb2-SH2−Ac-pYVNV Complex. Biochemistry 44, 13376-13382.
39. Schlaepfer, D.D., and Hunter, T. (1996). Evidence for In Vivo Phosphorylation of the Grb2 SH2-Domain Binding Site on Focal Adhesion Kinase by Src-Family Protein-Tyrosine Kinases. Molecular And Cellular Biology 16, 5623.
40. Schlaepfer, D.D., S.K.Hanks, Hunter, T., and P, v.d.G. (1994). Integrin-mediated signal transduction linked to Ras pathway by GRB2 binding to focal adhesion kinase. Nature 372, 786-791.
41. Schlessinger, J. (1994). SH2/SH3 signaling proteins. Current Opinion in Genetics and Development 4, 25-30.
42. Segawa, N., Nakamura, M., Nakamura, Y., Mori, I., Katsuoka, Y., and Kakudo, K. (2001). Phosphorylation of mitogen-activated protein kinase is inhibited by calcitonin in DU145 prostate cancer cells. Cancer Research 61, 6060-6053.
43. Skolnik, E.Y., Lee, C.H., Batzer, A., Vicentini, L.M., Zhou, M., Daly, R., Myers, M.J., Jr, B., J. M., Ullrich, A., White, M.F., et al. (1993). The SH2/SH3 domain-containing protein GRB2 interacts with tyrosine-phosphorylated IRS1 and Shc: implications for insulin control of ras signalling. The EMBO Journal 1 2 929 - 1936.
44. Songyang, Z., Shoelson, S.E., Mcglade, J., Olivier, P., Pawson, T., Bustelo, X.R., Barbacid, M., Sabe, H., Hanafusa, H., Yi, T., et al. (1994). Specific Motifs Recognized by the SH2 Domains of Csk, 3BP2, fps/fes, GRB-2, HCP, SHC, Syk, and Vav. Molecular And Cellular Biology 14, 2777-2785.
45. Thornton, K.H., Mueller, W.T., McConnell, P., Zhu, G., Saltiel, A.R., and Thanabal, V. (1996). Nuclear Magnetic Resonance Solution Structure of the Growth Factor Receptor-Bound Protein 2 Src Homology 2 Domain. Biochemistry 35, 11852-11864.
46. Tsuchiya, S., Ogura, K., Hatanaka, H., Nagata, K., Terasawa, H., Mandiyan, V., Schlessinger, J., Aimoto, S., Ohta, H., and Inagaki, F. (1999). Solution Structure of the SH2 Domain of Grb2/Ash Complexed with EGF Receptor-Derived Phosphotyrosine-Containing Peptide. Journal of Biochemistry 125, 1151-1169.

47. Yip, S.S., Crew, A.J., Gee, J.M.W., Hui, R., Blamey, R.W., Robertson, J.F.R., Nicholson, R.I., Sutherland, R.L., and Daly, R.J. (2000). Up-regulation of the protein tyrosine phosphatase SHP-1 in human breast cancer and correlation with GRB2 International Journal of Cancer 88, 363-368.