Tyrosine phosphorylation of the Gα-interacting protein GIV promotes activation of phosphoinositide 3-kinase during cell migration

Science Signaling

Volumn 4, Issue 192, 2011, Pages

  Lin, Changsheng ^a   Ear, Jason ^a   Pavlova, Yelena ^a   Mittal, Yash ^a   Kufareva, Irina ^b   Ghassemian, Majid ^b   Abagyan, Ruben ^b   Garcia Marcos, Mikel ^c   Ghosh, Pradipta ^a  

^a UNIVERSITY OF CALIFORNIA SAN DIEGO   (United States)

^b UNIVERSITY OF CALIFORNIA   (United States)

^c UNIVERSITY OF CALIFORNIA   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

ACTIN; G ALPHA INTERACTING VESICLE ASSOCIATED PROTEIN; G PROTEIN COUPLED RECEPTOR; GUANINE NUCLEOTIDE EXCHANGE FACTOR; PHOSPHATIDYLINOSITOL 3 KINASE; PROTEIN KINASE B; PROTEIN P85; PROTEIN SH2; UNCLASSIFIED DRUG; ACTIN BINDING PROTEIN; CCDC88A PROTEIN, HUMAN; TYROSINE; VESICULAR TRANSPORT PROTEIN;

BREAST CARCINOMA; CANCER GROWTH; CANCER INVASION; CELL MIGRATION; CONTROLLED STUDY; ENZYME ACTIVATION; HUMAN; HUMAN CELL; NONHUMAN; PRIORITY JOURNAL; PROTEIN BINDING; PROTEIN EXPRESSION; PROTEIN INTERACTION; PROTEIN MOTIF; PROTEIN PHOSPHORYLATION; REVIEW; ANALYSIS OF VARIANCE; ARTICLE; CELL MOTION; CHEMICAL STRUCTURE; FLUORESCENT ANTIBODY TECHNIQUE; IMMUNOPRECIPITATION; LIQUID CHROMATOGRAPHY; METABOLISM; PHOSPHORYLATION; PHYSIOLOGY; TANDEM MASS SPECTROMETRY; TUMOR CELL LINE;

GREAT ISLAND VIRUS;

ANALYSIS OF VARIANCE; CELL LINE, TUMOR; CELL MOVEMENT; CHROMATOGRAPHY, LIQUID; ENZYME ACTIVATION; FLUORESCENT ANTIBODY TECHNIQUE; HUMANS; IMMUNOPRECIPITATION; MICROFILAMENT PROTEINS; MODELS, MOLECULAR; PHOSPHATIDYLINOSITOL 3-KINASE; PHOSPHORYLATION; TANDEM MASS SPECTROMETRY; TYROSINE; VESICULAR TRANSPORT PROTEINS;

EID: 80053257932     PISSN: 19450877     EISSN: 19379145     Source Type: Journal    
DOI: 10.1126/scisignal.2002049     Document Type: Review

References (74)

1. A. Enomoto, H. Murakami, N. Asai, N. Morone, T. Watanabe, K. Kawai, Y. Murakumo, J. Usukura, K. Kaibuchi, M. Takahashi, Akt/PKB regulates actin organization and cell motility via Girdin/APE. Dev. Cell 9, 389-402 (2005).
2. P. Ghosh, A. O. Beas, S. J. Bornheimer, M. Garcia-Marcos, E. P. Forry, C. Johannson, J. Ear, B. H. Jung, B. Cabrera, J. M. Carethers, M. G. Farquhar, A Gαi-GIV molecular complex binds epidermal growth factor receptor and determines whether cells migrate or proliferate. Mol. Biol. Cell 21, 2338-2354 (2010).
3. P. Jiang, A. Enomoto, M. Jijiwa, T. Kato, T. Hasegawa, M. Ishida, T. Sato, N. Asai, Y. Murakumo, M. Takahashi, An actin-binding protein Girdin regulates the motility of breast cancer cells. Cancer Res. 68, 1310-1318 (2008).
4. T. Kitamura, N. Asai, A. Enomoto, K. Maeda, T. Kato, M. Ishida, P. Jiang, T. Watanabe, J. Usukura, T. Kondo, F. Costantini, T. Murohara, M. Takahashi, Regulation of VEGF-mediated angiogenesis by the Akt/PKB substrate Girdin. Nat. Cell Biol. 10, 329-337 (2008).
5. M. Anai, N. Shojima, H. Katagiri, T. Ogihara, H. Sakoda, Y. Onishi, H. Ono, M. Fujishiro, Y. Fukushima, N. Horike, A. Viana, M. Kikuchi, N. Noguchi, S. Takahashi, K. Takata, Y. Oka, Y. Uchijima, H. Kurihara, T. Asano, A novel protein kinase B (PKB)/AKT-binding protein enhances PKB kinase activity and regulates DNA synthesis. J. Biol. Chem. 280, 18525-18535 (2005).
6. M. Garcia-Marcos, J. Ear, M. G. Farquhar, P. Ghosh, A GDI (AGS3) and a GEF (GIV) regulate autophagy by balancing G protein activity and growth factor signals. Mol. Biol. Cell 22, 673-686 (2011).
7. M. Garcia-Marcos, P. Ghosh, M. G. Farquhar, GIV is a nonreceptor GEF for Gαi with a unique motif that regulates Akt signaling. Proc. Natl. Acad. Sci. U.S.A. 106, 3178-3183 (2009).
8. P. Ghosh, M. Garcia-Marcos, S. J. Bornheimer, M. G. Farquhar, Activation of Gαi3 triggers cell migration via regulation of GIV. J. Cell Biol. 182, 381-393 (2008).
9. M. Garcia-Marcos, B. H. Jung, J. Ear, B. Cabrera, J. M. Carethers, P. Ghosh, Expression of GIV/Girdin, a metastasis-related protein, predicts patient survival in colon cancer. FASEB J. 25, 590-599 (2011).
10. J. Rush, A. Moritz, K. A. Lee, A. Guo, V. L. Goss, E. J. Spek, H. Zhang, X. M. Zha, R. D. Polakiewicz, M. J. Comb, Immunoaffinity profiling of tyrosine phosphorylation in cancer cells. Nat. Biotechnol. 23, 94-101 (2005).
11. A. Moritz, Y. Li, A. Guo, J. Villén, Y. Wang, J. MacNeill, J. Kornhauser, K. Sprott, J. Zhou, A. Possemato, J. M. Ren, P. Hornbeck, L. C. Cantley, S. P. Gygi, J. Rush, M. J. Comb, Akt-RSK-S6 kinase signaling networks activated by oncogenic receptor tyrosine kinases. Sci. Signal. 3, ra64 (2010).
12. A. Guo, J. Villén, J. Kornhauser, K. A. Lee, M. P. Stokes, K. Rikova, A. Possemato, J. Nardone, G. Innocenti, R. Wetzel, Y. Wang, J. MacNeill, J. Mitchell, S. P. Gygi, J. Rush, R. D. Polakiewicz, M. J. Comb, Signaling networks assembled by oncogenic EGFR and c-Met. Proc. Natl. Acad. Sci. U.S.A. 105, 692-697 (2008).
13. C. Jørgensen, A. Sherman, G. I. Chen, A. Pasculescu, A. Poliakov, M. Hsiung, B. Larsen, D. G.Wilkinson, R. Linding, T. Pawson, Cell-specific information processing in segregating populations of Eph receptor ephrin-expressing cells. Science 326, 1502-1509 (2009).
14. J. R. St-Germain, P. Taylor, J. Tong, L. L. Jin, A. Nikolic, I. I. Stewart, R. M. Ewing, M. Dharsee, Z. Li, S. Trudel, M. F. Moran, Multiple myeloma phosphotyrosine proteomic profile associated with FGFR3 expression, ligand activation, and drug inhibition. Proc. Natl. Acad. Sci. U.S.A. 106, 20127-20132 (2009).
15. N. Osherov, A. Levitzki, Epidermal-growth-factor-dependent activation of the src-family kinases. Eur. J. Biochem. 225, 1047-1053 (1994).
16. J. H. Hanke, J. P. Gardner, R. L. Dow, P. S. Changelian, W. H. Brissette, E. J. Weringer, B. A. Pollok, P. A. Connelly, Discovery of a novel, potent, and Src family-selective tyrosine kinase inhibitor. Study of Lck- and FynT-dependent T cell activation. J. Biol. Chem. 271, 695-701 (1996).
17. J. Andreev, M. L. Galisteo, O. Kranenburg, S. K. Logan, E. S. Chiu, M. Okigaki, L. A. Cary, W. H. Moolenaar, J. Schlessinger, Src and Pyk2 mediate G-protein-coupled receptor activation of epidermal growth factor receptor (EGFR) but are not required for coupling to the mitogen-activated protein (MAP) kinase signaling cascade. J. Biol. Chem. 276, 20130-20135 (2001).
18. A. Enomoto, J. Ping, M. Takahashi, Girdin, a novel actin-binding protein, and its family of proteins possess versatile functions in the Akt and Wnt signaling pathways. Ann. N. Y. Acad. Sci. 1086, 169-184 (2006).
19. L. C. Cantley, The phosphoinositide 3-kinase pathway. Science 296, 1655-1657 (2002).
20. E. Piccione, R. D. Case, S. M. Domchek, P. Hu, M. Chaudhuri, J. M. Backer, J. Schlessinger, S. E. Shoelson, Phosphatidylinositol 3-kinase p85 SH2 domain specificity defined by direct phosphopeptide/SH2 domain binding. Biochemistry 32, 3197-3202 (1993).
21. Z. Songyang, S. E. Shoelson, M. Chaudhuri, G. Gish, T. Pawson, W. G. Haser, F. King, T. Roberts, S. Ratnofsky, R. J. Lechleider, B. G. Neel, R. B. Birge, J. E. Fajardo, M. M. Chou, H. Hanafusa, B. Schaffhausen, L. C. Cantley, SH2 domains recognize specific phosphopeptide sequences. Cell 72, 767-778 (1993).
22. R. Abagyan, M. Totrov, Biased probability Monte Carlo conformational searches and electrostatic calculations for peptides and proteins. J. Mol. Biol. 235, 983-1002 (1994).
23. R. Abagyan, M. Totrov, D. Kuznetsov, ICM - A new method for protein modeling and design: Applications to docking and structure prediction from the distorted native conformation. J. Comp. Chem. 15, 488-506 (1994).
24. R. A. Pauptit, C. A. Dennis, D. J. Derbyshire, A. L. Breeze, S. A. Weston, S. Rowsell, G. N. Murshudov, NMR trial models: Experiences with the colicin immunity protein Im7 and the p85α C-terminal SH2-peptide complex. Acta Crystallogr. D Biol. Crystallogr. 57, 1397-1404 (2001).
25. R. T. Nolte, M. J. Eck, J. Schlessinger, S. E. Shoelson, S. C. Harrison, Crystal structure of the PI 3-kinase p85 amino-terminal SH2 domain and its phosphopeptide complexes. Nat. Struct. Biol. 3, 364-374 (1996).
26. K. H. Holt, L. Olson, W. S. Moye-Rowley, J. E. Pessin, Phosphatidylinositol 3-kinase activation is mediated by high-affinity interactions between distinct domains within the p110 and p85 subunits. Mol. Cell. Biol. 14, 42-49 (1994).
27. B. Cuevas, Y. Lu, S. Watt, R. Kumar, J. Zhang, K. A. Siminovitch, G. B. Mills, SHP-1 regulates Lck-induced phosphatidylinositol 3-kinase phosphorylation and activity. J. Biol. Chem. 274, 27583-27589 (1999).
28. M. Whitman, C. P. Downes, M. Keeler, T. Keller, L. Cantley, Type I phosphatidylinositol kinase makes a novel inositol phospholipid, phosphatidylinositol-3-phosphate. Nature 332, 644-646 (1988).
29. M. J. Fry, A. Gebhardt, P. J. Parker, J. G. Foulkes, Phosphatidylinositol turnover and transformation of cells by Abelson murine leukaemia virus. EMBO J. 4, 3173-3178 (1985).
30. P. Wang, P. Kumar, C. Wang, K. A. Defea, Differential regulation of class IA phosphoinositide 3-kinase catalytic subunits p110α and β by protease-activated receptor 2 and b-arrestins. Biochem. J. 408, 221-230 (2007).
31. Z. Zhang, X. Y. Yang, S. P. Soltoff, D. M. Cohen, PI3K signaling in the murine kidney inner medullary cell response to urea. Am. J. Physiol. Renal Physiol. 278, F155-F164 (2000).
32. J. M. Kavran, D. E. Klein, A. Lee, M. Falasca, S. J. Isakoff, E. Y. Skolnik, M. A. Lemmon, Specificity and promiscuity in phosphoinositide binding by pleckstrin homology domains. J. Biol. Chem. 273, 30497-30508 (1998).
33. C. D. Kontos, T. P. Stauffer, W. P. Yang, J. D. York, L. Huang, M. A. Blanar, T. Meyer, K. G. Peters, Tyrosine 1101 of Tie2 is the major site of association of p85 and is required for activation of phosphatidylinositol 3-kinase and Akt. Mol. Cell. Biol. 18, 4131-4140 (1998).
34. G. Servant, O. D. Weiner, P. Herzmark, T. Balla, J. W. Sedat, H. R. Bourne, Polarization of chemoattractant receptor signaling during neutrophil chemotaxis. Science 287, 1037-1040 (2000).
35. S. J. Watton, J. Downward, Akt/PKB localisation and 3′ phosphoinositide generation at sites of epithelial cell-matrix and cell-cell interaction. Curr. Biol. 9, 433-436 (1999).
36. P. Várnai, K. I. Rother, T. Balla, Phosphatidylinositol 3-kinase-dependent membrane association of the Bruton's tyrosine kinase pleckstrin homology domain visualized in single living cells. J. Biol. Chem. 274, 10983-10989 (1999).
37. V. Band, D. Zajchowski, K. Swisshelm, D. Trask, V. Kulesa, C. Cohen, J. Connolly, R. Sager, Tumor progression in four mammary epithelial cell lines derived from the same patient. Cancer Res. 50, 7351-7357 (1990).
38. M. Qiao, J. D. Iglehart, A. B. Pardee, Metastatic potential of 21T human breast cancer cells depends on Akt/protein kinase B activation. Cancer Res. 67, 5293-5299 (2007).
39. A. Enomoto, N. Asai, T. Namba, Y. Wang, T. Kato, M. Tanaka, H. Tatsumi, S. Taya, D. Tsuboi, K. Kuroda, N. Kaneko, K. Sawamoto, R. Miyamoto, M. Jijiwa, Y. Murakumo, M. Sokabe, T. Seki, K. Kaibuchi, M. Takahashi, Roles of disrupted-in-schizophrenia 1-interacting protein girdin in postnatal development of the dentate gyrus. Neuron 63, 774-787 (2009).
40. H. Miyake, K. Maeda, N. Asai, R. Shibata, H. Ichimiya, M. Isotani-Sakakibara, Y. Yamamura, K. Kato, A. Enomoto, M. Takahashi, T. Murohara, The actin-binding protein Girdin and its Akt-mediated phosphorylation regulate neointima formation after vascular injury. Circ. Res. 108, 1170-1179 (2011).
41. M. Yamaguchi, O. Suyari, R. Nagai, M. Takahashi, dGirdin a new player of Akt/PKB signaling in Drosophila melanogaster. Front. Biosci. 15, 1164-1171 (2010).
42. Y. Wang, N. Kaneko, N. Asai, A. Enomoto, M. Isotani-Sakakibara, T. Kato, M. Asai, Y. Murakumo, H. Ota, T. Hikita, T. Namba, K. Kuroda, K. Kaibuchi, G. L. Ming, H. Song, K. Sawamoto, M. Takahashi, Girdin is an intrinsic regulator of neuroblast chain migration in the rostral migratory stream of the postnatal brain. J. Neurosci. 31, 8109-8122 (2011).
43. P. Ghosh, M. Garcia-Marcos, M. G. Farquhar, GIV/Girdin is a rheostat that fine-tunes growth factor signals during tumor progression. Cell Adh. Migr. 5, 237-248 (2011).
44. J. M. Haugh, F. Codazzi, M. Teruel, T. Meyer, Spatial sensing in fibroblasts mediated by 3′ phosphoinositides. J. Cell Biol. 151, 1269-1280 (2000).
45. G. Servant, O. D. Weiner, E. R. Neptune, J. W. Sedat, H. R. Bourne, Dynamics of a chemoattractant receptor in living neutrophils during chemotaxis. Mol. Biol. Cell 10, 1163-1178 (1999).
46. Z. Xiao, N. Zhang, D. B. Murphy, P. N. Devreotes, Dynamic distribution of chemoattractant receptors in living cells during chemotaxis and persistent stimulation. J. Cell Biol. 139, 365-374 (1997).
47. M. Bailly, J. Wyckoff, B. Bouzahzah, R. Hammerman, V. Sylvestre, M. Cammer, R. Pestell, J. E. Segall, Epidermal growth factor receptor distribution during chemotactic responses. Mol. Biol. Cell 11, 3873-3883 (2000).
48. C. Janetopoulos, T. Jin, P. Devreotes, Receptor-mediated activation of heterotrimeric G-proteins in living cells. Science 291, 2408-2411 (2001).
49. T. Jin, N. Zhang, Y. Long, C. A. Parent, P. N. Devreotes, Localization of the G protein βγ complex in living cells during chemotaxis. Science 287, 1034-1036 (2000).
50. T. C. He, H. Zhuang, N. Jiang, M. D. Waterfield, D. M. Wojchowski, Association of the p85 regulatory subunit of phosphatidylinositol 3-kinase with an essential erythropoietin receptor subdomain. Blood 82, 3530-3538 (1993).
51. K. D. Moon, C. B. Post, D. L. Durden, Q. Zhou, P. De, M. L. Harrison, R. L. Geahlen, Molecular basis for a direct interaction between the Syk protein-tyrosine kinase and phosphoinositide 3-kinase. J. Biol. Chem. 280, 1543-1551 (2005).
52. T. Rordorf-Nikolic, D. J. van Horn, D. Chen, M. F. White, J. M. Backer, Regulation of phosphatidylinositol 3′-kinase by tyrosyl phosphoproteins. Full activation requires occupancy of both SH2 domains in the 85-kDa regulatory subunit. J. Biol. Chem. 270, 3662-3666 (1995).
53. M. J. Layton, A. G. Harpur, G. Panayotou, P. I. Bastiaens, M. D. Waterfield, Binding of a diphosphotyrosine-containing peptide that mimics activated platelet-derived growth factor receptor β induces oligomerization of phosphatidylinositol 3-kinase. J. Biol. Chem. 273, 33379-33385 (1998).
54. E. A. Ottinger, M. C. Botfield, S. E. Shoelson, Tandem SH2 domains confer high specificity in tyrosine kinase signaling. J. Biol. Chem. 273, 729-735 (1998).
55. C. J. McGlade, C. Ellis, M. Reedijk, D. Anderson, G. Mbamalu, A. D. Reith, G. Panayotou, P. End, A. Bernstein, A. Kazlauskas, M. D. Waterfield, T. Pawson, SH2 domains of the p85a subunit of phosphatidylinositol 3-kinase regulate binding to growth factor receptors. Mol. Cell. Biol. 12, 991-997 (1992).
56. P. Hu, B. Margolis, E. Y. Skolnik, R. Lammers, A. Ullrich, J. Schlessinger, Interaction of phosphatidylinositol 3-kinase-associated p85 with epidermal growth factor and platelet-derived growth factor receptors. Mol. Cell. Biol. 12, 981-990 (1992).
57. D. Altschuler, K. Yamamoto, E. G. Lapetina, Insulin-like growth factor-1-mediated association of p85 phosphatidylinositol 3-kinase with pp 185: Requirement of SH2 domains for in vivo interaction. Mol. Endocrinol. 8, 1139-1146 (1994).
58. S. A. Cunningham, M. N. Waxham, P. M. Arrate, T. A. Brock, Interaction of the Flt-1 tyrosine kinase receptor with the p85 subunit of phosphatidylinositol 3-kinase. Mapping of a novel site involved in binding. J. Biol. Chem. 270, 20254-20257 (1995).
59. R. V. Rajala, R. E. Anderson, Interaction of the insulin receptor β-subunit with phosphatidylinositol 3-kinase in bovine ROS. Invest. Ophthalmol. Vis. Sci. 42, 3110-3117 (2001).
60. M. Cully, H. You, A. J. Levine, T. W. Mak, Beyond PTEN mutations: The PI3K pathway as an integrator of multiple inputs during tumorigenesis. Nat. Rev. Cancer 6, 184-192 (2006).
61. U. Maier, A. Babich, B. Nürnberg, Roles of non-catalytic subunits in Gβγ-induced activation of class I phosphoinositide 3-kinase isoforms β and γ. J. Biol. Chem. 274, 29311-29317 (1999).
62. L. Stephens, A. Eguinoa, S. Corey, T. Jackson, P. T. Hawkins, Receptor stimulated accumulation of phosphatidylinositol (3,4,5)-trisphosphate by G-protein mediated pathways in human myeloid derived cells. EMBO J. 12, 2265-2273 (1993).
63. J. Guillermet-Guibert, K. Bjorklof, A. Salpekar, C. Gonella, F. Ramadani, A. Bilancio, S. Meek, A. J. Smith, K. Okkenhaug, B. vanhaesebroeck, The p110β isoform of phosphoinositide 3-kinase signals downstream of G protein-coupled receptors and is functionally redundant with p110γ. Proc. Natl. Acad. Sci. U.S.A. 105, 8292-8297 (2008).
64. L. Larue, A. Bellacosa, Epithelial-mesenchymal transition in development and cancer: Role of phosphatidylinositol 3′ kinase/AKT pathways. Oncogene 24, 7443-7454 (2005).
65. G. N. Gill, T. Kawamoto, C. Cochet, A. Le, J. D. Sato, H. Masui, C. McLeod, J. Mendelsohn, Monoclonal anti-epidermal growth factor receptor antibodies which are inhibitors of epidermal growth factor binding and antagonists of epidermal growth factor binding and antagonists of epidermal growth factor-stimulated tyrosine protein kinase activity. J. Biol. Chem. 259, 7755-7760 (1984).
66. B. Zheng, C. Lavoie, T. D. Tang, P. Ma, T. Meerloo, A. Beas, M. G. Farquhar, Regulation of epidermal growth factor receptor degradation by heterotrimeric Gas protein. Mol. Biol. Cell 15, 5538-5550 (2004).
67. X. Lou, H. Yano, F. Lee, M. V. Chao, M. G. Farquhar, GIPC and GAIP form a complex with TrkA: A putative link between G protein and receptor tyrosine kinase pathways. Mol. Biol. Cell 12, 615-627 (2001).
68. E. D. Fixman, M. Holgado-Madruga, L. Nguyen, D. M. Kamikura, T. M. Fournier, A. J. Wong, M. Park, Efficient cellular transformation by the Met oncoprotein requires a functional Grb2 binding site and correlates with phosphorylation of the Grb2-associated proteins, Cbl and Gab1. J. Biol. Chem. 272, 20167-20172 (1997).
69. T. Fukazawa, K. A. Reedquist, G. Panchamoorthy, S. Soltoff, T. Trub, B. Druker, L. Cantley, S. E. Shoelson, H. Band, T cell activation-dependent association between the p85 subunit of the phosphatidylinositol 3-kinase and Grb2/phospholipase C-γ1-binding phosphotyrosyl protein pp36/38. J. Biol. Chem. 270, 20177-20182 (1995).
70. i sensitive to activation by GIV/girdin is required to promote cell migration. J. Biol. Chem. 285, 12765-12777 (2010).
71. M. Guttman, G. N. Betts, H. Barnes, M. Ghassemian, P. van der Geer, E. A. Komives, Interactions of the NPXY microdomains of the low density lipoprotein receptor-related protein 1. Proteomics 9, 5016-5028 (2009).
72. M. W. Pinkse, P. M. Uitto, M. J. Hilhorst, B. Ooms, A. J. Heck, Selective isolation at the femtomole level of phosphopeptides from proteolytic digests using 2D-NanoLCESI-MS/MS and titanium oxide precolumns. Anal. Chem. 76, 3935-3943 (2004).
73. A. L. McCormack, D. M. Schieltz, B. Goode, S. Yang, G. Barnes, D. Drubin, J. R. Yates III, Direct analysis and identification of proteins in mixtures by LC/MS/MS and database searching at the low-femtomole level. Anal. Chem. 69, 767-776 (1997).
74. Acknowledgments: We thank M. G. Farquhar and G. N. Gill (UCSD) for scientific advice and thoughtful comments during the preparation of this manuscript; J. Wong and A. Ong (undergraduate students, UCSD) for assistance with protein expression and purification; and the members of the UCSD Mass Spectrometry Core Facility, of which M. Ghassemian is the director. Funding: P.G. was supported by the Burroughs Wellcome Fund, the Doris Duke Charitable Foundation, and a Research Scholar Award from the American Gastroenterology Association Foundation for Digestive Health and Nutrition. M.G.-M. was supported by Susan G. Komen Postdoctoral Fellowship KG080079. M.G. was supported by the Superfund Research Program. R.A. was supported by R01 GM 071872. Y.M. was supported by the Sarah Rogers Fellowship (UCSD). J.E. was supported by the McNair Scholarship Program. Author contributions: C.L., J.E., Y.P., Y.M., M.G.-M., and P.G. performed the experiments. I.K. and R.A. performed the computational modeling. M.G. performed the MS studies. Y.P., C.L., and J.E. prepared and characterized the protein constructs. C.L., J.E., Y.M., M.G.-M., and P.G. designed the experiments and analyzed the data. C.L., M.G.-M., and P.G. wrote the paper. Competing interests: The authors declare that they have no competing interests. Data availability: The MS data associated with this manuscript may be downloaded from www.sciencesignaling.org/cgi/content/full/4/ 192/ra64/DC1.