Affinity-driven peptide selection of an NFAT inhibitor more selective than cyclosporin A

Science

Volumn 285, Issue 5436, 1999, Pages 2129-2133

  Aramburu, José ^a,b   Yaffe, Michael B ^c   López Rodríguez, Cristina ^a,b   Cantley, Lewis C ^c,d   Hogan, Patrick G ^b,e   Rao, Anjana ^a,b  

^a HARVARD MEDICAL SCHOOL   (United States)

^b CENTER FOR BLOOD RESEARCH   (United States)

^c BETH ISRAEL DEACONESS MEDICAL CENTER   (United States)

^d HARVARD INSTITUTES OF MEDICINE   (United States)

^e CBR Laboratories Inc   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

CALCINEURIN; CYCLOSPORIN A; CYTOKINE; NUCLEAR FACTOR; PEPTIDE; PEPTIDE LIBRARY; PHOSPHATASE; TRANSCRIPTION FACTOR;

AMINO ACID SEQUENCE; ARTICLE; CHEMICAL INTERACTION; CYTOKINE RELEASE; ENZYME ACTIVITY; HUMAN; HUMAN CELL; IMMUNOREGULATION; PRIORITY JOURNAL; PROTEIN BINDING; PROTEIN EXPRESSION; RECEPTOR AFFINITY; T LYMPHOCYTE ACTIVATION;

AMINO ACID SEQUENCE; BINDING SITES; CALCINEURIN; CELL NUCLEUS; CYCLOSPORINE; CYTOKINES; DNA-BINDING PROTEINS; GENE EXPRESSION REGULATION; GENES, REPORTER; HELA CELLS; HUMANS; IMMUNOSUPPRESSIVE AGENTS; JURKAT CELLS; MOLECULAR SEQUENCE DATA; NFATC TRANSCRIPTION FACTORS; NUCLEAR PROTEINS; OLIGOPEPTIDES; PEPTIDE LIBRARY; PEPTIDES; PHOSPHORYLATION; RECOMBINANT FUSION PROTEINS; T-LYMPHOCYTES; TRANSCRIPTION FACTORS; TRANSFECTION;

EID: 0032849010     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.285.5436.2129     Document Type: Article

References (47)

1. A. Rao, C. Luo, P. G. Hogan, Annu. Rev. Immunol. 15, 707 (1997); C. López-Rodríguez, J. Aramburu, A. S. Rakeman, A. Rao, Proc. Natl. Acad. Sci. U.S.A. 96, 7214 (1999); C. López-Rodríguez et al., Cold Spring Harbor Symp. Quant. Biol., in press; G. R. Crabtree, Cell 96, 611 (1999).
2. A. Rao, C. Luo, P. G. Hogan, Annu. Rev. Immunol. 15, 707 (1997); C. López-Rodríguez, J. Aramburu, A. S. Rakeman, A. Rao, Proc. Natl. Acad. Sci. U.S.A. 96, 7214 (1999); C. López-Rodríguez et al., Cold Spring Harbor Symp. Quant. Biol., in press; G. R. Crabtree, Cell 96, 611 (1999).
3. A. Rao, C. Luo, P. G. Hogan, Annu. Rev. Immunol. 15, 707 (1997); C. López-Rodríguez, J. Aramburu, A. S. Rakeman, A. Rao, Proc. Natl. Acad. Sci. U.S.A. 96, 7214 (1999); C. López-Rodríguez et al., Cold Spring Harbor Symp. Quant. Biol., in press; G. R. Crabtree, Cell 96, 611 (1999).
4. A. Rao, C. Luo, P. G. Hogan, Annu. Rev. Immunol. 15, 707 (1997); C. López-Rodríguez, J. Aramburu, A. S. Rakeman, A. Rao, Proc. Natl. Acad. Sci. U.S.A. 96, 7214 (1999); C. López-Rodríguez et al., Cold Spring Harbor Symp. Quant. Biol., in press; G. R. Crabtree, Cell 96, 611 (1999).
5. J. D. Molkentin et al., Cell 93, 215 (1998); J. D. Molkentin, Science 282, 1007 (1998) ; S. Izumo and H. Aoki, Nature Med. 4, 661 (1998).
6. J. D. Molkentin et al., Cell 93, 215 (1998); J. D. Molkentin, Science 282, 1007 (1998) ; S. Izumo and H. Aoki, Nature Med. 4, 661 (1998).
7. J. D. Molkentin et al., Cell 93, 215 (1998); J. D. Molkentin, Science 282, 1007 (1998) ; S. Izumo and H. Aoki, Nature Med. 4, 661 (1998).
8. E. R. Chin et al., Genes Dev. 12, 2499 (1998). C. Semsarian et al., Nature 400, 576 (1999); A. Musaro, K. J. McCullagh, F. J. Naya, E. N. Olson, N. Rosenthal, ibid., p. 581.
9. E. R. Chin et al., Genes Dev. 12, 2499 (1998). C. Semsarian et al., Nature 400, 576 (1999); A. Musaro, K. J. McCullagh, F. J. Naya, E. N. Olson, N. Rosenthal, ibid., p. 581.
10. E. R. Chin et al., Genes Dev. 12, 2499 (1998). C. Semsarian et al., Nature 400, 576 (1999); A. Musaro, K. J. McCullagh, F. J. Naya, E. N. Olson, N. Rosenthal, ibid., p. 581.
11. K. T.-Y. Shaw et al., Proc. Natl. Acad. Sci. U.S.A. 92, 11205 (1995); C. Luo et al., ibid. 93, 8907 (1996); F. Shibasaki, E. R. Price, D. Milan, F. McKeon, Nature 382, 370 (1996); L. A. Timmerman, N. A. Clipstone, S. N. Ho, J. P. Northrop, G. R. Crabtree, ibid. 383, 837 (1996); E. S. Masuda et al., Mol. Cell. Biol. 17, 2066 (1997).
12. K. T.-Y. Shaw et al., Proc. Natl. Acad. Sci. U.S.A. 92, 11205 (1995); C. Luo et al., ibid. 93, 8907 (1996); F. Shibasaki, E. R. Price, D. Milan, F. McKeon, Nature 382, 370 (1996); L. A. Timmerman, N. A. Clipstone, S. N. Ho, J. P. Northrop, G. R. Crabtree, ibid. 383, 837 (1996); E. S. Masuda et al., Mol. Cell. Biol. 17, 2066 (1997).
13. K. T.-Y. Shaw et al., Proc. Natl. Acad. Sci. U.S.A. 92, 11205 (1995); C. Luo et al., ibid. 93, 8907 (1996); F. Shibasaki, E. R. Price, D. Milan, F. McKeon, Nature 382, 370 (1996); L. A. Timmerman, N. A. Clipstone, S. N. Ho, J. P. Northrop, G. R. Crabtree, ibid. 383, 837 (1996); E. S. Masuda et al., Mol. Cell. Biol. 17, 2066 (1997).
14. K. T.-Y. Shaw et al., Proc. Natl. Acad. Sci. U.S.A. 92, 11205 (1995); C. Luo et al., ibid. 93, 8907 (1996); F. Shibasaki, E. R. Price, D. Milan, F. McKeon, Nature 382, 370 (1996); L. A. Timmerman, N. A. Clipstone, S. N. Ho, J. P. Northrop, G. R. Crabtree, ibid. 383, 837 (1996); E. S. Masuda et al., Mol. Cell. Biol. 17, 2066 (1997).
15. K. T.-Y. Shaw et al., Proc. Natl. Acad. Sci. U.S.A. 92, 11205 (1995); C. Luo et al., ibid. 93, 8907 (1996); F. Shibasaki, E. R. Price, D. Milan, F. McKeon, Nature 382, 370 (1996); L. A. Timmerman, N. A. Clipstone, S. N. Ho, J. P. Northrop, G. R. Crabtree, ibid. 383, 837 (1996); E. S. Masuda et al., Mol. Cell. Biol. 17, 2066 (1997).
16. J. Aramburu et al., Mol. Cell 1, 627 (1998); F. J. García-Cózar et al., J. Biol. Chem. 273, 23877 (1998).
17. J. Aramburu et al., Mol. Cell 1, 627 (1998); F. J. García-Cózar et al., J. Biol. Chem. 273, 23877 (1998).
18. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; x, any amino acid; and Y, Tyr.
19. M. B. Yaffe et al., Cell 91, 961 (1997).
20. 2, 100 μM sodium vanadate, 100 μM EGTA, 1 mM dithiothreitol, and 1% Triton X-100. After peptide binding, samples were washed twice with buffer and twice with phosphate-buffered saline, eluted with 30% acetic acid, and sequenced. Preference values for amino acids were determined by comparing the abundance of each amino acid at a particular position in the recovered peptides to that of the same amino acid at that position in the original peptide library mixture (7).
21. 32P-RII peptide. For each of these assays, the peptides used as inhibitors were SPRIEIT-13 and SPAIAIA-25 (5), and the 16-oligomer VIVIT peptide (Fig. 1D).
22. J. Aramburu, unpublished data.
23. 2-terminus of GFP. Twenty-four hours after transfection, cells were stimulated for 6 hours with phorbol 12-myristate 13-acetate (PMA) (20 nM) and ionomycin (1 μM) or with immobilized anti-CD3 (0.2 μg/ml) (HIT3a, Pharmingen) and soluble anti-CD28 (0.5 μg/ ml) (CD28.2, Pharmingen). CsA was added 30 min before the stimuli. Luciferase activity in cell lysates was normalized to levels of hGH.
24. The calcineurin targeting site of murine NFAT1, GPSPRIEITPSHELMQAGG (residues 108 through 126) was mutated to GPHPVIVITGPHELMQAGG (substitutions underlined) by replacing the DNA encoding the wild-type NFAT1 sequence between flanking Bsp 120I and Eco O109I sites with an oligonucleotide encoding the mutant sequence. Dephosphorylation of HA-NFAT1-GFP (9) was assessed in whole-cell extracts by protein immunoblotting, and subcellular localization was analyzed by GFP fluorescence (5).
25. 6 cells) were selected with magnetic beads coated with anti-mCD4 (Dynabeads L3T4, Dynal, Lake Success, NY) (22). The selected mCD4-expressing cells were >90% GFP-or GFP-VIVIT-positive, whereas mCD4-nonexpressing cells were <5% GFP-positive, as assessed by fluorescence microscopy. Multiprobe RNase protection assays were performed with the RiboQuant multiprobe kit (Pharmingen) (22).
26. A. M. Ranger et al., Nature 392, 186 (1998); J. L. de la Pompa et al., ibid., p. 182.
27. A. M. Ranger et al., Nature 392, 186 (1998); J. L. de la Pompa et al., ibid., p. 182.
28. I.-C. Ho, J. H.-J. Kim, J. W. Rooney, B. M. Spiegelman, L. H. Glimcher, Proc. Natl. Acad. Sci. U.S.A. 95, 15537 (1998).
29. A. C. Greenlund et al., Immunity 2, 677 (1995); U. Schindler, P. Wu, M. Rothe, M. Brasseur, S. L. McKnight, ibid., p. 689; T. Tsukazaki, T. A. Chiang, A. F. Davison, L. Attisano, J. L. Wrana, Cell 95, 779 (1998); L. Attisano and J. L. Wrana, Curr. Opin. Cell Biol. 10, 188 (1998); T. Kallunki, T. Deng, M. Hibi, M. Karin, Cell 87, 929 (1996); A. J. Whitmarsh, J. Cavanagh, C. Tournier, J. Yasuda, R. J. Davis, Science 281, 1671 (1998).
30. A. C. Greenlund et al., Immunity 2, 677 (1995); U. Schindler, P. Wu, M. Rothe, M. Brasseur, S. L. McKnight, ibid., p. 689; T. Tsukazaki, T. A. Chiang, A. F. Davison, L. Attisano, J. L. Wrana, Cell 95, 779 (1998); L. Attisano and J. L. Wrana, Curr. Opin. Cell Biol. 10, 188 (1998); T. Kallunki, T. Deng, M. Hibi, M. Karin, Cell 87, 929 (1996); A. J. Whitmarsh, J. Cavanagh, C. Tournier, J. Yasuda, R. J. Davis, Science 281, 1671 (1998).
31. A. C. Greenlund et al., Immunity 2, 677 (1995); U. Schindler, P. Wu, M. Rothe, M. Brasseur, S. L. McKnight, ibid., p. 689; T. Tsukazaki, T. A. Chiang, A. F. Davison, L. Attisano, J. L. Wrana, Cell 95, 779 (1998); L. Attisano and J. L. Wrana, Curr. Opin. Cell Biol. 10, 188 (1998); T. Kallunki, T. Deng, M. Hibi, M. Karin, Cell 87, 929 (1996); A. J. Whitmarsh, J. Cavanagh, C. Tournier, J. Yasuda, R. J. Davis, Science 281, 1671 (1998).
32. A. C. Greenlund et al., Immunity 2, 677 (1995); U. Schindler, P. Wu, M. Rothe, M. Brasseur, S. L. McKnight, ibid., p. 689; T. Tsukazaki, T. A. Chiang, A. F. Davison, L. Attisano, J. L. Wrana, Cell 95, 779 (1998); L. Attisano and J. L. Wrana, Curr. Opin. Cell Biol. 10, 188 (1998); T. Kallunki, T. Deng, M. Hibi, M. Karin, Cell 87, 929 (1996); A. J. Whitmarsh, J. Cavanagh, C. Tournier, J. Yasuda, R. J. Davis, Science 281, 1671 (1998).
33. A. C. Greenlund et al., Immunity 2, 677 (1995); U. Schindler, P. Wu, M. Rothe, M. Brasseur, S. L. McKnight, ibid., p. 689; T. Tsukazaki, T. A. Chiang, A. F. Davison, L. Attisano, J. L. Wrana, Cell 95, 779 (1998); L. Attisano and J. L. Wrana, Curr. Opin. Cell Biol. 10, 188 (1998); T. Kallunki, T. Deng, M. Hibi, M. Karin, Cell 87, 929 (1996); A. J. Whitmarsh, J. Cavanagh, C. Tournier, J. Yasuda, R. J. Davis, Science 281, 1671 (1998).
34. A. C. Greenlund et al., Immunity 2, 677 (1995); U. Schindler, P. Wu, M. Rothe, M. Brasseur, S. L. McKnight, ibid., p. 689; T. Tsukazaki, T. A. Chiang, A. F. Davison, L. Attisano, J. L. Wrana, Cell 95, 779 (1998); L. Attisano and J. L. Wrana, Curr. Opin. Cell Biol. 10, 188 (1998); T. Kallunki, T. Deng, M. Hibi, M. Karin, Cell 87, 929 (1996); A. J. Whitmarsh, J. Cavanagh, C. Tournier, J. Yasuda, R. J. Davis, Science 281, 1671 (1998).
35. N. H. Sigal et al., J. Exp. Med. 173, 619 (1991); M. Pascual et al., Immunol Today 11, 514 (1998). M. Hojo et al., Nature 397, 530 (1999); G. J. Nabel, ibid., p. 471; M. J. Mihatsch et al., Clin. Nephrol. 49, 356 (1998); M. Cecka, Surg. Clin. North. Am. 78, 133 (1998).
36. N. H. Sigal et al., J. Exp. Med. 173, 619 (1991); M. Pascual et al., Immunol Today 11, 514 (1998). M. Hojo et al., Nature 397, 530 (1999); G. J. Nabel, ibid., p. 471; M. J. Mihatsch et al., Clin. Nephrol. 49, 356 (1998); M. Cecka, Surg. Clin. North. Am. 78, 133 (1998).
37. N. H. Sigal et al., J. Exp. Med. 173, 619 (1991); M. Pascual et al., Immunol Today 11, 514 (1998). M. Hojo et al., Nature 397, 530 (1999); G. J. Nabel, ibid., p. 471; M. J. Mihatsch et al., Clin. Nephrol. 49, 356 (1998); M. Cecka, Surg. Clin. North. Am. 78, 133 (1998).
38. N. H. Sigal et al., J. Exp. Med. 173, 619 (1991); M. Pascual et al., Immunol Today 11, 514 (1998). M. Hojo et al., Nature 397, 530 (1999); G. J. Nabel, ibid., p. 471; M. J. Mihatsch et al., Clin. Nephrol. 49, 356 (1998); M. Cecka, Surg. Clin. North. Am. 78, 133 (1998).
39. N. H. Sigal et al., J. Exp. Med. 173, 619 (1991); M. Pascual et al., Immunol Today 11, 514 (1998). M. Hojo et al., Nature 397, 530 (1999); G. J. Nabel, ibid., p. 471; M. J. Mihatsch et al., Clin. Nephrol. 49, 356 (1998); M. Cecka, Surg. Clin. North. Am. 78, 133 (1998).
40. N. H. Sigal et al., J. Exp. Med. 173, 619 (1991); M. Pascual et al., Immunol Today 11, 514 (1998). M. Hojo et al., Nature 397, 530 (1999); G. J. Nabel, ibid., p. 471; M. J. Mihatsch et al., Clin. Nephrol. 49, 356 (1998); M. Cecka, Surg. Clin. North. Am. 78, 133 (1998).
41. C. Loh et al., J. Biol. Chem. 271, 10884 (1996).
42. C. Luo et al., Mol. Cell. Biol. 16, 3955 (1996).
43. F. Mercurio et al., Science 278, 860 (1997).
44. J. P. Northrop et al., Nature 369, 497 (1994); T. Hoey, Y. L. Sun, K. Williamson, X. Xu, Immunity 2, 461 (1995).
45. J. P. Northrop et al., Nature 369, 497 (1994); T. Hoey, Y. L. Sun, K. Williamson, X. Xu, Immunity 2, 461 (1995).
46. A. Kiani, J. P. B. Viola, A. H. Lichtman, A. Rao, Immunity 7, 849 (1997).
47. We thank M. Berne for peptide synthesis and sequencing; D. Littman, G. Crabtree, and T. Hoey for reagents; and members of the laboratory for helpful discussion and advice. Supported by NIH grants R01 AI 40127 (to A.R.), R43 AI 43726 (to P.G.H.), R01 HL 03601 (to M.B.Y.), and R01 GM 56203 (to L.C.C.); by the Ministerio de Educación y Ciencia, Spain (C.L.-R.); and by the Arthritis Foundation (J.A.).