Interaction of RAFT1 with gephyrin required for rapamycin-sensitive signaling

Science

Volumn 284, Issue 5417, 1999, Pages 1161-1164

  Sabatini, David M ^a,d   Barrow, Roxanne K ^a   Blackshaw, Seth ^a   Burnett, Patrick E ^a   Lai, Michael M ^a   Field, Michael E ^a   Bahr, Ben A ^b   Kirsch, Joachim ^c   Betz, Heinrich ^c   Snyder, Solomon H ^a  

^a JOHNS HOPKINS UNIVERSITY SCHOOL OF MEDICINE   (United States)

^b UNIVERSITY OF CONNECTICUT   (United States)

^c MAX PLANCK INSTITUTE FOR BRAIN RESEARCH   (Germany)

^d WHITEHEAD INSTITUTE FOR BIOMEDICAL RESEARCH   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

GEPHYRIN; RAPAMYCIN;

ARTICLE; HUMAN; HUMAN CELL; IMMUNOPRECIPITATION; PRIORITY JOURNAL; PROTEIN ANALYSIS; PROTEIN BINDING; PROTEIN EXPRESSION; PROTEIN PROTEIN INTERACTION; RNA TRANSLATION; SIGNAL TRANSDUCTION;

ADAPTOR PROTEINS, SIGNAL TRANSDUCING; AMINO ACID SEQUENCE; ANIMALS; CARRIER PROTEINS; CELL LINE; CELL MEMBRANE; CYTOPLASM; GENE EXPRESSION; HELA CELLS; HUMANS; MEMBRANE PROTEINS; MOLECULAR SEQUENCE DATA; MUTATION; PHOSPHOPROTEINS; PHOSPHORYLATION; PHOSPHOTRANSFERASES (ALCOHOL GROUP ACCEPTOR); RATS; RECEPTORS, GLYCINE; REPRESSOR PROTEINS; RIBOSOMAL PROTEIN S6 KINASES; SIGNAL TRANSDUCTION; SIROLIMUS;

ATAXIA; ATAXIA TELANGIECTASIA; MAMMALIA;

EID: 0033553624     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.284.5417.1161     Document Type: Article

References (45)

1. K. Savitsky et al., Science 268, 1749 (1995); K. O. Hartley et al., Cell 82, 849 (1995); E. J. Brown et al., Nature 369, 756 (1994); D. M. Sabatini et al., Cell 78, 35 (1994); C. J. Sabers et al., J. Biol. Chem. 270, 815 (1995); M. I. Chiu, H. Katz, V. Berlin, Proc. Natl. Acad. Sci. U.S.A. 91, 12574 (1994); J. Kunz et al., Cell 73, 585 (1993); R. Cafferkey et al., Mol. Cell. Biol. 13, 6012 (1993); D. M. Morrow et al., Cell 82, 831 (1995).
2. K. Savitsky et al., Science 268, 1749 (1995); K. O. Hartley et al., Cell 82, 849 (1995); E. J. Brown et al., Nature 369, 756 (1994); D. M. Sabatini et al., Cell 78, 35 (1994); C. J. Sabers et al., J. Biol. Chem. 270, 815 (1995); M. I. Chiu, H. Katz, V. Berlin, Proc. Natl. Acad. Sci. U.S.A. 91, 12574 (1994); J. Kunz et al., Cell 73, 585 (1993); R. Cafferkey et al., Mol. Cell. Biol. 13, 6012 (1993); D. M. Morrow et al., Cell 82, 831 (1995).
3. K. Savitsky et al., Science 268, 1749 (1995); K. O. Hartley et al., Cell 82, 849 (1995); E. J. Brown et al., Nature 369, 756 (1994); D. M. Sabatini et al., Cell 78, 35 (1994); C. J. Sabers et al., J. Biol. Chem. 270, 815 (1995); M. I. Chiu, H. Katz, V. Berlin, Proc. Natl. Acad. Sci. U.S.A. 91, 12574 (1994); J. Kunz et al., Cell 73, 585 (1993); R. Cafferkey et al., Mol. Cell. Biol. 13, 6012 (1993); D. M. Morrow et al., Cell 82, 831 (1995).
4. K. Savitsky et al., Science 268, 1749 (1995); K. O. Hartley et al., Cell 82, 849 (1995); E. J. Brown et al., Nature 369, 756 (1994); D. M. Sabatini et al., Cell 78, 35 (1994); C. J. Sabers et al., J. Biol. Chem. 270, 815 (1995); M. I. Chiu, H. Katz, V. Berlin, Proc. Natl. Acad. Sci. U.S.A. 91, 12574 (1994); J. Kunz et al., Cell 73, 585 (1993); R. Cafferkey et al., Mol. Cell. Biol. 13, 6012 (1993); D. M. Morrow et al., Cell 82, 831 (1995).
5. K. Savitsky et al., Science 268, 1749 (1995); K. O. Hartley et al., Cell 82, 849 (1995); E. J. Brown et al., Nature 369, 756 (1994); D. M. Sabatini et al., Cell 78, 35 (1994); C. J. Sabers et al., J. Biol. Chem. 270, 815 (1995); M. I. Chiu, H. Katz, V. Berlin, Proc. Natl. Acad. Sci. U.S.A. 91, 12574 (1994); J. Kunz et al., Cell 73, 585 (1993); R. Cafferkey et al., Mol. Cell. Biol. 13, 6012 (1993); D. M. Morrow et al., Cell 82, 831 (1995).
6. K. Savitsky et al., Science 268, 1749 (1995); K. O. Hartley et al., Cell 82, 849 (1995); E. J. Brown et al., Nature 369, 756 (1994); D. M. Sabatini et al., Cell 78, 35 (1994); C. J. Sabers et al., J. Biol. Chem. 270, 815 (1995); M. I. Chiu, H. Katz, V. Berlin, Proc. Natl. Acad. Sci. U.S.A. 91, 12574 (1994); J. Kunz et al., Cell 73, 585 (1993); R. Cafferkey et al., Mol. Cell. Biol. 13, 6012 (1993); D. M. Morrow et al., Cell 82, 831 (1995).
7. K. Savitsky et al., Science 268, 1749 (1995); K. O. Hartley et al., Cell 82, 849 (1995); E. J. Brown et al., Nature 369, 756 (1994); D. M. Sabatini et al., Cell 78, 35 (1994); C. J. Sabers et al., J. Biol. Chem. 270, 815 (1995); M. I. Chiu, H. Katz, V. Berlin, Proc. Natl. Acad. Sci. U.S.A. 91, 12574 (1994); J. Kunz et al., Cell 73, 585 (1993); R. Cafferkey et al., Mol. Cell. Biol. 13, 6012 (1993); D. M. Morrow et al., Cell 82, 831 (1995).
8. K. Savitsky et al., Science 268, 1749 (1995); K. O. Hartley et al., Cell 82, 849 (1995); E. J. Brown et al., Nature 369, 756 (1994); D. M. Sabatini et al., Cell 78, 35 (1994); C. J. Sabers et al., J. Biol. Chem. 270, 815 (1995); M. I. Chiu, H. Katz, V. Berlin, Proc. Natl. Acad. Sci. U.S.A. 91, 12574 (1994); J. Kunz et al., Cell 73, 585 (1993); R. Cafferkey et al., Mol. Cell. Biol. 13, 6012 (1993); D. M. Morrow et al., Cell 82, 831 (1995).
9. K. Savitsky et al., Science 268, 1749 (1995); K. O. Hartley et al., Cell 82, 849 (1995); E. J. Brown et al., Nature 369, 756 (1994); D. M. Sabatini et al., Cell 78, 35 (1994); C. J. Sabers et al., J. Biol. Chem. 270, 815 (1995); M. I. Chiu, H. Katz, V. Berlin, Proc. Natl. Acad. Sci. U.S.A. 91, 12574 (1994); J. Kunz et al., Cell 73, 585 (1993); R. Cafferkey et al., Mol. Cell. Biol. 13, 6012 (1993); D. M. Morrow et al., Cell 82, 831 (1995).
10. T. Jayaraman and A. R. Marks, J. Biol. Chem. 268, 25385 (1993); M. W. Albers et al., ibid., p. 22825; S. N. Sehgal, H. Baker, C. Vezina, J. Antibiot. (Tokyo) 28, 727 (1975); J. Heitman, N. R. Movva, M. N. Hall, Science 253, 905 (1991).
11. T. Jayaraman and A. R. Marks, J. Biol. Chem. 268, 25385 (1993); M. W. Albers et al., ibid., p. 22825; S. N. Sehgal, H. Baker, C. Vezina, J. Antibiot. (Tokyo) 28, 727 (1975); J. Heitman, N. R. Movva, M. N. Hall, Science 253, 905 (1991).
12. T. Jayaraman and A. R. Marks, J. Biol. Chem. 268, 25385 (1993); M. W. Albers et al., ibid., p. 22825; S. N. Sehgal, H. Baker, C. Vezina, J. Antibiot. (Tokyo) 28, 727 (1975); J. Heitman, N. R. Movva, M. N. Hall, Science 253, 905 (1991).
13. T. Jayaraman and A. R. Marks, J. Biol. Chem. 268, 25385 (1993); M. W. Albers et al., ibid., p. 22825; S. N. Sehgal, H. Baker, C. Vezina, J. Antibiot. (Tokyo) 28, 727 (1975); J. Heitman, N. R. Movva, M. N. Hall, Science 253, 905 (1991).
14. J. Chung, C. J. Kuo, G. R. Crabtree, J. Blenis, Cell 69, 1227 (1992); C. J. Kuo et al., Nature 358, 70 (1992); E. J. Brown et al., ibid. 377, 441 (1995); L. Beretta et al., EMBO J. 15, 658 (1996); S. R. von Manteuffel et al., Proc. Natl. Acad. Sci. U.S.A. 93, 4076 (1996).
15. J. Chung, C. J. Kuo, G. R. Crabtree, J. Blenis, Cell 69, 1227 (1992); C. J. Kuo et al., Nature 358, 70 (1992); E. J. Brown et al., ibid. 377, 441 (1995); L. Beretta et al., EMBO J. 15, 658 (1996); S. R. von Manteuffel et al., Proc. Natl. Acad. Sci. U.S.A. 93, 4076 (1996).
16. J. Chung, C. J. Kuo, G. R. Crabtree, J. Blenis, Cell 69, 1227 (1992); C. J. Kuo et al., Nature 358, 70 (1992); E. J. Brown et al., ibid. 377, 441 (1995); L. Beretta et al., EMBO J. 15, 658 (1996); S. R. von Manteuffel et al., Proc. Natl. Acad. Sci. U.S.A. 93, 4076 (1996).
17. J. Chung, C. J. Kuo, G. R. Crabtree, J. Blenis, Cell 69, 1227 (1992); C. J. Kuo et al., Nature 358, 70 (1992); E. J. Brown et al., ibid. 377, 441 (1995); L. Beretta et al., EMBO J. 15, 658 (1996); S. R. von Manteuffel et al., Proc. Natl. Acad. Sci. U.S.A. 93, 4076 (1996).
18. J. Chung, C. J. Kuo, G. R. Crabtree, J. Blenis, Cell 69, 1227 (1992); C. J. Kuo et al., Nature 358, 70 (1992); E. J. Brown et al., ibid. 377, 441 (1995); L. Beretta et al., EMBO J. 15, 658 (1996); S. R. von Manteuffel et al., Proc. Natl. Acad. Sci. U.S.A. 93, 4076 (1996).
19. G. J. Brunn et al., Science 277, 99 (1997).
20. N. C. Barbet et al., Mol. Biol. Cell 7, 25 (1996).
21. C. T. Keith and S. L. Schreiber, Science 270, 50 (1995).
22. P. E. Burnett et al., Proc. Natl. Acad. Sci. U.S.A. 95, 1432 (1998).
23. + colony. The rescued library plasmid encoded amino acids 209 to 685 of gephyrin. Truncations and mutations of RAFT1(944-1338) were made with polymerase chain reaction-based mutagenesis techniques.
24. P. Prior et al., Neuron 8, 1161 (1992); J. Kirsch, I. Wolters, A. Triller, H. Betz, Nature 366, 745 (1993).
25. P. Prior et al., Neuron 8, 1161 (1992); J. Kirsch, I. Wolters, A. Triller, H. Betz, Nature 366, 745 (1993).
26. G. Meyer, J. Kirsch, H. Betz, D. Langosch, Neuron 15, 563 (1995); J. Kirsch, J. Kuhse, H. Betz, Mol. Cell. Neurosci. 6, 450 (1995).
27. G. Meyer, J. Kirsch, H. Betz, D. Langosch, Neuron 15, 563 (1995); J. Kirsch, J. Kuhse, H. Betz, Mol. Cell. Neurosci. 6, 450 (1995).
28. GST and GST-gephyrin (residues 209 to 685) proteins were expressed in Escherichia coli BL21(DE3) and purified with glutathione-agarose [D. B. Smith and K. S. Johnson, Gene 67, 31 (1988)]. Plates (10 cm in diameter) of 80% confluent HEK293 cells were lysed (300 μl per plate) in buffer A [50 mM Hepes-KOH (pH 7.4), 40 mM NaCl, 0.5% Triton X-100, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, aprotinin (5 mg/ml), antipain (1 mg/ml), leupeptin (1 mg/ml), chymostatin (6 mg/ml), and pepstatin A (0.7 mg/ml)] and 100 μl added to 5 μg of GST or GST-gephyrin bound to 20 μl of glutathione-agarose beads. After a 1-hour incubation on ice, the beads were washed three times with buffer A containing 500 mM NaCl. Bound RAFT1 was eluted with 1.25× sample buffer and detected by immunoblotting with 782 antibody to RAFT1 (anti-782-RAFT1) (19).
29. 4, 50 mM NaF, 10 mM sodium pyrophosphate, and 10 mM sodium β-glycerophosphate. Immune complexes were prepared with 0.5 μl of rabbit antibody to HA (Babco) and 40 μl of protein A-Sepharose and washed three times with buffer A containing 500 mM NaCl. Bound proteins were eluted with 1.25× sample buffer, and RAFT1 was detected by immunoblotting with 9E10 antibody to myc (Calbiochem).
30. D. M. Sabatini and S. H. Snyder, unpublished observations.
31. In situ hybridization was done as described [S. Blackshaw and S. H. Snyder, J. Neurosci. 17, 8074 (1997)] with sense or antisense digoxigenin-labeled RNA probes corresponding to amino acids 209 to 685 of gephyrin or 944 to 1338 of RAFT1 (13).
32. Rat brain subcellular fractions were prepared as described [P. E. Burnett et al., Proc. Natl Acad. Sci. U.S.A. 95, 8351 (1998)]. The quality of the subcellular fractions was monitored by protein immunoblotting of 20 μg of each fraction with antibodies to mGluR1α (Pharmingen), PSD-95 (Santa Cruz), synaptophysin (Boehringer Mannheim), and FKBP12 [L. Walensky et al., J. Cell Biol. 141, 143 (1998)]. The gephyrin [B. T. Kawasaki, K. B. Hoffman, R. S. Yamamoto, B. A. Bahr, J. Neurosci. Res. 49, 381 (1997)] and RAFT1 antibodies (19) have been described.
33. Rat brain subcellular fractions were prepared as described [P. E. Burnett et al., Proc. Natl Acad. Sci. U.S.A. 95, 8351 (1998)]. The quality of the subcellular fractions was monitored by protein immunoblotting of 20 μg of each fraction with antibodies to mGluR1α (Pharmingen), PSD-95 (Santa Cruz), synaptophysin (Boehringer Mannheim), and FKBP12 [L. Walensky et al., J. Cell Biol. 141, 143 (1998)]. The gephyrin [B. T. Kawasaki, K. B. Hoffman, R. S. Yamamoto, B. A. Bahr, J. Neurosci. Res. 49, 381 (1997)] and RAFT1 antibodies (19) have been described.
34. Rat brain subcellular fractions were prepared as described [P. E. Burnett et al., Proc. Natl Acad. Sci. U.S.A. 95, 8351 (1998)]. The quality of the subcellular fractions was monitored by protein immunoblotting of 20 μg of each fraction with antibodies to mGluR1α (Pharmingen), PSD-95 (Santa Cruz), synaptophysin (Boehringer Mannheim), and FKBP12 [L. Walensky et al., J. Cell Biol. 141, 143 (1998)]. The gephyrin [B. T. Kawasaki, K. B. Hoffman, R. S. Yamamoto, B. A. Bahr, J. Neurosci. Res. 49, 381 (1997)] and RAFT1 antibodies (19) have been described.
35. D. Watters et al., Oncogene 14, 1911 (1997).
36. 56k (100 ng) is transfected with 10 μg of empty expression vector (13).
37. O. Steward and G. A. Banker, Trends Neurosci. 15, 180 (1992); F. B. Gao, BioEssays 20, 70 (1998).
38. O. Steward and G. A. Banker, Trends Neurosci. 15, 180 (1992); F. B. Gao, BioEssays 20, 70 (1998).
39. D. M. Sabatini et al., J. Biol. Chem. 270, 20875 (1995).
40. Single-letter abbreviations for 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; and Y, Tyr.
41. HeLa cells were fractionated [J. Kruppa and D. D. Sabatini, J. Cell Biol. 74, 414 (1977)], 50 μg of each fraction was resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (6% gels), and RAFT1 was detected with antibody 782 to RAFT1 (19).
42. HeLa cells grown on chamber slides were transfected with lipofectamine (Life Technologies) with expression vectors encoding mycGBD (residues 1010 to 1128 of RAFT1) (10 μg); mycRAFT1 (5 μg), and prk5 (5 μg); or mycRAFT1 (5 μg) and gephyrin P1 (5 μg) (9). After a 48-hour incubation, the cells were processed for immunofluorescence [L. D. Walensky and S. H. Snyder, J. Cell Biol. 130, 857 (1995)]. Antibodies 9E-10 to myc (2 μg/ml) or 782 to RAFT1 (5 μg/ml) (19) were used as the primary antibodies in an overnight incubation at 4°C.
43. 56k and 17% gels for 4E-BP1) and detected with immunoblotting.
44. RAFT1 kinase assays were done as described (7). The addition to the assays of 1.5 μg of GST-gephyrin (11) did not affect RAFT1 autophosphorylation nor was gephyrin a RAFT1 substrate (13).
45. We thank T. Morimoto for help with HeLa cell fractionation, A. Lanahan and P. Worley for the rat hippocampal library, and L. Walensky, N. Cohen, and J. Lawler for fruitful discussions. Supported by U.S. Public Health Service grant DA-00266 and Research Scientist Award DA-00074 to S.H.S., and training grant GM-07309 to D.M.S.