Association of Src tyrosine kinase with a human potassium channel mediated by SH3 domain

Science

Volumn 274, Issue 5295, 1996, Pages 2089-2091

  Holmes, Todd C ^a   Fadool, Debra A ^a   Ren, Ruibao ^a   Levitan, Irwin B ^a  

^a BRANDEIS UNIVERSITY   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

POTASSIUM CHANNEL;

ANIMAL CELL; ANTIBODY SPECIFICITY; ARTICLE; GENE TARGETING; IMMUNOPRECIPITATION; LIGAND BINDING; MUSCLE CONTRACTION; NONHUMAN; PRIORITY JOURNAL; PROTEIN DOMAIN; REPOLARIZATION; SEQUENCE HOMOLOGY;

AMINO ACID SEQUENCE; CELL LINE; CLONING, MOLECULAR; HUMANS; KV1.5 POTASSIUM CHANNEL; MOLECULAR SEQUENCE DATA; MYOCARDIUM; ONCOGENE PROTEIN PP60(V-SRC); PATCH-CLAMP TECHNIQUES; PHOSPHORYLATION; PHOSPHOTYROSINE; POTASSIUM CHANNELS; POTASSIUM CHANNELS, VOLTAGE-GATED; RECOMBINANT FUSION PROTEINS; SIGNAL TRANSDUCTION; SRC HOMOLOGY DOMAINS; SRC-FAMILY KINASES; TRANSFECTION;

ANIMALIA;

EID: 0030473947     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.274.5295.2089     Document Type: Article

References (38)

1. I.B. Levitan, Annu. Rev. Physiol. 56, 193 (1994).
2. X.-Y. Huang, A. D. Morielli, E. G. Peralta, Cell 75, 1145 (1993); S. Lev et al., Nature 376, 737 (1995).
3. X.-Y. Huang, A. D. Morielli, E. G. Peralta, Cell 75, 1145 (1993); S. Lev et al., Nature 376, 737 (1995).
4. T. C. Holmes, D. A. Fadool, I. B. Levitan, J. Neurosci. 16, 1581 (1996).
5. S. Chung, P. H. Reinhart, B. L. Martin, D. Brautigan, I. B. Levitan, Science 253, 560 (1991); K. Bielefeldt and M. B. Jackson, Biophys. J. 66, 1904 (1994); C. Rosenmund et al., Nature 368, 853 (1994); S. Swope and R. Huganir, J. Biol. Chem. 269, 29817 (1994); D. Rotin et al., EMBO J. 13, 4440 (1994); P. H. Reinhart and I. B. Levitan, J. Neurosci. 15, 4572 (1995).
6. S. Chung, P. H. Reinhart, B. L. Martin, D. Brautigan, I. B. Levitan, Science 253, 560 (1991); K. Bielefeldt and M. B. Jackson, Biophys. J. 66, 1904 (1994); C. Rosenmund et al., Nature 368, 853 (1994); S. Swope and R. Huganir, J. Biol. Chem. 269, 29817 (1994); D. Rotin et al., EMBO J. 13, 4440 (1994); P. H. Reinhart and I. B. Levitan, J. Neurosci. 15, 4572 (1995).
7. S. Chung, P. H. Reinhart, B. L. Martin, D. Brautigan, I. B. Levitan, Science 253, 560 (1991); K. Bielefeldt and M. B. Jackson, Biophys. J. 66, 1904 (1994); C. Rosenmund et al., Nature 368, 853 (1994); S. Swope and R. Huganir, J. Biol. Chem. 269, 29817 (1994); D. Rotin et al., EMBO J. 13, 4440 (1994); P. H. Reinhart and I. B. Levitan, J. Neurosci. 15, 4572 (1995).
8. S. Chung, P. H. Reinhart, B. L. Martin, D. Brautigan, I. B. Levitan, Science 253, 560 (1991); K. Bielefeldt and M. B. Jackson, Biophys. J. 66, 1904 (1994); C. Rosenmund et al., Nature 368, 853 (1994); S. Swope and R. Huganir, J. Biol. Chem. 269, 29817 (1994); D. Rotin et al., EMBO J. 13, 4440 (1994); P. H. Reinhart and I. B. Levitan, J. Neurosci. 15, 4572 (1995).
9. S. Chung, P. H. Reinhart, B. L. Martin, D. Brautigan, I. B. Levitan, Science 253, 560 (1991); K. Bielefeldt and M. B. Jackson, Biophys. J. 66, 1904 (1994); C. Rosenmund et al., Nature 368, 853 (1994); S. Swope and R. Huganir, J. Biol. Chem. 269, 29817 (1994); D. Rotin et al., EMBO J. 13, 4440 (1994); P. H. Reinhart and I. B. Levitan, J. Neurosci. 15, 4572 (1995).
10. S. Chung, P. H. Reinhart, B. L. Martin, D. Brautigan, I. B. Levitan, Science 253, 560 (1991); K. Bielefeldt and M. B. Jackson, Biophys. J. 66, 1904 (1994); C. Rosenmund et al., Nature 368, 853 (1994); S. Swope and R. Huganir, J. Biol. Chem. 269, 29817 (1994); D. Rotin et al., EMBO J. 13, 4440 (1994); P. H. Reinhart and I. B. Levitan, J. Neurosci. 15, 4572 (1995).
11. G. Cohen, R. Ren, D. Baltimore, Cell 80, 237 (1995).
12. M. Tamkun et al., FASEB J. 5, 331 (1991); K. Overturf et al., Am. J. Physiol. 267, C1231 (1994); Y. Sasaki, K. Ishii, K. Nunoki, T. Yamagishi, N. Taira, FEBS Lett. 372, 20 (1995).
13. M. Tamkun et al., FASEB J. 5, 331 (1991); K. Overturf et al., Am. J. Physiol. 267, C1231 (1994); Y. Sasaki, K. Ishii, K. Nunoki, T. Yamagishi, N. Taira, FEBS Lett. 372, 20 (1995).
14. M. Tamkun et al., FASEB J. 5, 331 (1991); K. Overturf et al., Am. J. Physiol. 267, C1231 (1994); Y. Sasaki, K. Ishii, K. Nunoki, T. Yamagishi, N. Taira, FEBS Lett. 372, 20 (1995).
15. R. Rickles et al., EMBO J. 13, 5598 (1994); H. Yu et al., Cell 76, 933 (1994).
16. R. Rickles et al., EMBO J. 13, 5598 (1994); H. Yu et al., Cell 76, 933 (1994).
17. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; D, Asp; E, Glu; G, Gly; L, Leu; M, Met; P, Pro; R, Arg; S, Ser; and V, Val. Unspecified amino acid residues are indicated by X.
18. The sequences of proline-rich motifs in 15 mammalian potassium channels and two N-methyl-D-aspartate receptor subunits, together with their GenBank/ European Molecular Biology Laboratory accession numbers, can be retrieved from Science's Beyond the Printed Page site on the World Wide Web: http: //sciencemag.org/science/feature/beyond/#holmes.
19. All cDNA mammalian expression vectors contained the cytomegalovirus (CMV) promoter sequence up-stream from the coding region for channels and kinases. A vector encoding hKv1.5 fused to an epitope tag (18) was provided by L. Philipson (University of Chicago), and one encoding v-Src was provided by R. Huganir (Johns Hopkins University, Baltimore, MD). HEK 293 cells were lipofectamine transfected and lysed as described (3). Tissue lysate was prepared from human myocardium with a glass-teflon homogenizer. Proteins were immunoprecipitated overnight at 4°C from lysates with 4 μg of primary antibody and 25 μl of protein A/G (Pierce, Rockford, IL) per 400 μg of lysate protein. The lysates and washed immunoprecipitated proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE), and protein immunoblots were probed with an antibody (19) that recognizes both rKv1.5 and hKv1.5 [anti-Kv1.5; provided by J. Trimmer (State University of New York, Stony Brook, NY)], an antibody (18) to an epitope tag sequence fused to hKv1.5 (anti-tag-hKv1.5; provided by L. Philipson), or antibody to Src (MAB 327; Oncogene Science, Cambridge, MA). Antibody binding was visualized by enhanced chemiluminescence (ECL; Amersham, Arlington Heights, IL). Silver stain (20) was used to detect immunoprecipitated proteins separated by SDS-PAGE. Src activity was measured under standard conditions (21) with the substrate G10A, a GST fusion protein that contains an Src SH3 domain binding motif and a tyrosine phosphorylation site
20. The reaction products were separated by SDS-PAGE, and protein immunoblots were probed with an antibody to phosphotyrosine (4G10; Upstate Biotechnology, Lake Placid, NY) and visualized by ECL.
21. K. Alexandropoulos, G. Cheng, D. Baltimore, Proc. Natl. Acad. Sci. U.S.A. 92, 3110 (1995).
22. D. Mays, J. Foose, L. Philipson, M. Tamkun, J. Clin. Invest. 96, 282 (1995); S. Tsukita et al., J. Cell Biol. 113, 867 (1991).
23. D. Mays, J. Foose, L. Philipson, M. Tamkun, J. Clin. Invest. 96, 282 (1995); S. Tsukita et al., J. Cell Biol. 113, 867 (1991).
24. R. Swanson et al., Neuron 4, 929 (1990).
25. T. C. Holmes and I. B. Levitan, unpublished results.
26. 62-83; Bio-Synthesis, Lewisville, TX) (8). The proteins bound to the washed agarose beads were separated by SDS-PAGE, and immunoblots were probed with anti-tag-hKv1.5.
27. Patch-clamp recordings of macroscopic currents were made in the cell-attached patch configuration (3). Patches were held at -90 mV and stepped to depolarizing potentials in 5-mV increments to 0 mV. The pulse duration was 400 ms and the interpulse interval was 10 s.
28. E. Kim, M. Neithammer, A. Rothschild, Y. N. Jan, M. Sheng, Nature 378, 85 (1995); H.-C. Kornau, L. T. Schenker, M. B. Kennedy, P. H. Seeburg, Science 269, 1737 (1995); M. D. Ehlers, S. Zhang, J. P. Bernhardt, R. L. Huganir, Cell 84, 745 (1996); M. C. Faux and J. D. Scott, ibid. 85, 9 (1996); C. Fuhrer and Z. W. Hall, J. Biol. Chem., in press.
29. E. Kim, M. Neithammer, A. Rothschild, Y. N. Jan, M. Sheng, Nature 378, 85 (1995); H.-C. Kornau, L. T. Schenker, M. B. Kennedy, P. H. Seeburg, Science 269, 1737 (1995); M. D. Ehlers, S. Zhang, J. P. Bernhardt, R. L. Huganir, Cell 84, 745 (1996); M. C. Faux and J. D. Scott, ibid. 85, 9 (1996); C. Fuhrer and Z. W. Hall, J. Biol. Chem., in press.
30. E. Kim, M. Neithammer, A. Rothschild, Y. N. Jan, M. Sheng, Nature 378, 85 (1995); H.-C. Kornau, L. T. Schenker, M. B. Kennedy, P. H. Seeburg, Science 269, 1737 (1995); M. D. Ehlers, S. Zhang, J. P. Bernhardt, R. L. Huganir, Cell 84, 745 (1996); M. C. Faux and J. D. Scott, ibid. 85, 9 (1996); C. Fuhrer and Z. W. Hall, J. Biol. Chem., in press.
31. E. Kim, M. Neithammer, A. Rothschild, Y. N. Jan, M. Sheng, Nature 378, 85 (1995); H.-C. Kornau, L. T. Schenker, M. B. Kennedy, P. H. Seeburg, Science 269, 1737 (1995); M. D. Ehlers, S. Zhang, J. P. Bernhardt, R. L. Huganir, Cell 84, 745 (1996); M. C. Faux and J. D. Scott, ibid. 85, 9 (1996); C. Fuhrer and Z. W. Hall, J. Biol. Chem., in press.
32. E. Kim, M. Neithammer, A. Rothschild, Y. N. Jan, M. Sheng, Nature 378, 85 (1995); H.-C. Kornau, L. T. Schenker, M. B. Kennedy, P. H. Seeburg, Science 269, 1737 (1995); M. D. Ehlers, S. Zhang, J. P. Bernhardt, R. L. Huganir, Cell 84, 745 (1996); M. C. Faux and J. D. Scott, ibid. 85, 9 (1996); C. Fuhrer and Z. W. Hall, J. Biol. Chem., in press.
33. L. Philipson, A. Malayev, A. Kuznetsov, C. Chang, D. Nelson, Biochim. Biophys. Acta 1153, 111 (1993).
34. M. Maletic-Savatic, N. Lenn, J. Trimmer, J. Neurosci. 15, 3840 (1995).
35. R. Middleton, D. Pheasant, C. Miller, Biochemistry 33, 13189 (1994).
36. H. Cheng, H. Nishio, O. Hatase, S. Ralph, J. Wang, J. Biol. Chem. 267, 9248 (1992).
37. R. Ren, B. J. Mayer, P. Cicchetti, D. Baltimore, Science 259, 1157 (1993).
38. We thank R. Huganir, L. Philipson, and J. Trimmer for antibodies and cDNA constructs; M. Mendelsohn for samples of human myocardial tissue; and C. Miller for comments on the manuscript. Supported by grants to I.B.L. and R.R. from NIH. T.C.H. and D.A.F. were supported by National Research Service Awards.