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. 1995 Apr 11;92(8):3110–3114. doi: 10.1073/pnas.92.8.3110

Proline-rich sequences that bind to Src homology 3 domains with individual specificities.

K Alexandropoulos 1, G Cheng 1, D Baltimore 1
PMCID: PMC42114  PMID: 7536925

Abstract

To study the binding specificity of Src homology 3 (SH3) domains, we have screened a mouse embryonic expression library for peptide fragments that interact with them. Several clones were identified that express fragments of proteins which, through proline-rich binding sites, exhibit differential binding specificity to various SH3 domains. Src-SH3-specific binding uses a sequence of 7 aa of the consensus RPLPXXP, in which the N-terminal arginine is very important. The SH3 domains of the Src-related kinases Fyn, Lyn, and Hck bind to this sequence with the same affinity as that of the Src SH3. In contrast, a quite different proline-rich sequence from the Btk protein kinase binds to the Fyn, Lyn, and Hck SH3 domains, but not to the Src SH3. Specific binding of the Abl SH3 requires a longer, more proline-rich sequence but no arginine. One clone that binds to both Src and Abl SH3 domains through a common site exhibits reversed binding orientation, in that an arginine indispensable for binding to all tested SH3 domains occurs at the C terminus. Another clone contains overlapping yet distinct Src and Abl SH3 binding sites. Binding to the SH3 domains is mediated by a common PXXP amino acid sequence motif present on all ligands, and specificity comes about from other interactions, often ones involving arginine. The rules governing in vivo usage of particular sites by particular SH3 domains are not clear, but one binding orientation may be more specific than another.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bar-Sagi D., Rotin D., Batzer A., Mandiyan V., Schlessinger J. SH3 domains direct cellular localization of signaling molecules. Cell. 1993 Jul 16;74(1):83–91. doi: 10.1016/0092-8674(93)90296-3. [DOI] [PubMed] [Google Scholar]
  2. Booker G. W., Gout I., Downing A. K., Driscoll P. C., Boyd J., Waterfield M. D., Campbell I. D. Solution structure and ligand-binding site of the SH3 domain of the p85 alpha subunit of phosphatidylinositol 3-kinase. Cell. 1993 May 21;73(4):813–822. doi: 10.1016/0092-8674(93)90259-s. [DOI] [PubMed] [Google Scholar]
  3. Cheng G., Ye Z. S., Baltimore D. Binding of Bruton's tyrosine kinase to Fyn, Lyn, or Hck through a Src homology 3 domain-mediated interaction. Proc Natl Acad Sci U S A. 1994 Aug 16;91(17):8152–8155. doi: 10.1073/pnas.91.17.8152. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cicchetti P., Mayer B. J., Thiel G., Baltimore D. Identification of a protein that binds to the SH3 region of Abl and is similar to Bcr and GAP-rho. Science. 1992 Aug 7;257(5071):803–806. doi: 10.1126/science.1379745. [DOI] [PubMed] [Google Scholar]
  5. Eck M. J., Atwell S. K., Shoelson S. E., Harrison S. C. Structure of the regulatory domains of the Src-family tyrosine kinase Lck. Nature. 1994 Apr 21;368(6473):764–769. doi: 10.1038/368764a0. [DOI] [PubMed] [Google Scholar]
  6. Feng S., Chen J. K., Yu H., Simon J. A., Schreiber S. L. Two binding orientations for peptides to the Src SH3 domain: development of a general model for SH3-ligand interactions. Science. 1994 Nov 18;266(5188):1241–1247. doi: 10.1126/science.7526465. [DOI] [PubMed] [Google Scholar]
  7. Goudreau N., Cornille F., Duchesne M., Parker F., Tocqué B., Garbay C., Roques B. P. NMR structure of the N-terminal SH3 domain of GRB2 and its complex with a proline-rich peptide from Sos. Nat Struct Biol. 1994 Dec;1(12):898–907. doi: 10.1038/nsb1294-898. [DOI] [PubMed] [Google Scholar]
  8. Gout I., Dhand R., Hiles I. D., Fry M. J., Panayotou G., Das P., Truong O., Totty N. F., Hsuan J., Booker G. W. The GTPase dynamin binds to and is activated by a subset of SH3 domains. Cell. 1993 Oct 8;75(1):25–36. [PubMed] [Google Scholar]
  9. Haslam R. J., Koide H. B., Hemmings B. A. Pleckstrin domain homology. Nature. 1993 May 27;363(6427):309–310. doi: 10.1038/363309b0. [DOI] [PubMed] [Google Scholar]
  10. Hirai H., Varmus H. E. Site-directed mutagenesis of the SH2- and SH3-coding domains of c-src produces varied phenotypes, including oncogenic activation of p60c-src. Mol Cell Biol. 1990 Apr;10(4):1307–1318. doi: 10.1128/mcb.10.4.1307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Jackson P., Baltimore D. N-terminal mutations activate the leukemogenic potential of the myristoylated form of c-abl. EMBO J. 1989 Feb;8(2):449–456. doi: 10.1002/j.1460-2075.1989.tb03397.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kohda D., Hatanaka H., Odaka M., Mandiyan V., Ullrich A., Schlessinger J., Inagaki F. Solution structure of the SH3 domain of phospholipase C-gamma. Cell. 1993 Mar 26;72(6):953–960. doi: 10.1016/0092-8674(93)90583-c. [DOI] [PubMed] [Google Scholar]
  13. Koyama S., Yu H., Dalgarno D. C., Shin T. B., Zydowsky L. D., Schreiber S. L. Structure of the PI3K SH3 domain and analysis of the SH3 family. Cell. 1993 Mar 26;72(6):945–952. doi: 10.1016/0092-8674(93)90582-b. [DOI] [PubMed] [Google Scholar]
  14. Lim W. A., Richards F. M. Critical residues in an SH3 domain from Sem-5 suggest a mechanism for proline-rich peptide recognition. Nat Struct Biol. 1994 Apr;1(4):221–225. doi: 10.1038/nsb0494-221. [DOI] [PubMed] [Google Scholar]
  15. Lim W. A., Richards F. M., Fox R. O. Structural determinants of peptide-binding orientation and of sequence specificity in SH3 domains. Nature. 1994 Nov 24;372(6504):375–379. doi: 10.1038/372375a0. [DOI] [PubMed] [Google Scholar]
  16. Lowenstein E. J., Daly R. J., Batzer A. G., Li W., Margolis B., Lammers R., Ullrich A., Skolnik E. Y., Bar-Sagi D., Schlessinger J. The SH2 and SH3 domain-containing protein GRB2 links receptor tyrosine kinases to ras signaling. Cell. 1992 Aug 7;70(3):431–442. doi: 10.1016/0092-8674(92)90167-b. [DOI] [PubMed] [Google Scholar]
  17. Mayer B. J., Hamaguchi M., Hanafusa H. A novel viral oncogene with structural similarity to phospholipase C. Nature. 1988 Mar 17;332(6161):272–275. doi: 10.1038/332272a0. [DOI] [PubMed] [Google Scholar]
  18. Mayer B. J., Jackson P. K., Van Etten R. A., Baltimore D. Point mutations in the abl SH2 domain coordinately impair phosphotyrosine binding in vitro and transforming activity in vivo. Mol Cell Biol. 1992 Feb;12(2):609–618. doi: 10.1128/mcb.12.2.609. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Mayer B. J., Ren R., Clark K. L., Baltimore D. A putative modular domain present in diverse signaling proteins. Cell. 1993 May 21;73(4):629–630. doi: 10.1016/0092-8674(93)90244-k. [DOI] [PubMed] [Google Scholar]
  20. Musacchio A., Noble M., Pauptit R., Wierenga R., Saraste M. Crystal structure of a Src-homology 3 (SH3) domain. Nature. 1992 Oct 29;359(6398):851–855. doi: 10.1038/359851a0. [DOI] [PubMed] [Google Scholar]
  21. Musacchio A., Saraste M., Wilmanns M. High-resolution crystal structures of tyrosine kinase SH3 domains complexed with proline-rich peptides. Nat Struct Biol. 1994 Aug;1(8):546–551. doi: 10.1038/nsb0894-546. [DOI] [PubMed] [Google Scholar]
  22. Noble M. E., Musacchio A., Saraste M., Courtneidge S. A., Wierenga R. K. Crystal structure of the SH3 domain in human Fyn; comparison of the three-dimensional structures of SH3 domains in tyrosine kinases and spectrin. EMBO J. 1993 Jul;12(7):2617–2624. doi: 10.2210/pdb1shf/pdb. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Pleiman C. M., Hertz W. M., Cambier J. C. Activation of phosphatidylinositol-3' kinase by Src-family kinase SH3 binding to the p85 subunit. Science. 1994 Mar 18;263(5153):1609–1612. doi: 10.1126/science.8128248. [DOI] [PubMed] [Google Scholar]
  24. Ren R., Mayer B. J., Cicchetti P., Baltimore D. Identification of a ten-amino acid proline-rich SH3 binding site. Science. 1993 Feb 19;259(5098):1157–1161. doi: 10.1126/science.8438166. [DOI] [PubMed] [Google Scholar]
  25. Ren R., Ye Z. S., Baltimore D. Abl protein-tyrosine kinase selects the Crk adapter as a substrate using SH3-binding sites. Genes Dev. 1994 Apr 1;8(7):783–795. doi: 10.1101/gad.8.7.783. [DOI] [PubMed] [Google Scholar]
  26. Rickles R. J., Botfield M. C., Weng Z., Taylor J. A., Green O. M., Brugge J. S., Zoller M. J. Identification of Src, Fyn, Lyn, PI3K and Abl SH3 domain ligands using phage display libraries. EMBO J. 1994 Dec 1;13(23):5598–5604. doi: 10.1002/j.1460-2075.1994.tb06897.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Sadowski I., Stone J. C., Pawson T. A noncatalytic domain conserved among cytoplasmic protein-tyrosine kinases modifies the kinase function and transforming activity of Fujinami sarcoma virus P130gag-fps. Mol Cell Biol. 1986 Dec;6(12):4396–4408. doi: 10.1128/mcb.6.12.4396. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Seidel-Dugan C., Meyer B. E., Thomas S. M., Brugge J. S. Effects of SH2 and SH3 deletions on the functional activities of wild-type and transforming variants of c-Src. Mol Cell Biol. 1992 Apr;12(4):1835–1845. doi: 10.1128/mcb.12.4.1835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Terasawa H., Kohda D., Hatanaka H., Tsuchiya S., Ogura K., Nagata K., Ishii S., Mandiyan V., Ullrich A., Schlessinger J. Structure of the N-terminal SH3 domain of GRB2 complexed with a peptide from the guanine nucleotide releasing factor Sos. Nat Struct Biol. 1994 Dec;1(12):891–897. doi: 10.1038/nsb1294-891. [DOI] [PubMed] [Google Scholar]
  30. Yu H., Chen J. K., Feng S., Dalgarno D. C., Brauer A. W., Schreiber S. L. Structural basis for the binding of proline-rich peptides to SH3 domains. Cell. 1994 Mar 11;76(5):933–945. doi: 10.1016/0092-8674(94)90367-0. [DOI] [PubMed] [Google Scholar]
  31. Yu H., Rosen M. K., Shin T. B., Seidel-Dugan C., Brugge J. S., Schreiber S. L. Solution structure of the SH3 domain of Src and identification of its ligand-binding site. Science. 1992 Dec 4;258(5088):1665–1668. doi: 10.1126/science.1280858. [DOI] [PubMed] [Google Scholar]

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