Skip to main content

Some NLM-NCBI services and products are experiencing heavy traffic, which may affect performance and availability. We apologize for the inconvenience and appreciate your patience. For assistance, please contact our Help Desk at info@ncbi.nlm.nih.gov.

Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1994 Sep;14(9):5929–5938. doi: 10.1128/mcb.14.9.5929

Genetic analysis of a phosphatidylinositol 3-kinase SH2 domain reveals determinants of specificity.

M Yoakim 1, W Hou 1, Z Songyang 1, Y Liu 1, L Cantley 1, B Schaffhausen 1
PMCID: PMC359119  PMID: 8065326

Abstract

Phosphatidylinositol 3-kinase is an important element in both normal and oncogenic signal transduction. Polyomavirus middle T antigen transforms cells in a manner depending on association of its tyrosine 315 phosphorylation site with Src homology 2 (SH2) domains on the p85 subunit of the phosphatidylinositol 3-kinase. Both nonselective and site-directed mutagenesis have been used to probe the interaction of middle T with the N-terminal SH2 domain of p85. Most of the 24 mutants obtained showed reduced middle T binding. However, mutations that showed increased binding were also found. Comparison of middle T binding to that of the platelet-derived growth factor receptor showed that some mutations altered the specificity of recognition by the SH2 domain. Mutations altering S-393, D-394, and P-395 were shown to affect the ability of the SH2 domain to select peptides from a degenerate phosphopeptide library. These results focus attention on the role of the EF loop in the SH2 domain in determining binding selectivity at the third position after the phosphotyrosine.

Full text

PDF
5929

Images in this article

Selected References

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

  1. Bibbins K. B., Boeuf H., Varmus H. E. Binding of the Src SH2 domain to phosphopeptides is determined by residues in both the SH2 domain and the phosphopeptides. Mol Cell Biol. 1993 Dec;13(12):7278–7287. doi: 10.1128/mcb.13.12.7278. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bolen J. B., Thiele C. J., Israel M. A., Yonemoto W., Lipsich L. A., Brugge J. S. Enhancement of cellular src gene product associated tyrosyl kinase activity following polyoma virus infection and transformation. Cell. 1984 Oct;38(3):767–777. doi: 10.1016/0092-8674(84)90272-1. [DOI] [PubMed] [Google Scholar]
  3. Booker G. W., Breeze A. L., Downing A. K., Panayotou G., Gout I., Waterfield M. D., Campbell I. D. Structure of an SH2 domain of the p85 alpha subunit of phosphatidylinositol-3-OH kinase. Nature. 1992 Aug 20;358(6388):684–687. doi: 10.1038/358684a0. [DOI] [PubMed] [Google Scholar]
  4. Cantley L. C., Auger K. R., Carpenter C., Duckworth B., Graziani A., Kapeller R., Soltoff S. Oncogenes and signal transduction. Cell. 1991 Jan 25;64(2):281–302. doi: 10.1016/0092-8674(91)90639-g. [DOI] [PubMed] [Google Scholar]
  5. Carmichael G., Schaffhausen B. S., Mandel G., Liang T. J., Benjamin T. L. Transformation by polyoma virus is drastically reduced by substitution of phenylalanine for tyrosine at residue 315 of middle-sized tumor antigen. Proc Natl Acad Sci U S A. 1984 Feb;81(3):679–683. doi: 10.1073/pnas.81.3.679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cartwright C. A., Hutchinson M. A., Eckhart W. Structural and functional modification of pp60c-src associated with polyoma middle tumor antigen from infected or transformed cells. Mol Cell Biol. 1985 Oct;5(10):2647–2652. doi: 10.1128/mcb.5.10.2647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cohen B., Liu Y. X., Druker B., Roberts T. M., Schaffhausen B. S. Characterization of pp85, a target of oncogenes and growth factor receptors. Mol Cell Biol. 1990 Jun;10(6):2909–2915. doi: 10.1128/mcb.10.6.2909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cohen B., Yoakim M., Piwnica-Worms H., Roberts T. M., Schaffhausen B. S. Tyrosine phosphorylation is a signal for the trafficking of pp85, an 85-kDa phosphorylated polypeptide associated with phosphatidylinositol kinase activity. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4458–4462. doi: 10.1073/pnas.87.12.4458. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Courtneidge S. A. Activation of the pp60c-src kinase by middle T antigen binding or by dephosphorylation. EMBO J. 1985 Jun;4(6):1471–1477. doi: 10.1002/j.1460-2075.1985.tb03805.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Courtneidge S. A., Heber A. An 81 kd protein complexed with middle T antigen and pp60c-src: a possible phosphatidylinositol kinase. Cell. 1987 Sep 25;50(7):1031–1037. doi: 10.1016/0092-8674(87)90169-3. [DOI] [PubMed] [Google Scholar]
  11. Eck M. J., Shoelson S. E., Harrison S. C. Recognition of a high-affinity phosphotyrosyl peptide by the Src homology-2 domain of p56lck. Nature. 1993 Mar 4;362(6415):87–91. doi: 10.1038/362087a0. [DOI] [PubMed] [Google Scholar]
  12. Eckhart W., Hutchinson M. A., Hunter T. An activity phosphorylating tyrosine in polyoma T antigen immunoprecipitates. Cell. 1979 Dec;18(4):925–933. doi: 10.1016/0092-8674(79)90205-8. [DOI] [PubMed] [Google Scholar]
  13. Escobedo J. A., Kaplan D. R., Kavanaugh W. M., Turck C. W., Williams L. T. A phosphatidylinositol-3 kinase binds to platelet-derived growth factor receptors through a specific receptor sequence containing phosphotyrosine. Mol Cell Biol. 1991 Feb;11(2):1125–1132. doi: 10.1128/mcb.11.2.1125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fantl W. J., Escobedo J. A., Martin G. A., Turck C. W., del Rosario M., McCormick F., Williams L. T. Distinct phosphotyrosines on a growth factor receptor bind to specific molecules that mediate different signaling pathways. Cell. 1992 May 1;69(3):413–423. doi: 10.1016/0092-8674(92)90444-h. [DOI] [PubMed] [Google Scholar]
  15. Feig L. A., Pan B. T., Roberts T. M., Cooper G. M. Isolation of ras GTP-binding mutants using an in situ colony-binding assay. Proc Natl Acad Sci U S A. 1986 Jul;83(13):4607–4611. doi: 10.1073/pnas.83.13.4607. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Freund R., Dawe C. J., Carroll J. P., Benjamin T. L. Changes in frequency, morphology, and behavior of tumors induced in mice by a polyoma virus mutant with a specifically altered oncogene. Am J Pathol. 1992 Dec;141(6):1409–1425. [PMC free article] [PubMed] [Google Scholar]
  17. Kaplan D. R., Whitman M., Schaffhausen B., Pallas D. C., White M., Cantley L., Roberts T. M. Common elements in growth factor stimulation and oncogenic transformation: 85 kd phosphoprotein and phosphatidylinositol kinase activity. Cell. 1987 Sep 25;50(7):1021–1029. doi: 10.1016/0092-8674(87)90168-1. [DOI] [PubMed] [Google Scholar]
  18. Kaplan D. R., Whitman M., Schaffhausen B., Raptis L., Garcea R. L., Pallas D., Roberts T. M., Cantley L. Phosphatidylinositol metabolism and polyoma-mediated transformation. Proc Natl Acad Sci U S A. 1986 Jun;83(11):3624–3628. doi: 10.1073/pnas.83.11.3624. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kashishian A., Kazlauskas A., Cooper J. A. Phosphorylation sites in the PDGF receptor with different specificities for binding GAP and PI3 kinase in vivo. EMBO J. 1992 Apr;11(4):1373–1382. doi: 10.1002/j.1460-2075.1992.tb05182.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Katzav S. Single point mutations in the SH2 domain impair the transforming potential of vav and fail to activate proto-vav. Oncogene. 1993 Jul;8(7):1757–1763. [PubMed] [Google Scholar]
  21. Koch C. A., Anderson D., Moran M. F., Ellis C., Pawson T. SH2 and SH3 domains: elements that control interactions of cytoplasmic signaling proteins. Science. 1991 May 3;252(5006):668–674. doi: 10.1126/science.1708916. [DOI] [PubMed] [Google Scholar]
  22. Mahadevan D., Thanki N., McPhie P., Beeler J. F., Yu J. C., Wlodawer A., Heidaran M. A. Comparison of calcium-dependent conformational changes in the N-terminal SH2 domains of p85 and GAP defines distinct properties for SH2 domains. Biochemistry. 1994 Jan 25;33(3):746–754. doi: 10.1021/bi00169a016. [DOI] [PubMed] [Google Scholar]
  23. Marengere L. E., Pawson T. Identification of residues in GTPase-activating protein Src homology 2 domains that control binding to tyrosine phosphorylated growth factor receptors and p62. J Biol Chem. 1992 Nov 15;267(32):22779–22786. [PubMed] [Google Scholar]
  24. Marengere L. E., Songyang Z., Gish G. D., Schaller M. D., Parsons J. T., Stern M. J., Cantley L. C., Pawson T. SH2 domain specificity and activity modified by a single residue. Nature. 1994 Jun 9;369(6480):502–505. doi: 10.1038/369502a0. [DOI] [PubMed] [Google Scholar]
  25. Mayer B. J., Baltimore D. Signalling through SH2 and SH3 domains. Trends Cell Biol. 1993 Jan;3(1):8–13. doi: 10.1016/0962-8924(93)90194-6. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Neu H. C., Heppel L. A. The release of enzymes from Escherichia coli by osmotic shock and during the formation of spheroplasts. J Biol Chem. 1965 Sep;240(9):3685–3692. [PubMed] [Google Scholar]
  28. Nishimura R., Li W., Kashishian A., Mondino A., Zhou M., Cooper J., Schlessinger J. Two signaling molecules share a phosphotyrosine-containing binding site in the platelet-derived growth factor receptor. Mol Cell Biol. 1993 Nov;13(11):6889–6896. doi: 10.1128/mcb.13.11.6889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Overduin M., Rios C. B., Mayer B. J., Baltimore D., Cowburn D. Three-dimensional solution structure of the src homology 2 domain of c-abl. Cell. 1992 Aug 21;70(4):697–704. doi: 10.1016/0092-8674(92)90437-h. [DOI] [PubMed] [Google Scholar]
  30. Pallas D. C., Schley C., Mahoney M., Harlow E., Schaffhausen B. S., Roberts T. M. Polyomavirus small t antigen: overproduction in bacteria, purification, and utilization for monoclonal and polyclonal antibody production. J Virol. 1986 Dec;60(3):1075–1084. doi: 10.1128/jvi.60.3.1075-1084.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Pascal S. M., Singer A. U., Gish G., Yamazaki T., Shoelson S. E., Pawson T., Kay L. E., Forman-Kay J. D. Nuclear magnetic resonance structure of an SH2 domain of phospholipase C-gamma 1 complexed with a high affinity binding peptide. Cell. 1994 May 6;77(3):461–472. doi: 10.1016/0092-8674(94)90160-0. [DOI] [PubMed] [Google Scholar]
  32. Pawson T., Gish G. D. SH2 and SH3 domains: from structure to function. Cell. 1992 Oct 30;71(3):359–362. doi: 10.1016/0092-8674(92)90504-6. [DOI] [PubMed] [Google Scholar]
  33. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Songyang Z., Shoelson S. E., Chaudhuri M., Gish G., Pawson T., Haser W. G., King F., Roberts T., Ratnofsky S., Lechleider R. J. SH2 domains recognize specific phosphopeptide sequences. Cell. 1993 Mar 12;72(5):767–778. doi: 10.1016/0092-8674(93)90404-e. [DOI] [PubMed] [Google Scholar]
  35. Songyang Z., Shoelson S. E., McGlade J., Olivier P., Pawson T., Bustelo X. R., Barbacid M., Sabe H., Hanafusa H., Yi T. Specific motifs recognized by the SH2 domains of Csk, 3BP2, fps/fes, GRB-2, HCP, SHC, Syk, and Vav. Mol Cell Biol. 1994 Apr;14(4):2777–2785. doi: 10.1128/mcb.14.4.2777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Stern M. J., Marengere L. E., Daly R. J., Lowenstein E. J., Kokel M., Batzer A., Olivier P., Pawson T., Schlessinger J. The human GRB2 and Drosophila Drk genes can functionally replace the Caenorhabditis elegans cell signaling gene sem-5. Mol Biol Cell. 1993 Nov;4(11):1175–1188. doi: 10.1091/mbc.4.11.1175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Talmage D. A., Freund R., Young A. T., Dahl J., Dawe C. J., Benjamin T. L. Phosphorylation of middle T by pp60c-src: a switch for binding of phosphatidylinositol 3-kinase and optimal tumorigenesis. Cell. 1989 Oct 6;59(1):55–65. doi: 10.1016/0092-8674(89)90869-6. [DOI] [PubMed] [Google Scholar]
  38. Taylor J. W., Schmidt W., Cosstick R., Okruszek A., Eckstein F. The use of phosphorothioate-modified DNA in restriction enzyme reactions to prepare nicked DNA. Nucleic Acids Res. 1985 Dec 20;13(24):8749–8764. doi: 10.1093/nar/13.24.8749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Valius M., Kazlauskas A. Phospholipase C-gamma 1 and phosphatidylinositol 3 kinase are the downstream mediators of the PDGF receptor's mitogenic signal. Cell. 1993 Apr 23;73(2):321–334. doi: 10.1016/0092-8674(93)90232-f. [DOI] [PubMed] [Google Scholar]
  40. Waksman G., Kominos D., Robertson S. C., Pant N., Baltimore D., Birge R. B., Cowburn D., Hanafusa H., Mayer B. J., Overduin M. Crystal structure of the phosphotyrosine recognition domain SH2 of v-src complexed with tyrosine-phosphorylated peptides. Nature. 1992 Aug 20;358(6388):646–653. doi: 10.1038/358646a0. [DOI] [PubMed] [Google Scholar]
  41. Waksman G., Shoelson S. E., Pant N., Cowburn D., Kuriyan J. Binding of a high affinity phosphotyrosyl peptide to the Src SH2 domain: crystal structures of the complexed and peptide-free forms. Cell. 1993 Mar 12;72(5):779–790. doi: 10.1016/0092-8674(93)90405-f. [DOI] [PubMed] [Google Scholar]
  42. Whitman M., Kaplan D. R., Schaffhausen B., Cantley L., Roberts T. M. Association of phosphatidylinositol kinase activity with polyoma middle-T competent for transformation. Nature. 1985 May 16;315(6016):239–242. doi: 10.1038/315239a0. [DOI] [PubMed] [Google Scholar]
  43. Yoakim M., Hou W., Liu Y., Carpenter C. L., Kapeller R., Schaffhausen B. S. Interactions of polyomavirus middle T with the SH2 domains of the pp85 subunit of phosphatidylinositol-3-kinase. J Virol. 1992 Sep;66(9):5485–5491. doi: 10.1128/jvi.66.9.5485-5491.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES