PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of molcellbPermissionsJournals.ASM.orgJournalMCB ArticleJournal InfoAuthorsReviewers
 
Mol Cell Biol. 1994 September; 14(9): 5929–5938.
PMCID: PMC359119

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

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

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (1.9M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Click on the image to see a larger version.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Bibbins KB, Boeuf H, Varmus HE. 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. [PMC free article] [PubMed]
  • Bolen JB, Thiele CJ, Israel MA, Yonemoto W, Lipsich LA, Brugge JS. Enhancement of cellular src gene product associated tyrosyl kinase activity following polyoma virus infection and transformation. Cell. 1984 Oct;38(3):767–777. [PubMed]
  • Booker GW, Breeze AL, Downing AK, Panayotou G, Gout I, Waterfield MD, Campbell ID. Structure of an SH2 domain of the p85 alpha subunit of phosphatidylinositol-3-OH kinase. Nature. 1992 Aug 20;358(6388):684–687. [PubMed]
  • Cantley LC, Auger KR, Carpenter C, Duckworth B, Graziani A, Kapeller R, Soltoff S. Oncogenes and signal transduction. Cell. 1991 Jan 25;64(2):281–302. [PubMed]
  • Carmichael G, Schaffhausen BS, Mandel G, Liang TJ, Benjamin TL. 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. [PubMed]
  • Cartwright CA, Hutchinson MA, 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. [PMC free article] [PubMed]
  • Cohen B, Liu YX, Druker B, Roberts TM, Schaffhausen BS. Characterization of pp85, a target of oncogenes and growth factor receptors. Mol Cell Biol. 1990 Jun;10(6):2909–2915. [PMC free article] [PubMed]
  • Cohen B, Yoakim M, Piwnica-Worms H, Roberts TM, Schaffhausen BS. 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. [PubMed]
  • Courtneidge SA. Activation of the pp60c-src kinase by middle T antigen binding or by dephosphorylation. EMBO J. 1985 Jun;4(6):1471–1477. [PubMed]
  • Courtneidge SA, 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. [PubMed]
  • Eck MJ, Shoelson SE, Harrison SC. Recognition of a high-affinity phosphotyrosyl peptide by the Src homology-2 domain of p56lck. Nature. 1993 Mar 4;362(6415):87–91. [PubMed]
  • Eckhart W, Hutchinson MA, Hunter T. An activity phosphorylating tyrosine in polyoma T antigen immunoprecipitates. Cell. 1979 Dec;18(4):925–933. [PubMed]
  • Escobedo JA, Kaplan DR, Kavanaugh WM, Turck CW, Williams LT. 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. [PMC free article] [PubMed]
  • Fantl WJ, Escobedo JA, Martin GA, Turck CW, del Rosario M, McCormick F, Williams LT. Distinct phosphotyrosines on a growth factor receptor bind to specific molecules that mediate different signaling pathways. Cell. 1992 May 1;69(3):413–423. [PubMed]
  • Feig LA, Pan BT, Roberts TM, Cooper GM. 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. [PubMed]
  • Freund R, Dawe CJ, Carroll JP, Benjamin TL. 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. [PubMed]
  • Kaplan DR, Whitman M, Schaffhausen B, Pallas DC, White M, Cantley L, Roberts TM. Common elements in growth factor stimulation and oncogenic transformation: 85 kd phosphoprotein and phosphatidylinositol kinase activity. Cell. 1987 Sep 25;50(7):1021–1029. [PubMed]
  • Kaplan DR, Whitman M, Schaffhausen B, Raptis L, Garcea RL, Pallas D, Roberts TM, Cantley L. Phosphatidylinositol metabolism and polyoma-mediated transformation. Proc Natl Acad Sci U S A. 1986 Jun;83(11):3624–3628. [PubMed]
  • Kashishian A, Kazlauskas A, Cooper JA. 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. [PubMed]
  • 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]
  • Koch CA, Anderson D, Moran MF, Ellis C, Pawson T. SH2 and SH3 domains: elements that control interactions of cytoplasmic signaling proteins. Science. 1991 May 3;252(5006):668–674. [PubMed]
  • Mahadevan D, Thanki N, McPhie P, Beeler JF, Yu JC, Wlodawer A, Heidaran MA. 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. [PubMed]
  • Marengere LE, 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]
  • Marengere LE, Songyang Z, Gish GD, Schaller MD, Parsons JT, Stern MJ, Cantley LC, Pawson T. SH2 domain specificity and activity modified by a single residue. Nature. 1994 Jun 9;369(6480):502–505. [PubMed]
  • Mayer BJ, Baltimore D. Signalling through SH2 and SH3 domains. Trends Cell Biol. 1993 Jan;3(1):8–13. [PubMed]
  • Mayer BJ, Jackson PK, Van Etten RA, 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. [PMC free article] [PubMed]
  • Neu HC, Heppel LA. 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]
  • 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. [PMC free article] [PubMed]
  • Overduin M, Rios CB, Mayer BJ, 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. [PubMed]
  • Pallas DC, Schley C, Mahoney M, Harlow E, Schaffhausen BS, Roberts TM. Polyomavirus small t antigen: overproduction in bacteria, purification, and utilization for monoclonal and polyclonal antibody production. J Virol. 1986 Dec;60(3):1075–1084. [PMC free article] [PubMed]
  • Pascal SM, Singer AU, Gish G, Yamazaki T, Shoelson SE, Pawson T, Kay LE, Forman-Kay JD. 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. [PubMed]
  • Pawson T, Gish GD. SH2 and SH3 domains: from structure to function. Cell. 1992 Oct 30;71(3):359–362. [PubMed]
  • Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. [PubMed]
  • Songyang Z, Shoelson SE, Chaudhuri M, Gish G, Pawson T, Haser WG, King F, Roberts T, Ratnofsky S, Lechleider RJ, et al. SH2 domains recognize specific phosphopeptide sequences. Cell. 1993 Mar 12;72(5):767–778. [PubMed]
  • Songyang Z, Shoelson SE, McGlade J, Olivier P, Pawson T, Bustelo XR, Barbacid M, Sabe H, Hanafusa H, Yi T, et al. 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. [PMC free article] [PubMed]
  • Stern MJ, Marengere LE, Daly RJ, Lowenstein EJ, 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. [PMC free article] [PubMed]
  • Talmage DA, Freund R, Young AT, Dahl J, Dawe CJ, Benjamin TL. 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. [PubMed]
  • Taylor JW, 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. [PMC free article] [PubMed]
  • 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. [PubMed]
  • Waksman G, Kominos D, Robertson SC, Pant N, Baltimore D, Birge RB, Cowburn D, Hanafusa H, Mayer BJ, Overduin M, et al. Crystal structure of the phosphotyrosine recognition domain SH2 of v-src complexed with tyrosine-phosphorylated peptides. Nature. 1992 Aug 20;358(6388):646–653. [PubMed]
  • Waksman G, Shoelson SE, 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. [PubMed]
  • Whitman M, Kaplan DR, Schaffhausen B, Cantley L, Roberts TM. Association of phosphatidylinositol kinase activity with polyoma middle-T competent for transformation. Nature. 1985 May 16;315(6016):239–242. [PubMed]
  • Yoakim M, Hou W, Liu Y, Carpenter CL, Kapeller R, Schaffhausen BS. 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. [PMC free article] [PubMed]

Articles from Molecular and Cellular Biology are provided here courtesy of American Society for Microbiology (ASM)