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Molecular Biology Reports

, Volume 36, Issue 4, pp 631–639 | Cite as

Epidermal growth factor receptors: function modulation by phosphorylation and glycosylation interplay

  • Afshan Kaleem
  • Ishtiaq Ahmad
  • Daniel C. Hoessli
  • Evelyne Walker-Nasir
  • Muhammad Saleem
  • Abdul Rauf Shakoori
  • Nasir-ud-Din
Article

Abstract

Post-translational modifications (PTMs) of proteins induce structural and functional changes that are most often transitory and difficult to follow and investigate in vivo. In silico prediction procedures for PTMs are very valuable to foresee and define such transitory changes responsible for the multifunctionality of proteins. Epidermal growth factor receptor (EGFR) is such a multifunctional transmembrane protein with intrinsic tyrosine kinase activity that is regulated primarily by ligand-stimulated transphosphorylation of dimerized receptors. In human EGFR, potential phosphorylation sites on Ser, Thr and Tyr residues including five autophosphorylation sites on Tyr were investigated using in silico procedures. In addition to phosphorylation, O-GlcNAc modifications and interplay between these two modifications was also predicted. The interplay of phosphorylation and O-GlcNAc modification on same or neighboring Ser/Thr residues is termed as Yin Yang hypothesis and the interplay sites are named as Yin Yang sites. Amongst these modification sites, one residue is localized in the juxtamembrane (Thr 654) and two are found in the catalytic domain (Ser 1046/1047) of the EGFR. We propose that, when EGFR is O-GlcNAc modified on Thr 654, EGFR may be transferred from early to late endosomes, whereas when EGFR is O-GlcNAc modified on Ser 1046/1047 desensitization of the receptor may be prevented. These findings suggest a complex interplay between phosphorylation and O-GlcNAc modification resulting in modulation of EGFR’s functionality.

Keywords

Epidermal growth factor receptor Phosphorylation O-GlcNAc modification Lysosomal targeting Desensitization 

Notes

Acknowledgement

Nasir-ud-Din acknowledges support from Pakistan Academy of Sciences for this work.

References

  1. 1.
    Tang PA, Moore MJ (2006) Epidermal growth factor receptor antagonists in pancreatic cancer: what is their role? Am J Cancer 5:213–221CrossRefGoogle Scholar
  2. 2.
    Hida K, Klagsbrun M (2005) A new perspective on tumor endothelial cells: unexpected chromosome and centrosome abnormalities. Cancer Res 65:2507–2510PubMedCrossRefGoogle Scholar
  3. 3.
    Schlessinger J (2000) Cell signaling by receptor tyrosine kinases. Cell 103:211–225PubMedCrossRefGoogle Scholar
  4. 4.
    Downward J, Parker P, Waterfield MD (1984) Autophosphorylation sites on the epidermal growth factor receptor. Nature 311:483–485PubMedCrossRefGoogle Scholar
  5. 5.
    Walton GM, Chen WS, Rosenfeld MG et al (1990) Analysis of deletions of the carboxyl terminus of the epidermal growth factor receptor reveals self-phosphorylation at tyrosine 992 and enhanced in vivo tyrosine phosphorylation of cell substrates. J Biol Chem 265:1750–1754PubMedGoogle Scholar
  6. 6.
    Margolis BL, Lax I, Kris R et al (1989) All autophosphorylation sites of epidermal growth factor (EGF) receptor and HER2/neu are located in their carboxyl-terminal tails. Identification of a novel site in EGF receptor. J Biol Chem 264:10667–10671PubMedGoogle Scholar
  7. 7.
    Tebar F, Lladó A, Enrich C (2002) Role of calmodulin in the modulation of the MAPK signalling pathway and the transactivation of epidermal growth factor receptor mediated by PKC. FEBS Lett 517:206–210PubMedCrossRefGoogle Scholar
  8. 8.
    Countaway JL, Nairn AC, Davis RJ (1992) Mechanism of desensitization of the epidermal growth factor receptor protein-tyrosine kinase. J Biol Chem 267:1129–1140PubMedGoogle Scholar
  9. 9.
    Aifa S, Frikha F, Miled N et al (2006) Phosphorylation of Thr654 but not Thr669 within the juxtamembrane domain of the EGF receptor inhibits CaM binding. Biochem Biophys Res Commun 347:381–387PubMedCrossRefGoogle Scholar
  10. 10.
    Aifa S, Johansen K, Nilsson UK et al (2002) Interactions between the juxtamembrane domain of the EGFR and calmodulin measured by surface plasmon resonance. Cell Signal 14:1005–1013PubMedCrossRefGoogle Scholar
  11. 11.
    Bao J, Alroy I, Waterman H et al (2000) Threonine phosphorylation diverts internalized epidermal growth factor receptors from a degradative pathway to the recycling endosome. J Biol Chem 275:26178–26186PubMedCrossRefGoogle Scholar
  12. 12.
    Barbier AJ, Poppleton HM, Yigzaw Y et al (1999) Transmodulation of epidermal growth factor receptor function by cyclic AMP-dependent protein kinase. J Biol Chem 274:14067–14073PubMedCrossRefGoogle Scholar
  13. 13.
    Morrison P, Saltiel AR, Rosner MR (1996) Role of mitogen-activated protein kinase kinase in regulation of the epidermal growth factor receptor by protein kinase C. J Biol Chem 271:12891–12896PubMedCrossRefGoogle Scholar
  14. 14.
    Morrison P, Takishima K, Rosner MR (1993) Role of threonine residues in regulation of epidermal growth factor receptor by protein kinase C and mitogen-activated protein kinase. J Biol Chem 268:15536–15543PubMedGoogle Scholar
  15. 15.
    Northwood IC, Gonzalez FA, Wartmann M et al (1991) Isolation and characterization of two growth factor-stimulated protein kinases that phosphorylate the epidermal growth factor receptor at threonine 669. J Biol Chem 266:15266–15276PubMedGoogle Scholar
  16. 16.
    Takishima K, Griswold-Prenner I, Ingebritsen T et al (1991) Epidermal growth factor (EGF) receptor T669 peptide kinase from 3T3-L1 cells is an EGF-stimulated “MAP” kinase. Proc Natl Acad Sci 88:2520–2524PubMedCrossRefGoogle Scholar
  17. 17.
    Chen N, Ma W-Y, She Q-B et al (2001) Transactivation of the epidermal growth factor receptor is involved in 12-O-tetradecanoylphorbol-13-acetate-induced signal transduction. J Biol Chem 276:46722–46728PubMedCrossRefGoogle Scholar
  18. 18.
    Comer FI, Hart GW (2000) O-Glycosylation of nuclear and cytosolic proteins: dynamic interplay between O-GlcNAc and O-Phosphate. J Biol Chem 275:29179–29182PubMedCrossRefGoogle Scholar
  19. 19.
    Wells L, Whelan SA, Hart GW (2003) O-GlcNAc: a regulatory post-translational modification. Biochem Biophys Res Com 302:435–441PubMedCrossRefGoogle Scholar
  20. 20.
    Sprung R, Nandi A, Chen Y et al (2005) Tagging-via-substrate strategy for probing O-GlcNAc modified proteins. J Proteome Res 4:950–957PubMedCrossRefGoogle Scholar
  21. 21.
    Nielsen H, Brunak S, VonHeijne G (1999) Machine learning approach for prediction of signal peptide and other protein signals. Protein Eng 12:3–9PubMedCrossRefGoogle Scholar
  22. 22.
    Schueler-Furman O, Baker D (2003) Conserved residue clustering and protein structure prediction. Proteins 52:225–235PubMedCrossRefGoogle Scholar
  23. 23.
    Kaleem A, Hoessli DH, Ahmad I et al (2008) Immediate-early gene regulation by interplay between different post-translational modifications on human histone H3. J Cell Biochem 103:835–851. doi: 10.1002/jcb.21454 Google Scholar
  24. 24.
    Khwaja TA, Wajahat T, Ahmad I et al (2008) In silico modulation of apoptotic Bcl-2 proteins by mistletoe lectin-1: functional consequences of protein modifications. J Cell Biochem 103:479–491. doi: 10.1002/jcb.21412 Google Scholar
  25. 25.
    Ahmad I, Hoessli DC, Walker-Nasir E et al (2006) Oct-2 DNA binding transcription factor: functional consequences of phosphorylation and glycosylation. Nucleic Acids Res 34:175–184PubMedCrossRefGoogle Scholar
  26. 26.
    Boeckmann B, Bairoch A, Apweiler R et al (2003) The Swiss-Prot protein knowledge base and its supplement TrEMBL in 2003. Nucleic Acids Res 31:365–370PubMedCrossRefGoogle Scholar
  27. 27.
    Altschul SF, Madden TL, Schäffer AA et al (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402PubMedCrossRefGoogle Scholar
  28. 28.
    Thompson JD, Higgins DG, Gibson TJ (1994) ClustalW: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680PubMedCrossRefGoogle Scholar
  29. 29.
    Blom N, Gammeltoft S, Brunak S (1999) Sequence- and structure-based prediction of eukaryotic protein phosphorylation sites. J Mol Biol 294:1351–1362PubMedCrossRefGoogle Scholar
  30. 30.
    Kreegipuu A, Blom N, Brunak S (1999) PhosphoBase, a database of phosphorylation sites: release 2.0. Nucleic Acids Res 27:237–239PubMedCrossRefGoogle Scholar
  31. 31.
    Blom N, Sicheritz-Ponten T, Gupta R et al (2004) Prediction of post-translational glycosylation and phosphorylation of proteins from the amino acid sequence. Proteomics 4:1633–1649PubMedCrossRefGoogle Scholar
  32. 32.
    Stover DR, Becker M, Liebetanz J et al (1995) Src phosphorylation of the epidermal growth factor receptor at novel sites mediates receptor interaction with Src and P85 alpha. J Biol Chem 270:15591–15597PubMedCrossRefGoogle Scholar
  33. 33.
    Wang Y, Pennock S, Chen X et al (2002) Endosomal signaling of epidermal growth factor receptor stimulates signal transduction pathways leading to cell survival. Mol Cell Biol 22:7279–7290PubMedCrossRefGoogle Scholar
  34. 34.
    Burke P, Schooler K, Wiley HS (2001) Regulation of epidermal growth factor receptor signaling by endocytosis and intracellular trafficking. Mol Biol Cell 12:897–1910Google Scholar
  35. 35.
    Levkowitz G, Waterman H, Ettenberg SA et al (1999) Ubiquitin ligase activity and tyrosine phosphorylation underlie suppression of growth factor signaling by c-Cbl/Sli-1. Mol Cell 4:1029–1040PubMedCrossRefGoogle Scholar
  36. 36.
    Huang F, Kirkpatrick D, Jiang X et al (2006) Differential regulation of EGF receptor internalization and degradation by multiubiquitination within the kinase domain. Mol Cell 21:737–748PubMedCrossRefGoogle Scholar
  37. 37.
    Huang F, Goh LK, Sorkin A (2007) EGF receptor ubiquitination is not necessary for its internalization. PNAS 104:16904–16909PubMedCrossRefGoogle Scholar
  38. 38.
    de Melker AA, van der Horst G, Borst J (2004) Ubiquitin ligase activity of c-Cbl guides the epidermal growth factor receptor into clathrin-coated pits by two distinct modes of Eps15 recruitment. J Biol Chem 279:55465–55473PubMedCrossRefGoogle Scholar
  39. 39.
    Griffith LS, Schmitz B (1999) O-linked N-acetylglucosamine levels in cerebellar neurons respond reciprocally to pertubations of phosphorylation. Eur J Biochem 262:824–831PubMedCrossRefGoogle Scholar
  40. 40.
    Feinmesser RL, Wicks SJ, Taverner CJ et al (1999) Ca2+/Calmodulin-dependent kinase II phosphorylates the epidermal growth factor receptor on multiple sites in the cytoplasmic tail and serine 744 within the kinase domain to regulate signal generation. J Biol Chem 274:16168–16173PubMedCrossRefGoogle Scholar
  41. 41.
    Liu J, Pang Y, Chang T et al (2006) Increased hexosamine biosynthesis and protein O-GlcNAc levels associated with myocardial protection against calcium paradox and ischemia. J Mol Cell Cardiol 40:1303–1312Google Scholar
  42. 42.
    Cole RN, Hart GW (1999) Glycosylation sites flank phosphorylation sites on synapsin I: O-linked N-acetylglucosamine residues are localized within domains mediating synapsin I interactions. J Neurochem 73:418–428PubMedCrossRefGoogle Scholar
  43. 43.
    Matthews JA, Acevedo-Duncan M, Potter RL (2005) Selective decrease of membrane-associated PKC-a and PKC-e in response to elevated intracellular O-GlcNAc levels in transformed human glial cells. Biochim Biophys Acta 1743:305–315PubMedCrossRefGoogle Scholar
  44. 44.
    Love DC, Hanover JA (2005) The hexosamine signaling pathway: deciphering the “O-GlcNAc code”. Sci STKE 312:re13CrossRefGoogle Scholar
  45. 45.
    Kreppel LK, Blomberg MA, Hart GW (1997) Dynamic glycosylation of nuclear and cytosolic proteins cloning and characterization of a unique O-GlcNAc transferase with multiple tetratricopeptide repeats. J Biol Chem 272:9308–9315PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2008

Authors and Affiliations

  • Afshan Kaleem
    • 1
  • Ishtiaq Ahmad
    • 1
  • Daniel C. Hoessli
    • 2
  • Evelyne Walker-Nasir
    • 1
  • Muhammad Saleem
    • 3
  • Abdul Rauf Shakoori
    • 4
  • Nasir-ud-Din
    • 1
    • 5
    • 6
  1. 1.Institute of Molecular Sciences and BioinformaticsLahorePakistan
  2. 2.Department of Pathology and Immunology, CMUUniversity of GenevaGenevaSwitzerland
  3. 3.Department of BotanyUniversity of the PunjabLahorePakistan
  4. 4.School of Biological SciencesUniversity of the PunjabLahorePakistan
  5. 5.HEJ Research Institute of ChemistryUniversity of KarachiKarachiPakistan
  6. 6.Institute of Management Sciences, University of GenevaGenevaSwitzerland

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