Skip to main content
SpringerLink
Log in

Phosphatidylinositol-4,5-bisphosphate regulates epidermal growth factor receptor activation

  • Signaling and Cell Physiology
  • Published:
Pflügers Archiv - European Journal of Physiology Aims and scope Submit manuscript

Abstract

Phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2 or PIP2] is a direct modulator of a diverse array of proteins in eukaryotic cells. The functional integrity of transmembrane proteins, such as ion channels and transporters, is critically dependent on specific interactions with PIP2 and other phosphoinositides. Here, we report a novel requirement for PIP2 in the activation of the epidermal growth factor receptor (EGFR). Down-regulation of PIP2 levels either via pharmacological inhibition of PI kinase activity, or via manipulation of the levels of the lipid kinase PIP5K1α and the lipid phosphatase synaptojanin, reduced EGFR tyrosine phosphorylation, whereas up-regulation of PIP2 levels via overexpression of PIP5K1α had the opposite effect. A cluster of positively charged residues in the juxtamembrane domain (basic JD) of EGFR is likely to mediate binding of EGFR to PIP2 and PIP2-dependent regulation of EGFR activation. A peptide mimicking the EGFR juxtamembrane domain that was assayed by surface plasmon resonance displayed strong binding to PIP2. Neutralization of positively charged amino acids abolished EGFR/PIP2 interaction in the context of this peptide and down-regulated epidermal growth factor (EGF)-induced EGFR auto-phosphorylation and EGF-induced EGFR signaling to ion channels in the context of the full-length receptor. These results suggest that EGFR activation and downstream signaling depend on interactions of EGFR with PIP2 and point to the basic JD’s critical involvement in these interactions. The addition of this very different class of membrane proteins to ion channels and transporters suggests that PIP2 may serve as a general modulator of the activity of many diverse eukaryotic transmembrane proteins through their basic JDs.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Aifa S, Aydin J, Nordvall G, Lundstrom I, Svensson SP, Hermanson O (2005) A basic peptide within the juxtamembrane region is required for EGF receptor dimerization. Exp Cell Res 302:108–114

    Article  CAS  PubMed  Google Scholar 

  2. Choi SY, Chang J, Jiang B, Seol GH, Min SS, Han JS, Shin HS, Gallagher M, Kirkwood A (2005) Multiple receptors coupled to phospholipase C gate long-term depression in visual cortex. J Neurosci 25:11433–11443

    Article  CAS  PubMed  Google Scholar 

  3. Chuang HH, Prescott ED, Kong H, Shields S, Jordt SE, Basbaum AI, Chao MV, Julius D (2001) Bradykinin and nerve growth factor release the capsaicin receptor from PtdIns(4, 5)P2-mediated inhibition. Nature 411:957–962

    Article  CAS  PubMed  Google Scholar 

  4. Cochet C, Filhol O, Payrastre B, Hunter T, Gill GN (1991) Interaction between the epidermal growth factor receptor and phosphoinositide kinases. J Biol Chem 266:637–644

    CAS  PubMed  Google Scholar 

  5. Czech MP (2000) PIP2 and PIP3: complex roles at the cell surface. Cell 100:603–606

    Article  CAS  PubMed  Google Scholar 

  6. Di Paolo G, Moskowitz HS, Gipson K, Wenk MR, Voronov S, Obayashi M, Flavell R, Fitzsimonds RM, Ryan TA, De Camilli P (2004) Impaired PtdIns(4, 5)P2 synthesis in nerve terminals produces defects in synaptic vesicle trafficking. Nature 431:415–422

    Article  PubMed  Google Scholar 

  7. Downes CP, Gray A, Lucocq JM (2005) Probing phosphoinositide functions in signaling and membrane trafficking. Trends Cell Biol 15:259–268

    Article  CAS  PubMed  Google Scholar 

  8. Fan Z, Makielski JC (1997) Anionic phospholipids activate ATP-sensitive potassium channels. J Biol Chem 272:5388–5395

    Article  CAS  PubMed  Google Scholar 

  9. Fleishman SJ, Schlessinger J, Ben-Tal N (2002) A putative molecular-activation switch in the transmembrane domain of erbB2. Proc Natl Acad Sci USA 99:15937–15940

    Article  CAS  PubMed  Google Scholar 

  10. Gamper N, Shapiro MS (2007) Regulation of ion transport proteins by membrane phosphoinositides. Nat Rev Neurosci 8:921–934

    Article  CAS  PubMed  Google Scholar 

  11. Griffith J, Black J, Faerman C, Swenson L, Wynn M, Lu F, Lippke J, Saxena K (2004) The structural basis for autoinhibition of FLT3 by the juxtamembrane domain. Mol Cell 13:169–178

    Article  CAS  PubMed  Google Scholar 

  12. Horne EA, Dell’Acqua ML (2007) Phospholipase C is required for changes in postsynaptic structure and function associated with NMDA receptor-dependent long-term depression. J Neurosci 27:3523–3534

    Article  CAS  PubMed  Google Scholar 

  13. Huang CL, Feng S, Hilgemann DW (1998) Direct activation of inward rectifier potassium channels by PIP2 and its stabilization by Gbetagamma. Nature 391:803–806

    Article  CAS  PubMed  Google Scholar 

  14. Janmey PA, Lindberg U (2004) Cytoskeletal regulation: rich in lipids. Nat Rev Mol Cell Biol 5:658–666

    Article  CAS  PubMed  Google Scholar 

  15. Jura N, Endres NF, Engel K, Deindl S, Das R, Lamers MH, Wemmer DE, Zhang X, Kuriyan J (2009) Mechanism for activation of the EGF receptor catalytic domain by the juxtamembrane segment. Cell 137:1293–1307

    Article  PubMed  Google Scholar 

  16. Keselman I, Fribourg M, Felsenfeld DP, Logothetis DE (2007) Mechanism of PLC-mediated Kir3 current inhibition. Channels (Austin) 1:113–123

    Google Scholar 

  17. Kobrinsky E, Mirshahi T, Zhang H, Jin T, Logothetis DE (2000) Receptor-mediated hydrolysis of plasma membrane messenger PIP2 leads to K+-current desensitization. Nat Cell Biol 2:507–514

    Article  CAS  PubMed  Google Scholar 

  18. Logothetis DE, Jin T, Lupyan D, Rosenhouse-Dantsker A (2007) Phosphoinositide-mediated gating of inwardly rectifying K(+) channels. Pflugers Archiv 455:83–95

    Article  CAS  PubMed  Google Scholar 

  19. Logothetis DE, Movahedi S, Satler C, Lindpaintner K, Nadal-Ginard B (1992) Incremental reductions of positive charge within the S4 region of a voltage-gated K+ channel result in corresponding decreases in gating charge. Neuron 8:531–540

    Article  CAS  PubMed  Google Scholar 

  20. Logothetis DE, Petrou VI, Adney SK, Mahajan R (2010) Channelopathies linked to plasma membrane phosphoinositides. Pflugers Arch 460:321–341

    Article  CAS  PubMed  Google Scholar 

  21. Lopes CM, Zhang H, Rohacs T, Jin T, Yang J, Logothetis DE (2002) Alterations in conserved Kir channel-PIP2 interactions underlie channelopathies. Neuron 34:933–944

    Article  CAS  PubMed  Google Scholar 

  22. Lund KA, Lazar CS, Chen WS, Walsh BJ, Welsh JB, Herbst JJ, Walton GM, Rosenfeld MG, Gill GN, Wiley HS (1990) Phosphorylation of the epidermal growth factor receptor at threonine 654 inhibits ligand-induced internalization and down-regulation. J Biol Chem 265:20517–20523

    CAS  PubMed  Google Scholar 

  23. Mandal M, Yan Z (2009) Phosphatidylinositol (4, 5)-bisphosphate regulation of N-methyl-d-aspartate receptor channels in cortical neurons. Mol Pharmacol 76:1349–1359

    Article  CAS  PubMed  Google Scholar 

  24. Martin TF (2001) PI(4, 5)P(2) regulation of surface membrane traffic. Curr Opin Cell Biol 13:493–499

    Article  CAS  PubMed  Google Scholar 

  25. McLaughlin S, Smith SO, Hayman MJ, Murray D (2005) An electrostatic engine model for autoinhibition and activation of the epidermal growth factor receptor (EGFR/ErbB) family. J Gen Physiol 126:41–53

    Article  CAS  PubMed  Google Scholar 

  26. Michailidis IE, Helton TD, Petrou VI, Mirshahi T, Ehlers MD, Logothetis DE (2007) Phosphatidylinositol-4, 5-bisphosphate regulates NMDA receptor activity through alpha-actinin. J Neurosci 27:5523–5532

    Article  CAS  PubMed  Google Scholar 

  27. Miettinen PJ, Berger JE, Meneses J, Phung Y, Pedersen RA, Werb Z, Derynck R (1995) Epithelial immaturity and multiorgan failure in mice lacking epidermal growth factor receptor. Nature 376:337–341

    Article  CAS  PubMed  Google Scholar 

  28. Nakanishi S, Catt KJ, Balla T (1995) A Wortmannin-sensitive phosphatidylinositol 4-kinase that regulates hormone-sensitive pools of inositolphospholipids. Proc Natl Acad Sci USA 92:5317–5321

    Article  CAS  PubMed  Google Scholar 

  29. Opresko LK, Wiley HS (1990) Functional reconstitutional of the human epidermal growth factor receptor system in Xenopus oocytes. J Cell Biol 111:1661–1671

    Article  CAS  PubMed  Google Scholar 

  30. Rauch ME, Ferguson CG, Prestwich GD, Cafiso DS (2002) Myristoylated alanine-rich C kinase substrate (MARCKS) sequesters spin-labeled phosphatidylinositol 4, 5-bisphosphate in lipid bilayers. J Biol Chem 277:14068–14076

    Article  CAS  PubMed  Google Scholar 

  31. Red Brewer M, Choi SH, Alvarado D, Moravcevic K, Pozzi A, Lemmon MA, Carpenter G (2009) The juxtamembrane region of the EGF receptor functions as an activation domain. Mol Cell 34:641–651

    Article  PubMed  Google Scholar 

  32. Rhee SG (2001) Regulation of phosphoinositide-specific phospholipase C. Annu Rev Biochem 70:281–312

    Article  CAS  PubMed  Google Scholar 

  33. Rhee SG, Choi KD (1992) Multiple forms of phospholipase C isozymes and their activation mechanisms. Adv Second Messenger Phosphoprotein Res 26:35–61

    CAS  PubMed  Google Scholar 

  34. Rohacs T, Lopes CM, Jin T, Ramdya PP, Molnar Z, Logothetis DE (2003) Specificity of activation by phosphoinositides determines lipid regulation of Kir channels. Proc Natl Acad Sci USA 100:745–750

    Article  CAS  PubMed  Google Scholar 

  35. Rohacs T, Lopes CM, Michailidis I, Logothetis DE (2005) PI(4, 5)P2 regulates the activation and desensitization of TRPM8 channels through the TRP domain. Nat Neurosci 8:626–634

    Article  CAS  PubMed  Google Scholar 

  36. Sakisaka T, Itoh T, Miura K, Takenawa T (1997) Phosphatidylinositol 4, 5-bisphosphate phosphatase regulates the rearrangement of actin filaments. Mol Cell Biol 17:3841–3849

    CAS  PubMed  Google Scholar 

  37. Santiskulvong C, Rozengurt E (2007) Protein kinase Calpha mediates feedback inhibition of EGF receptor transactivation induced by Gq-coupled receptor agonists. Cell Signal 19:1348–1357

    Article  CAS  PubMed  Google Scholar 

  38. Schlessinger J (2002) Ligand-induced, receptor-mediated dimerization and activation of EGF receptor. Cell 110:669–672

    Article  CAS  PubMed  Google Scholar 

  39. Sengupta P, Bosis E, Nachliel E, Gutman M, Smith SO, Mihalyne G, Zaitseva I, McLaughlin S (2009) EGFR juxtamembrane domain, membranes, and calmodulin: kinetics of their interaction. Biophys J 96:4887–4895

    Article  CAS  PubMed  Google Scholar 

  40. Sibilia M, Steinbach JP, Stingl L, Aguzzi A, Wagner EF (1998) A strain-independent postnatal neurodegeneration in mice lacking the EGF receptor. EMBO J 17:719–731

    Article  CAS  PubMed  Google Scholar 

  41. Sorkin A, Carpenter G (1993) Interaction of activated EGF receptors with coated pit adaptins. Science 261:612–615

    Article  CAS  PubMed  Google Scholar 

  42. Suh BC, Hille B (2005) Regulation of ion channels by phosphatidylinositol 4, 5-bisphosphate. Curr Opin Neurobiol 15:370–378

    Article  CAS  PubMed  Google Scholar 

  43. Suh BC, Hille B (2007) Regulation of KCNQ channels by manipulation of phosphoinositides. J Physiol 582:911–916

    Article  CAS  PubMed  Google Scholar 

  44. Thiel KW, Carpenter G (2007) Epidermal growth factor receptor juxtamembrane region regulates allosteric tyrosine kinase activation. Proc Natl Acad Sci USA 104:19238–19243

    Article  CAS  PubMed  Google Scholar 

  45. Toker A (1998) The synthesis and cellular roles of phosphatidylinositol 4, 5-bisphosphate. Curr Opin Cell Biol 10:254–261

    Article  CAS  PubMed  Google Scholar 

  46. Weber W (1999) Ion currents of Xenopus laevis oocytes: state of the art. Biochim Biophys Acta 1421:213–233

    Article  CAS  PubMed  Google Scholar 

  47. Wenk MR, De Camilli P (2004) Protein-lipid interactions and phosphoinositide metabolism in membrane traffic: insights from vesicle recycling in nerve terminals. Proc Natl Acad Sci USA 101:8262–8269

    Article  CAS  PubMed  Google Scholar 

  48. Willars GB, Nahorski SR, Challiss RA (1998) Differential regulation of muscarinic acetylcholine receptor-sensitive polyphosphoinositide pools and consequences for signaling in human neuroblastoma cells. J Biol Chem 273:5037–5046

    Article  CAS  PubMed  Google Scholar 

  49. Wu L, Bauer CS, Zhen XG, Xie C, Yang J (2002) Dual regulation of voltage-gated calcium channels by PtdIns(4, 5)P2. Nature 419:947–952

    Article  CAS  PubMed  Google Scholar 

  50. Yamane K, Toyoshima C, Nishimura S (1992) Ligand-induced functions of the epidermal growth factor receptor require the positively charged region asymmetrically distributed across plasma membrane. Biochem Biophys Res Commun 184:1301–1310

    Article  CAS  PubMed  Google Scholar 

  51. Yin HL, Janmey PA (2003) Phosphoinositide regulation of the actin cytoskeleton. Annu Rev Physiol 65:761–789

    Article  CAS  PubMed  Google Scholar 

  52. Zhang H, Craciun LC, Mirshahi T, Rohacs T, Lopes CM, Jin T, Logothetis DE (2003) PIP(2) activates KCNQ channels, and its hydrolysis underlies receptor-mediated inhibition of M currents. Neuron 37:963–975

    Article  CAS  PubMed  Google Scholar 

  53. Zhang X, Gureasko J, Shen K, Cole PA, Kuriyan J (2006) An allosteric mechanism for activation of the kinase domain of epidermal growth factor receptor. Cell 125:1137–1149

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by a National Institutes of Health grant (HL-59949) to DEL. Support for RI and YC was provided by NIH grant DK-38761. We would like to thank Heikki Vaananen, Sophia Gruszecki, Samantha Lee, and Dr. Mei Zhang for excellent technical assistance, Drs. Pietro DeCamilli (Yale University, CT) for the gift of synaptojanin, and Drs. Show-Ling Shyng and Colin Nichols (Oregon Health and Science University, OR, USA and Washington University, MO, USA) for the gift of PI(5)P kinase. We thank Dr. Qi Zhao for help with the analysis of the FACS data. We would also like to thank Drs. Giorgos Panayotou (Fleming Institute, Athens, Greece), Mitchell Goldfarb (Hunter College, NY, USA), Michael Ehlers (Duke Neurobiology, NC, USA), Julia Sable (Columbia University, NY, USA), Tibor Rohacs (UMDNJ), Stuart Aaronson (Department of Oncological Sciences, MSSM) and the Logothetis laboratory members for insightful discussions and comments on the manuscript.

Author information

Author notes

  1. Ioannis E. Michailidis

    Present address: Department of Biological Sciences, Columbia University, New York, NY, 10027, USA

  2. Radda Rusinova

    Present address: Department of Physiology and Biophysics, Weill Medical College of Cornell University, New York, NY, 10065, USA

Authors and Affiliations

  1. Department of Structural and Chemical Biology, Mount Sinai School of Medicine, New York, NY, USA

    Ioannis E. Michailidis & Radda Rusinova

  2. Department of Psychiatry, Mount Sinai School of Medicine, New York, NY, USA

    Anastasios Georgakopoulos & Nikolaos K. Robakis

  3. Department of Pharmacology and Systems Therapeutics, Mount Sinai School of Medicine, New York, NY, USA

    Yibang Chen & Ravi Iyengar

  4. Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, 1101 E Marshall St, Richmond, VA, 23298, USA

    Diomedes E. Logothetis & Lia Baki

Authors
  1. Ioannis E. Michailidis
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Radda Rusinova
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Anastasios Georgakopoulos
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yibang Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ravi Iyengar
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Nikolaos K. Robakis
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Diomedes E. Logothetis
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Lia Baki
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Diomedes E. Logothetis or Lia Baki.

Electronic Supplementary Material

Below is the link to the electronic supplementary material.

Figure S1

Mutations in the JD of EGFR that impair its phosphorylation do not interfere with the surface expression of the receptor. Data summary (mean ± S.E.) of relative (% of wild-type) surface expression levels for wild-type and mutant EGFR from 2 independent experiments (number of oocytes analyzed in each experiment is indicated on the top of each bar). Total EGFR levels in the membrane fraction of these oocytes were similar to those presented in Figure 2B. (TIFF 1.45 MB)

Figure S2

Wortmannin treatment reduces PIP 2 levels. Oocytes were incubated for 1h in the presence and absence of wortmannin (15µM) and subsequently lysed for preparation of the membrane fraction. Extraction of membrane lipids and PIP2 ELISA assay are described under Supplemental Methods. Data points corresponding to known amounts of PIP2 were fitted to the equation y = A2 + (A1-A2)/(1 + (x/x0)p) with A1=2.14965±0.42526, A2=-0.05576±0.75398, x0=20.3308524.0873 and p=0.52726±0.39675. PIP2 levels in each sample, calculated from the standard curve (left panel), were normalized for PIP2 levels in the control non-treated sample from the same experiment. Data from 3 experiments are presented as mean ± S.E (right panel). Statistical significance is indicated. (TIFF 3.10 MB)

Figure S3

Downregulation of PI(5)PKIα does not affect cell surface expression of EGFR a) Histograms of mean EGFR extracellular fluorescence intensity measured by FACS analysis, as described in Supplementary Methods. Background was estimated by performing the experiment in the absence of primary antibody (Negative, neg). The rest of the experimental conditions correspond to Fig. 4c. b) Bars representing the effect of PIPKIα siRNA transfection on surface EGFR in the presence and absence of EGF (%±SE) were calculated from three experiments for each condition. The means were not significantly different in the presence of siRNA (t-test). (TIFF 3.14 MB)

ESM 4

(DOC 34 KB)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Michailidis, I.E., Rusinova, R., Georgakopoulos, A. et al. Phosphatidylinositol-4,5-bisphosphate regulates epidermal growth factor receptor activation. Pflugers Arch - Eur J Physiol 461, 387–397 (2011). https://doi.org/10.1007/s00424-010-0904-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00424-010-0904-3

Keywords

Search

Navigation