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EHDS are serine phosphoproteins: EHD1 phosphorylation is enhanced by serum stimulation

Cellular & Molecular Biology Letters volume 13, pages 632–648 (2008)Cite this article

Abstract

Endocytic processes are mediated by multiple protein-protein interacting modules and regulated by phosphorylation and dephosphorylation. The Eps15 homology domain containing protein 1 (EHD1) has been implicated in regulating recycling of proteins, internalized both in clathrin-dependent and clathrin-independent endocytic pathways, from the recycling compartment to the plasma membrane. EHD1 was found in a complex with clathrin, adaptor protein complex-2 (AP-2) and insulin-like growth factor-1 receptor (IGF-1R), and was shown to interact with Rabenosyn-5, SNAP29, EHBP1 (EH domain binding protein 1) and syndapin I and II. In this study, we show that EHD1, like the other human EHDs, undergoes serine-phosphorylation. Our results also indicate that EHD1 is a serum-inducible serine-phosphoprotein and that PKC (protein kinase C) is one of its kinases. In addition, we show that inhibitors of clathrin-mediated endocytosis decrease EHD1 phosphorylation, while inhibitors of caveolinmediated endocytosis do not affect EHD1 phosphorylation. The results of experiments in which inhibitors of endocytosis were employed strongly suggest that EHD1 phosphorylation occurs between early endosomes and the endocytic recycling compartment.

Abbreviations

AP-2:

adaptor protein complex-2

BSA:

bovine serum albumin

CHO:

Chinese hamster ovary

cpm:

counts per minute

dFCS:

dialyzed FCS

DMEM:

Dulbecco modified eagle medium

EHBP1:

EH domain binding protein 1

FCS:

fetal calf serum

FYVE:

phenylalanine-tyrosine-valine-glutamate

IGF-1R:

insulin-like growth factor-1 receptor

mDHFR:

mouse dihydrofolate reductase

MEM-Alpha:

minimal essential medium-alpha

ORF:

open reading frame

PBS:

phosphate buffered saline

PKC:

protein kinase C

PVDF:

polyvinidilen difluoride

TLC:

thin-layer chromatography

TLE:

thin-layer electrophoresis

References

  1. Wong, W.T., Kraus, M.H., Carlomagno, F., Zelano, A., Druck, T., Croce, C. M., Huebner, K. and Di Fiore, P.P. The human eps15 gene, encoding a tyrosine kinase substrate, is conserved in evolution and maps to 1p31–p32. Oncogene l9 (1994) 1591–1597.

    Google Scholar 

  2. Slepnev, V.I., Ochoa, G.C., Butler, M.H., Grabs, D. and De Camilli, P. Role of phosphorylation in regulation of the assembly of endocytic coat complexes. Science 281 (1998) 821–824.

    Article  PubMed  CAS  Google Scholar 

  3. Di Fiore, P.P., Pelicci, P.G. and Sorkin, A. EH: a novel protein-protein interaction domain potentially involved in intracellular sorting. Trends Biochem. Sci. 22 (1997) 411–413.

    Article  PubMed  Google Scholar 

  4. Santolini, E., Salcini, A.E., Kay, B.K., Yamabhai, M. and Di Fiore, P.P. The EH network. Exp. Cell Res. 253 (1999) 186–209.

    Article  PubMed  CAS  Google Scholar 

  5. Lee, D.W., Zhao, X., Scarselletta, S., Schweinsberg, P.J., Eisenberg, E., Grant, B.D. and Greene, L. E. ATP binding regulates oligomerization and endosome association of RME-1 family proteins. J. Biol. Chem. 280 (2005) 17213–17220.

    Article  PubMed  CAS  Google Scholar 

  6. Vetter, I.R. and Wittinghofer, A. The guanine nucleotide-binding switch in three dimensions. Science 294 (2001) 1299–1304.

    Article  PubMed  CAS  Google Scholar 

  7. Mintz, L., Galperin, E., Pasmanik-Chor, M., Tulzinsky, S., Bromberg, Y., Kozak, C.A., Joyner, A., Fein, A. and Horowitz, M. EHD1-an EH-domaincontaining protein with a specific expression pattern. Genomics 59 (1999) 66–76.

    Article  PubMed  CAS  Google Scholar 

  8. Pohl, U., Smith, J.S., Tachibana, I., Ueki, K., Lee, H.K., Ramaswamy, S., Wu, Q., Mohrenweiser, H.W., Jenkins, R.B. and Louis, D.N. EHD2, EHD3, and EHD4 encode novel members of a highly conserved family of EH domain-containing proteins. Genomics 63 (2000) 255–262.

    Article  PubMed  CAS  Google Scholar 

  9. Grant, B., Zhang, Y., Paupard, M.C., Lin, S.X., Hall, D.H. and Hirsh, D. Evidence that RME-1, a conserved C. elegans EH-domain protein, functions in endocytic recycling. Nat. Cell Biol. 3 (2001) 573–579.

    CAS  Google Scholar 

  10. Smith, C.A., Dho, S.E., Donaldson, J., Tepass, U. and McGlade, C.J. The Cell Fate Determinant Numb Interacts with EHD/Rme-1 Family Proteins and Has a Role in Endocytic Recycling. Mol. Biol. Cell 15 (2004) 2698–3708.

    Google Scholar 

  11. Blume, J.J., Halbach, A., Behrendt, D., Paulsson, M. and Plomann, M. EHD proteins are associated with tubular and vesicular compartments and interact with specific phospholipids. Exp. Cell Res. 313 (2007) 219–231.

    Article  PubMed  CAS  Google Scholar 

  12. Galperin, E., Benjamin, S., Rapaport, D., Rotem-Yehudar, R., Tolchinsky, S. and Horowitz, M. EHD3: a protein that resides in recycling tubular and vesicular membrane structures and interacts with EHD1. Traffic 3 (2002) 575–589.

    Article  PubMed  CAS  Google Scholar 

  13. George, M., Ying, G., Rainey, M.A., Solomon, A., Parikh, P.T., Gao, Q., Band, V. and Band, H. Shared as well as distinct roles of EHD proteins revealed by biochemical and functional comparisons in mammalian cells and C. elegans. BMC Cell Biol. 8 (2007) 3.

    Article  PubMed  Google Scholar 

  14. Naslavsky, N. and Caplan, S. C-terminal EH-domain-containing proteins: consensus for a role in endocytic trafficking, EH? J. Cell Sci. 118 (2005) 4093–4101.

    Article  CAS  Google Scholar 

  15. Rapaport, D., Auerbach, W., Naslavsky, N., Pasmanik-Chor, M., Galperin, E., Fein, A., Caplan, S., Joyner, A.L. and Horowitz, M. Recycling to the plasma membrane is delayed in EHD1 knockout mice. Traffic 7 (2006) 52–60.

    Article  PubMed  CAS  Google Scholar 

  16. Guilherme, A., Soriano, N.A., Bose, S., Holik, J., Bose, A., Pomerleau, D.P., Furcinitti, P., Leszyk, J., Corvera, S. and Czech, M.P. EHD2 and the novel EH domain binding protein EHBP1 couple endocytosis to the actin cytoskeleton. J. Biol. Chem. 279 (2004) 10593–10605.

    Article  PubMed  CAS  Google Scholar 

  17. Daumke, O., Lundmark, R., Vallis, Y., Martens, S., Butler, P.J. and McMahon, H.T. Architectural and mechanistic insights into an EHD ATPase involved in membrane remodelling. Nature 449 (2007) 923–927.

    Article  PubMed  CAS  Google Scholar 

  18. Naslavsky, N., Rahajeng, J., Sharma, M., Jovic, M. and Caplan, S. Interactions between EHD Proteins and Rab11-FIP2: A Role for EHD3 in Early Endosomal Transport. Mol. Biol. Cell 17 (2006) 163–177.

    Article  PubMed  CAS  Google Scholar 

  19. Shao, Y., Akmentin, W., Toledo-Aral, J.J., Rosenbaum, J., Valdez, G., Cabot, J.B., Hilbush, B.S. and Halegoua, S. Pincher, a pinocytic chaperone for nerve growth factor/TrkA signaling endosomes. J. Cell Biol. 157 (2002) 679–691.

    Article  PubMed  CAS  Google Scholar 

  20. Sharma, M., Naslavsky, N. and Caplan, S. A role for EHD4 in the regulation of early endosomal transport. Traffic 9 (2008) 995–1018.

    Article  PubMed  CAS  Google Scholar 

  21. Lin, S.X., Grant, B., Hirsh, D. and Maxfield, F.R. Rme-1 regulates the distribution and function of the endocytic recycling compartment in mammalian cells. Nat. Cell Biol. 3 (2001) 567–572.

    Article  PubMed  CAS  Google Scholar 

  22. Picciano, J.A., Ameen, N., Grant, B. and Bradbury, N.A. Rme-1 regulates the recycling of the cystic fibrosis transmembrane conductance regulator. Am. J. Physiol. Cell Physiol. 285 (2003) 1009–1018.

    Google Scholar 

  23. Guilherme, A., Soriano, N.A., Furcinitti, P.S. and Czech, M.P. Role of EHD1 and EHBP1 in perinuclear sorting and insulin-regulated GLUT4 recycling in 3T3-L1 adipocytes. J. Biol. Chem. 279 (2004) 40062–40075.

    Article  PubMed  CAS  Google Scholar 

  24. Caplan, S., Naslavsky, N., Hartnell, L.M., Lodge, R., Polishchuk, R.S., Donaldson, J.G. and Bonifacino, J.S. A tubular EHD1-containing compartment involved in the recycling of major histocompatibility complex class I molecules to the plasma membrane. Embo J. 21 (2002) 2557–2567.

    Article  PubMed  CAS  Google Scholar 

  25. Park, M., Penick, E.C., Edwards, J.G., Kauer, J.A. and Ehlers, M.D. Recycling endosomes supply AMPA receptors for LTP. Science 305 (2004) 1972–1975.

    Article  PubMed  CAS  Google Scholar 

  26. Rotem-Yehudar, R., Galperin, E. and Horowitz, M. Association of insulinlike growth factor 1 receptor with EHD1 and SNAP29. J. Biol. Chem. 276 (2001) 33054–33060.

    Article  PubMed  CAS  Google Scholar 

  27. Naslavsky, N., Rahajeng, J., Chenavas, S., Sorgen, P.L. and Caplan, S. EHD1 and Eps15 interact with phosphatidylinositols via their EH-domains. J. Biol. Chem. 282 (2007) 16612–16622.

    Article  PubMed  CAS  Google Scholar 

  28. Naslavsky, N., Boehm, M., Backlund, P.S., Jr. and Caplan, S. Rabenosyn-5 and EHD1 interact and sequentially regulate protein recycling to the plasma membrane. Mol. Biol. Cell 15 (2004) 2410–2422.

    Article  PubMed  CAS  Google Scholar 

  29. Braun, A., Pinyol, R., Dahlhaus, R., Koch, D., Fonarev, P., Grant, B.D., Kessels, M.M. and Qualmann, B. EHD proteins associate with syndapin I and II and such interactions play a crucial role in endosomal recycling. Mol. Biol. Cell 16 (2005) 3642–3658.

    Article  PubMed  CAS  Google Scholar 

  30. Xu, Y., Shi, H., Wei, S., Heng Wong, S. and Hong, W. Mutually exclusive interactions of EHD1 with GS32 and Syndapin II. Mol. Membr. Biol. 21 (2004) 269–277.

    Article  PubMed  CAS  Google Scholar 

  31. Nielsen, E., Christoforidis, S., Uttenweiler-Joseph, S., Miaczynska, M., Dewitte, F., Wilm, M., Hoflack, B. and Zerial, M. Rabenosyn-5, a novel Rab5 effector, is complexed with hVPS45 and recruited to endosomes through a FYVE finger domain. J. Cell Biol. 151 (2000) 601–612.

    Article  PubMed  CAS  Google Scholar 

  32. Kessels, M.M. and Qualmann, B. The syndapin protein family: linking membrane trafficking with the cytoskeleton. J. Cell Sci. 117 (2004) 3077–3086.

    Article  PubMed  CAS  Google Scholar 

  33. Cullis, D.N., Philip, B., Baleja, J.D. and Feig, L.A. Rab11-FIP2, an adaptor protein connecting cellular components involved in internalization and recycling of epidermal growth factor receptors. J. Biol. Chem. 277 (2002) 49158–49166.

    Article  PubMed  CAS  Google Scholar 

  34. Steegmaier, M., Yang, B., Yoo, J.S., Huang, B., Shen, M., Yu, S., Luo, Y. and Scheller, R.H. Three novel proteins of the syntaxin/SNAP-25 family. J. Biol. Chem. 273 (1998) 34171–34179.

    Article  PubMed  CAS  Google Scholar 

  35. Ricotta, D., Conner, S.D., Schmid, S.L., von Figura, K. and Honing, S. Phosphorylation of the AP2 mu subunit by AAK1 mediates high affinity binding to membrane protein sorting signals. J. Cell Biol. 156 (2002) 791–795.

    Article  PubMed  CAS  Google Scholar 

  36. Wilde, A. and Brodsky, F.M. In vivo phosphorylation of adaptors regulates their interaction with clathrin. J. Cell Biol. 135 (1996) 635–645.

    Article  PubMed  CAS  Google Scholar 

  37. Ihara, Y., Yasuoka, C., Kageyama, K., Wada, Y. and Kondo, T. Tyrosine phosphorylation of clathrin heavy chain under oxidative stress. Biochem. Biophys. Res. Commun. 297 (2002) 353–360.

    Article  PubMed  CAS  Google Scholar 

  38. Confalonieri, S., Salcini, A.E., Puri, C., Tacchetti, C. and Di Fiore, P.P. Tyrosine phosphorylation of Eps15 is required for ligand-regulated, but not constitutive, endocytosis. J. Cell Biol. 150 (2000) 905–912.

    Article  PubMed  CAS  Google Scholar 

  39. Kamps, M.P. and Sefton, B.M. Acid and base hydrolysis of phosphoproteins bound to immobilon facilitates analysis of phosphoamino acids in gelfractionated proteins. Anal. Biochem. 176 (1989) 22–27.

    Article  PubMed  CAS  Google Scholar 

  40. Boyle, W.J., van der Geer, P. and Hunter, T. Phosphopeptide mapping and phosphoamino acid analysis by two-dimensional separation on thin-layer cellulose plates. Methods Enzymol. 201 (1991) 110–149.

    Article  PubMed  CAS  Google Scholar 

  41. Lin, F.T., Krueger, K.M., Kendall, H.E., Daaka, Y., Fredericks, Z.L., Pitcher, J.A. and Lefkowitz, R.J. Clathrin-mediated endocytosis of the betaadrenergic receptor is regulated by phosphorylation/dephosphorylation of beta-arrestin1. J. Biol. Chem. 272 (1997) 31051–31057.

    Article  PubMed  CAS  Google Scholar 

  42. Simonsen, A., Lippe, R., Christoforidis, S., Gaullier, J.M., Brech, A., Callaghan, J., Toh, B.H., Murphy, C., Zerial, M. and Stenmark, H. EEA1 links PI(3)K function to Rab5 regulation of endosome fusion. Nature 394 (1998) 494–498.

    Article  PubMed  CAS  Google Scholar 

  43. Jones, D.T., Ganeshaguru, K., Anderson, R.J., Jackson, T.R., Bruckdorfer, K.R., Low, S.Y., Palmqvist, L., Prentice, H.G., Hoffbrand, A.V., Mehta, A.B. and Wickremasinghe, R.G. Albumin activates the AKT signaling pathway and protects B-chronic lymphocytic leukemia cells from chlorambucil-and radiation-induced apoptosis. Blood 101 (2003) 3174–3180.

    Article  PubMed  CAS  Google Scholar 

  44. Heuser, J.E. and Anderson, R.G. Hypertonic media inhibit receptormediated endocytosis by blocking clathrin-coated pit formation. J. Cell Biol. 108 (1989) 389–400.

    Article  PubMed  CAS  Google Scholar 

  45. Phonphok, Y. and Rosenthal, K.S. Stabilization of clathrin coated vesicles by amantadine, tromantadine and other hydrophobic amines. FEBS Lett. 281 (1991) 188–190.

    Article  PubMed  CAS  Google Scholar 

  46. Puri, V., Watanabe, R., Singh, R.D., Dominguez, M., Brown, J.C., Wheatley, C.L., Marks, D.L. and Pagano, R.E. Clathrin-dependent and-independent internalization of plasma membrane sphingolipids initiates two Golgi targeting pathways. J. Cell Biol. 154 (2001) 535–547.

    Article  PubMed  CAS  Google Scholar 

  47. Arcaro, A. and Wymann, M.P. Wortmannin is a potent phosphatidylinositol 3-kinase inhibitor: the role of phosphatidylinositol 3,4,5-trisphosphate in neutrophil responses. Biochem. J. 296 ( Pt 2) (1993) 297–301.

    PubMed  CAS  Google Scholar 

  48. Jones, A.T. and Clague, M.J. Phosphatidylinositol 3-kinase activity is required for early endosome fusion. Biochem. J. 311 (1995) 31–34.

    PubMed  CAS  Google Scholar 

  49. Li, G., D’Souza-Schorey, C., Barbieri, M.A., Roberts, R.L., Klippel, A., Williams, L.T. and Stahl, P.D. Evidence for phosphatidylinositol 3-kinase as a regulator of endocytosis via activation of Rab5. Proc. Natl. Acad. Sci. USA 92 (1995) 10207–10211.

    Article  PubMed  CAS  Google Scholar 

  50. Sheff, D.R., Daro, E.A., Hull, M. and Mellman, I. The receptor recycling pathway contains two distinct populations of early endosomes with different sorting functions. J. Cell Biol. 145 (1999) 123–139.

    Article  PubMed  CAS  Google Scholar 

  51. Welsh, G.I., Griffiths, M.R., Webster, K.J., Page, M.J. and Tavare, J.M. Proteome analysis of adipogenesis. Proteomics 4 (2004) 1042–1051.

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. University of California San Diego, San Diego, CA, 92093, USA

    Boris Fichtman

  2. The Weizmann Institute of Science, Rehovot, 76100, Israel

    Liat Ravid

  3. Department of Cell Research and Immunology, Faculty of Life Sciences, Tel-Aviv University, Ramat Aviv, 69978, Israel

    Debora Rapaport & Mia Horowitz

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  1. Boris Fichtman
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  2. Liat Ravid
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  3. Debora Rapaport
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  4. Mia Horowitz
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Correspondence to Mia Horowitz.

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Fichtman, B., Ravid, L., Rapaport, D. et al. EHDS are serine phosphoproteins: EHD1 phosphorylation is enhanced by serum stimulation. Cell Mol Biol Lett 13, 632–648 (2008). https://doi.org/10.2478/s11658-008-0027-4

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Cellular & Molecular Biology Letters

ISSN: 1689-1392

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