Skip to main content
The EMBO Journal logoLink to The EMBO Journal
. 1992 Dec;11(12):4379–4389. doi: 10.1002/j.1460-2075.1992.tb05538.x

Reversible phosphorylation--dephosphorylation determines the localization of rab4 during the cell cycle.

P van der Sluijs 1, M Hull 1, L A Huber 1, P Mâle 1, B Goud 1, I Mellman 1
PMCID: PMC557012  PMID: 1425574

Abstract

The ras-like GTP binding protein rab4 is the only known rab protein on endosomes that is phosphorylated during mitosis. Since a large fraction of rab4 accumulates in the cytosol in mitotic cells, we investigated the molecular mechanism controlling membrane association of rab4. We first show that human rab4 is phosphorylated by recombinant mammalian p34cdc2 kinase in vitro. Next, the actual site of phosphorylation and its functional significance were determined using stably transfected CHO cell lines producing high levels of wild type rab4 or rab4 mutants bearing alterations at Ser196, which occurs within a consensus site for p34cdc2 kinase phosphorylation (S196PRR). Mutation of Ser196 to glutamine or aspartic acid completely prevented rab4 phosphorylation in mitotic cells and also blocked its appearance in the cytosol. Neither C-terminal isoprenylation nor carboxymethylation of rab4 was affected by the mutations or by phosphorylation. Finally, dephosphorylation and reassociation of soluble rab4 with membranes occurred upon exit of cells from mitosis. Thus, phosphorylation of Ser196 is directly responsible for the reversible translocation of rab4 into the cytosol of mitotic cells.

Full text

PDF
4379

Images in this article

Selected References

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

  1. Bailly E., McCaffrey M., Touchot N., Zahraoui A., Goud B., Bornens M. Phosphorylation of two small GTP-binding proteins of the Rab family by p34cdc2. Nature. 1991 Apr 25;350(6320):715–718. doi: 10.1038/350715a0. [DOI] [PubMed] [Google Scholar]
  2. Bokoch G. M., Quilliam L. A., Bohl B. P., Jesaitis A. J., Quinn M. T. Inhibition of Rap1A binding to cytochrome b558 of NADPH oxidase by phosphorylation of Rap1A. Science. 1991 Dec 20;254(5039):1794–1796. doi: 10.1126/science.1763330. [DOI] [PubMed] [Google Scholar]
  3. Bourne H. R. Do GTPases direct membrane traffic in secretion? Cell. 1988 Jun 3;53(5):669–671. doi: 10.1016/0092-8674(88)90081-5. [DOI] [PubMed] [Google Scholar]
  4. Bourne H. R., Sanders D. A., McCormick F. The GTPase superfamily: conserved structure and molecular mechanism. Nature. 1991 Jan 10;349(6305):117–127. doi: 10.1038/349117a0. [DOI] [PubMed] [Google Scholar]
  5. Bucci C., Parton R. G., Mather I. H., Stunnenberg H., Simons K., Hoflack B., Zerial M. The small GTPase rab5 functions as a regulatory factor in the early endocytic pathway. Cell. 1992 Sep 4;70(5):715–728. doi: 10.1016/0092-8674(92)90306-w. [DOI] [PubMed] [Google Scholar]
  6. Casanova J. E., Breitfeld P. P., Ross S. A., Mostov K. E. Phosphorylation of the polymeric immunoglobulin receptor required for its efficient transcytosis. Science. 1990 May 11;248(4956):742–745. doi: 10.1126/science.2110383. [DOI] [PubMed] [Google Scholar]
  7. Celis J. E., Gesser B., Rasmussen H. H., Madsen P., Leffers H., Dejgaard K., Honore B., Olsen E., Ratz G., Lauridsen J. B. Comprehensive two-dimensional gel protein databases offer a global approach to the analysis of human cells: the transformed amnion cells (AMA) master database and its link to genome DNA sequence data. Electrophoresis. 1990 Dec;11(12):989–1071. doi: 10.1002/elps.1150111202. [DOI] [PubMed] [Google Scholar]
  8. Chavrier P., Gorvel J. P., Stelzer E., Simons K., Gruenberg J., Zerial M. Hypervariable C-terminal domain of rab proteins acts as a targeting signal. Nature. 1991 Oct 24;353(6346):769–772. doi: 10.1038/353769a0. [DOI] [PubMed] [Google Scholar]
  9. Chavrier P., Parton R. G., Hauri H. P., Simons K., Zerial M. Localization of low molecular weight GTP binding proteins to exocytic and endocytic compartments. Cell. 1990 Jul 27;62(2):317–329. doi: 10.1016/0092-8674(90)90369-p. [DOI] [PubMed] [Google Scholar]
  10. Chelsky D., Olson J. F., Koshland D. E., Jr Cell cycle-dependent methyl esterification of lamin B. J Biol Chem. 1987 Mar 25;262(9):4303–4309. [PubMed] [Google Scholar]
  11. Dunphy W. G., Brizuela L., Beach D., Newport J. The Xenopus cdc2 protein is a component of MPF, a cytoplasmic regulator of mitosis. Cell. 1988 Jul 29;54(3):423–431. doi: 10.1016/0092-8674(88)90205-x. [DOI] [PubMed] [Google Scholar]
  12. Farnsworth C. C., Kawata M., Yoshida Y., Takai Y., Gelb M. H., Glomset J. A. C terminus of the small GTP-binding protein smg p25A contains two geranylgeranylated cysteine residues and a methyl ester. Proc Natl Acad Sci U S A. 1991 Jul 15;88(14):6196–6200. doi: 10.1073/pnas.88.14.6196. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Featherstone C., Griffiths G., Warren G. Newly synthesized G protein of vesicular stomatitis virus is not transported to the Golgi complex in mitotic cells. J Cell Biol. 1985 Dec;101(6):2036–2046. doi: 10.1083/jcb.101.6.2036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fischer T. H., Gatling M. N., Lacal J. C., White G. C., 2nd rap1B, a cAMP-dependent protein kinase substrate, associates with the platelet cytoskeleton. J Biol Chem. 1990 Nov 15;265(32):19405–19408. [PubMed] [Google Scholar]
  15. Freeman R. S., Meyer A. N., Li J., Donoghue D. J. Phosphorylation of conserved serine residues does not regulate the ability of mosxe protein kinase to induce oocyte maturation or function as cytostatic factor. J Cell Biol. 1992 Feb;116(3):725–735. doi: 10.1083/jcb.116.3.725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Geuze H. J., Slot J. W., Schwartz A. L. Membranes of sorting organelles display lateral heterogeneity in receptor distribution. J Cell Biol. 1987 Jun;104(6):1715–1723. doi: 10.1083/jcb.104.6.1715. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gibbs J. B. Ras C-terminal processing enzymes--new drug targets? Cell. 1991 Apr 5;65(1):1–4. doi: 10.1016/0092-8674(91)90352-y. [DOI] [PubMed] [Google Scholar]
  18. Gorvel J. P., Chavrier P., Zerial M., Gruenberg J. rab5 controls early endosome fusion in vitro. Cell. 1991 Mar 8;64(5):915–925. doi: 10.1016/0092-8674(91)90316-q. [DOI] [PubMed] [Google Scholar]
  19. Goud B., McCaffrey M. Small GTP-binding proteins and their role in transport. Curr Opin Cell Biol. 1991 Aug;3(4):626–633. doi: 10.1016/0955-0674(91)90033-u. [DOI] [PubMed] [Google Scholar]
  20. Goud B., Zahraoui A., Tavitian A., Saraste J. Small GTP-binding protein associated with Golgi cisternae. Nature. 1990 Jun 7;345(6275):553–556. doi: 10.1038/345553a0. [DOI] [PubMed] [Google Scholar]
  21. Hancock J. F., Cadwallader K., Marshall C. J. Methylation and proteolysis are essential for efficient membrane binding of prenylated p21K-ras(B). EMBO J. 1991 Mar;10(3):641–646. doi: 10.1002/j.1460-2075.1991.tb07992.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hancock J. F., Magee A. I., Childs J. E., Marshall C. J. All ras proteins are polyisoprenylated but only some are palmitoylated. Cell. 1989 Jun 30;57(7):1167–1177. doi: 10.1016/0092-8674(89)90054-8. [DOI] [PubMed] [Google Scholar]
  23. Hancock J. F., Paterson H., Marshall C. J. A polybasic domain or palmitoylation is required in addition to the CAAX motif to localize p21ras to the plasma membrane. Cell. 1990 Oct 5;63(1):133–139. doi: 10.1016/0092-8674(90)90294-o. [DOI] [PubMed] [Google Scholar]
  24. Hata Y., Kaibuchi K., Kawamura S., Hiroyoshi M., Shirataki H., Takai Y. Enhancement of the actions of smg p21 GDP/GTP exchange protein by the protein kinase A-catalyzed phosphorylation of smg p21. J Biol Chem. 1991 Apr 5;266(10):6571–6577. [PubMed] [Google Scholar]
  25. Heald R., McKeon F. Mutations of phosphorylation sites in lamin A that prevent nuclear lamina disassembly in mitosis. Cell. 1990 May 18;61(4):579–589. doi: 10.1016/0092-8674(90)90470-y. [DOI] [PubMed] [Google Scholar]
  26. Hopkins C. R., Gibson A., Shipman M., Miller K. Movement of internalized ligand-receptor complexes along a continuous endosomal reticulum. Nature. 1990 Jul 26;346(6282):335–339. doi: 10.1038/346335a0. [DOI] [PubMed] [Google Scholar]
  27. Hunziker W., Whitney J. A., Mellman I. Selective inhibition of transcytosis by brefeldin A in MDCK cells. Cell. 1991 Nov 1;67(3):617–627. doi: 10.1016/0092-8674(91)90535-7. [DOI] [PubMed] [Google Scholar]
  28. Jans D. A., Ackermann M. J., Bischoff J. R., Beach D. H., Peters R. p34cdc2-mediated phosphorylation at T124 inhibits nuclear import of SV-40 T antigen proteins. J Cell Biol. 1991 Dec;115(5):1203–1212. doi: 10.1083/jcb.115.5.1203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Johnston P. A., Archer B. T., 3rd, Robinson K., Mignery G. A., Jahn R., Südhof T. C. rab3A attachment to the synaptic vesicle membrane mediated by a conserved polyisoprenylated carboxy-terminal sequence. Neuron. 1991 Jul;7(1):101–109. doi: 10.1016/0896-6273(91)90078-e. [DOI] [PubMed] [Google Scholar]
  30. Kinsella B. T., Maltese W. A. rab GTP-binding proteins with three different carboxyl-terminal cysteine motifs are modified in vivo by 20-carbon isoprenoids. J Biol Chem. 1992 Feb 25;267(6):3940–3945. [PubMed] [Google Scholar]
  31. Kitten G. T., Nigg E. A. The CaaX motif is required for isoprenylation, carboxyl methylation, and nuclear membrane association of lamin B2. J Cell Biol. 1991 Apr;113(1):13–23. doi: 10.1083/jcb.113.1.13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kornfeld S., Mellman I. The biogenesis of lysosomes. Annu Rev Cell Biol. 1989;5:483–525. doi: 10.1146/annurev.cb.05.110189.002411. [DOI] [PubMed] [Google Scholar]
  33. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Lapetina E. G., Lacal J. C., Reep B. R., Molina y Vedia L. A ras-related protein is phosphorylated and translocated by agonists that increase cAMP levels in human platelets. Proc Natl Acad Sci U S A. 1989 May;86(9):3131–3134. doi: 10.1073/pnas.86.9.3131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Lapetina E. G., Reep B. R. Specific binding of [alpha-32P]GTP to cytosolic and membrane-bound proteins of human platelets correlates with the activation of phospholipase C. Proc Natl Acad Sci U S A. 1987 Apr;84(8):2261–2265. doi: 10.1073/pnas.84.8.2261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Lippincott-Schwartz J., Yuan L., Tipper C., Amherdt M., Orci L., Klausner R. D. Brefeldin A's effects on endosomes, lysosomes, and the TGN suggest a general mechanism for regulating organelle structure and membrane traffic. Cell. 1991 Nov 1;67(3):601–616. doi: 10.1016/0092-8674(91)90534-6. [DOI] [PubMed] [Google Scholar]
  37. Miettinen H. M., Rose J. K., Mellman I. Fc receptor isoforms exhibit distinct abilities for coated pit localization as a result of cytoplasmic domain heterogeneity. Cell. 1989 Jul 28;58(2):317–327. doi: 10.1016/0092-8674(89)90846-5. [DOI] [PubMed] [Google Scholar]
  38. Moreno S., Nurse P. Substrates for p34cdc2: in vivo veritas? Cell. 1990 May 18;61(4):549–551. doi: 10.1016/0092-8674(90)90463-o. [DOI] [PubMed] [Google Scholar]
  39. Murray A. W., Solomon M. J., Kirschner M. W. The role of cyclin synthesis and degradation in the control of maturation promoting factor activity. Nature. 1989 May 25;339(6222):280–286. doi: 10.1038/339280a0. [DOI] [PubMed] [Google Scholar]
  40. Parker L. L., Atherton-Fessler S., Lee M. S., Ogg S., Falk J. L., Swenson K. I., Piwnica-Worms H. Cyclin promotes the tyrosine phosphorylation of p34cdc2 in a wee1+ dependent manner. EMBO J. 1991 May;10(5):1255–1263. doi: 10.1002/j.1460-2075.1991.tb08067.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Peter M., Nakagawa J., Dorée M., Labbé J. C., Nigg E. A. In vitro disassembly of the nuclear lamina and M phase-specific phosphorylation of lamins by cdc2 kinase. Cell. 1990 May 18;61(4):591–602. doi: 10.1016/0092-8674(90)90471-p. [DOI] [PubMed] [Google Scholar]
  42. Pfeffer S. R. GTP-binding proteins in intracellular transport. Trends Cell Biol. 1992 Feb;2(2):41–46. doi: 10.1016/0962-8924(92)90161-f. [DOI] [PubMed] [Google Scholar]
  43. Plutner H., Cox A. D., Pind S., Khosravi-Far R., Bourne J. R., Schwaninger R., Der C. J., Balch W. E. Rab1b regulates vesicular transport between the endoplasmic reticulum and successive Golgi compartments. J Cell Biol. 1991 Oct;115(1):31–43. doi: 10.1083/jcb.115.1.31. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Quilliam L. A., Mueller H., Bohl B. P., Prossnitz V., Sklar L. A., Der C. J., Bokoch G. M. Rap1A is a substrate for cyclic AMP-dependent protein kinase in human neutrophils. J Immunol. 1991 Sep 1;147(5):1628–1635. [PubMed] [Google Scholar]
  45. Sager P. R., Brown P. A., Berlin R. D. Analysis of transferrin recycling in mitotic and interphase HeLa cells by quantitative fluorescence microscopy. Cell. 1984 Dec;39(2 Pt 1):275–282. doi: 10.1016/0092-8674(84)90005-9. [DOI] [PubMed] [Google Scholar]
  46. 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]
  47. Sasaki T., Kaibuchi K., Kabcenell A. K., Novick P. J., Takai Y. A mammalian inhibitory GDP/GTP exchange protein (GDP dissociation inhibitor) for smg p25A is active on the yeast SEC4 protein. Mol Cell Biol. 1991 May;11(5):2909–2912. doi: 10.1128/mcb.11.5.2909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Suprynowicz F. A., Gerace L. A fractionated cell-free system for analysis of prophase nuclear disassembly. J Cell Biol. 1986 Dec;103(6 Pt 1):2073–2081. doi: 10.1083/jcb.103.6.2073. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Tuomikoski T., Felix M. A., Dorée M., Gruenberg J. Inhibition of endocytic vesicle fusion in vitro by the cell-cycle control protein kinase cdc2. Nature. 1989 Dec 21;342(6252):942–945. doi: 10.1038/342942a0. [DOI] [PubMed] [Google Scholar]
  50. Van Der Sluijs P., Hull M., Zahraoui A., Tavitian A., Goud B., Mellman I. The small GTP-binding protein rab4 is associated with early endosomes. Proc Natl Acad Sci U S A. 1991 Jul 15;88(14):6313–6317. doi: 10.1073/pnas.88.14.6313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Wandinger-Ness A., Bennett M. K., Antony C., Simons K. Distinct transport vesicles mediate the delivery of plasma membrane proteins to the apical and basolateral domains of MDCK cells. J Cell Biol. 1990 Sep;111(3):987–1000. doi: 10.1083/jcb.111.3.987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Warren G. Cell biology: mitosis and membranes. Nature. 1989 Dec 21;342(6252):857–858. doi: 10.1038/342857a0. [DOI] [PubMed] [Google Scholar]
  53. Warren G., Davoust J., Cockcroft A. Recycling of transferrin receptors in A431 cells is inhibited during mitosis. EMBO J. 1984 Oct;3(10):2217–2225. doi: 10.1002/j.1460-2075.1984.tb02119.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Woodman P. G., Mundy D. I., Cohen P., Warren G. Cell-free fusion of endocytic vesicles is regulated by phosphorylation. J Cell Biol. 1992 Jan;116(2):331–338. doi: 10.1083/jcb.116.2.331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. van der Sluijs P., Hull M., Webster P., Mâle P., Goud B., Mellman I. The small GTP-binding protein rab4 controls an early sorting event on the endocytic pathway. Cell. 1992 Sep 4;70(5):729–740. doi: 10.1016/0092-8674(92)90307-x. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES