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. 1995 Oct 24;92(22):10207–10211. doi: 10.1073/pnas.92.22.10207

Evidence for phosphatidylinositol 3-kinase as a regulator of endocytosis via activation of Rab5.

G Li 1, C D'Souza-Schorey 1, M A Barbieri 1, R L Roberts 1, A Klippel 1, L T Williams 1, P D Stahl 1
PMCID: PMC40765  PMID: 7479754

Abstract

Phosphatidylinositol (PI) 3-kinases have been implicated in several aspects of intracellular membrane trafficking, although a detailed mechanism is yet to be established. In this study we demonstrated that wortmannin, a specific inhibitor of PI 3-kinases, inhibited constitutive endocytosis of horseradish peroxidase and transferrin in BHK-21 and TRVb-1 cells. The IC50 was approximately 40 ng/ml (93 nM). In addition, wortmannin blocked the stimulation of horseradish peroxidase uptake by the small GTPase Rab5 but not the stimulation by the GTPase-defective, constitutively activated Rab5 Gln79-->Leu mutant (Rab5:Q79L), providing further evidence that PI 3-kinase activity is essential for the early endocytic process. To further investigate the mechanism, we examined the effect of wortmannin on early endosome fusion in vitro. Wortmannin decreased endosome fusion by 80% with an IC50 value similar to that in intact cells. Addition of Rab5:Q79L but not wild-type Rab5 reversed the inhibitory effect of wortmannin. Furthermore, addition of a constitutively activated PI 3-kinase but not its inactive counterpart stimulated early endosome fusion in vitro. These results strongly indicate that PI 3-kinase plays an important role in regulation of early endosome fusion, probably via activation of Rab5.

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Selected References

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  1. Arcaro A., Wymann M. P. Wortmannin is a potent phosphatidylinositol 3-kinase inhibitor: the role of phosphatidylinositol 3,4,5-trisphosphate in neutrophil responses. Biochem J. 1993 Dec 1;296(Pt 2):297–301. doi: 10.1042/bj2960297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Auger K. R., Carpenter C. L., Cantley L. C., Varticovski L. Phosphatidylinositol 3-kinase and its novel product, phosphatidylinositol 3-phosphate, are present in Saccharomyces cerevisiae. J Biol Chem. 1989 Dec 5;264(34):20181–20184. [PubMed] [Google Scholar]
  3. Barbieri M. A., Li G., Colombo M. I., Stahl P. D. Rab5, an early acting endosomal GTPase, supports in vitro endosome fusion without GTP hydrolysis. J Biol Chem. 1994 Jul 22;269(29):18720–18722. [PubMed] [Google Scholar]
  4. 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]
  5. D'Souza-Schorey C., Li G., Colombo M. I., Stahl P. D. A regulatory role for ARF6 in receptor-mediated endocytosis. Science. 1995 Feb 24;267(5201):1175–1178. doi: 10.1126/science.7855600. [DOI] [PubMed] [Google Scholar]
  6. Diaz R., Mayorga L., Stahl P. In vitro fusion of endosomes following receptor-mediated endocytosis. J Biol Chem. 1988 May 5;263(13):6093–6100. [PubMed] [Google Scholar]
  7. 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]
  8. Gould G. W., Jess T. J., Andrews G. C., Herbst J. J., Plevin R. J., Gibbs E. M. Evidence for a role of phosphatidylinositol 3-kinase in the regulation of glucose transport in Xenopus oocytes. J Biol Chem. 1994 Oct 28;269(43):26622–26625. [PubMed] [Google Scholar]
  9. Herman P. K., Emr S. D. Characterization of VPS34, a gene required for vacuolar protein sorting and vacuole segregation in Saccharomyces cerevisiae. Mol Cell Biol. 1990 Dec;10(12):6742–6754. doi: 10.1128/mcb.10.12.6742. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hoffenberg S., Sanford J. C., Liu S., Daniel D. S., Tuvin M., Knoll B. J., Wessling-Resnick M., Dickey B. F. Biochemical and functional characterization of a recombinant GTPase, Rab5, and two of its mutants. J Biol Chem. 1995 Mar 10;270(10):5048–5056. doi: 10.1074/jbc.270.10.5048. [DOI] [PubMed] [Google Scholar]
  11. Horazdovsky B. F., Busch G. R., Emr S. D. VPS21 encodes a rab5-like GTP binding protein that is required for the sorting of yeast vacuolar proteins. EMBO J. 1994 Mar 15;13(6):1297–1309. doi: 10.1002/j.1460-2075.1994.tb06382.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hu Q., Klippel A., Muslin A. J., Fantl W. J., Williams L. T. Ras-dependent induction of cellular responses by constitutively active phosphatidylinositol-3 kinase. Science. 1995 Apr 7;268(5207):100–102. doi: 10.1126/science.7701328. [DOI] [PubMed] [Google Scholar]
  13. Joly M., Kazlauskas A., Fay F. S., Corvera S. Disruption of PDGF receptor trafficking by mutation of its PI-3 kinase binding sites. Science. 1994 Feb 4;263(5147):684–687. doi: 10.1126/science.8303278. [DOI] [PubMed] [Google Scholar]
  14. Kanai F., Ito K., Todaka M., Hayashi H., Kamohara S., Ishii K., Okada T., Hazeki O., Ui M., Ebina Y. Insulin-stimulated GLUT4 translocation is relevant to the phosphorylation of IRS-1 and the activity of PI3-kinase. Biochem Biophys Res Commun. 1993 Sep 15;195(2):762–768. doi: 10.1006/bbrc.1993.2111. [DOI] [PubMed] [Google Scholar]
  15. Kapeller R., Cantley L. C. Phosphatidylinositol 3-kinase. Bioessays. 1994 Aug;16(8):565–576. doi: 10.1002/bies.950160810. [DOI] [PubMed] [Google Scholar]
  16. Li G., Barbieri M. A., Colombo M. I., Stahl P. D. Structural features of the GTP-binding defective Rab5 mutants required for their inhibitory activity on endocytosis. J Biol Chem. 1994 May 20;269(20):14631–14635. [PubMed] [Google Scholar]
  17. Li G., Stahl P. D. Structure-function relationship of the small GTPase rab5. J Biol Chem. 1993 Nov 15;268(32):24475–24480. [PubMed] [Google Scholar]
  18. Okada T., Kawano Y., Sakakibara T., Hazeki O., Ui M. Essential role of phosphatidylinositol 3-kinase in insulin-induced glucose transport and antilipolysis in rat adipocytes. Studies with a selective inhibitor wortmannin. J Biol Chem. 1994 Feb 4;269(5):3568–3573. [PubMed] [Google Scholar]
  19. Panayotou G., Waterfield M. D. Phosphatidyl-inositol 3-kinase: a key enzyme in diverse signalling processes. Trends Cell Biol. 1992 Dec;2(12):358–360. doi: 10.1016/0962-8924(92)90042-l. [DOI] [PubMed] [Google Scholar]
  20. Schu P. V., Takegawa K., Fry M. J., Stack J. H., Waterfield M. D., Emr S. D. Phosphatidylinositol 3-kinase encoded by yeast VPS34 gene essential for protein sorting. Science. 1993 Apr 2;260(5104):88–91. doi: 10.1126/science.8385367. [DOI] [PubMed] [Google Scholar]
  21. Stahl P., Schlesinger P. H., Sigardson E., Rodman J. S., Lee Y. C. Receptor-mediated pinocytosis of mannose glycoconjugates by macrophages: characterization and evidence for receptor recycling. Cell. 1980 Jan;19(1):207–215. doi: 10.1016/0092-8674(80)90402-x. [DOI] [PubMed] [Google Scholar]
  22. Stenmark H., Parton R. G., Steele-Mortimer O., Lütcke A., Gruenberg J., Zerial M. Inhibition of rab5 GTPase activity stimulates membrane fusion in endocytosis. EMBO J. 1994 Mar 15;13(6):1287–1296. doi: 10.1002/j.1460-2075.1994.tb06381.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Stephens L., Cooke F. T., Walters R., Jackson T., Volinia S., Gout I., Waterfield M. D., Hawkins P. T. Characterization of a phosphatidylinositol-specific phosphoinositide 3-kinase from mammalian cells. Curr Biol. 1994 Mar 1;4(3):203–214. doi: 10.1016/s0960-9822(00)00049-x. [DOI] [PubMed] [Google Scholar]
  24. Stephens L., Smrcka A., Cooke F. T., Jackson T. R., Sternweis P. C., Hawkins P. T. A novel phosphoinositide 3 kinase activity in myeloid-derived cells is activated by G protein beta gamma subunits. Cell. 1994 Apr 8;77(1):83–93. doi: 10.1016/0092-8674(94)90237-2. [DOI] [PubMed] [Google Scholar]
  25. Thelen M., Wymann M. P., Langen H. Wortmannin binds specifically to 1-phosphatidylinositol 3-kinase while inhibiting guanine nucleotide-binding protein-coupled receptor signaling in neutrophil leukocytes. Proc Natl Acad Sci U S A. 1994 May 24;91(11):4960–4964. doi: 10.1073/pnas.91.11.4960. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Trowbridge I. S., Collawn J. F., Hopkins C. R. Signal-dependent membrane protein trafficking in the endocytic pathway. Annu Rev Cell Biol. 1993;9:129–161. doi: 10.1146/annurev.cb.09.110193.001021. [DOI] [PubMed] [Google Scholar]
  27. Yano H., Nakanishi S., Kimura K., Hanai N., Saitoh Y., Fukui Y., Nonomura Y., Matsuda Y. Inhibition of histamine secretion by wortmannin through the blockade of phosphatidylinositol 3-kinase in RBL-2H3 cells. J Biol Chem. 1993 Dec 5;268(34):25846–25856. [PubMed] [Google Scholar]

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