Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1996 Dec 1;135(5):1249–1260. doi: 10.1083/jcb.135.5.1249

A role for phosphoinositide 3-kinase in the completion of macropinocytosis and phagocytosis by macrophages

PMCID: PMC2121091  PMID: 8947549

Abstract

Phosphoinositide 3-kinase (PI 3-kinase) has been implicated in growth factor signal transduction and vesicular membrane traffic. It is thought to mediate the earliest steps leading from ligation of cell surface receptors to increased cell surface ruffling. We show here that inhibitors of PI 3-kinase inhibit endocytosis in macrophages, not by interfering with the initiation of the process but rather by preventing its completion. Consistent with earlier studies, the inhibitors wortmannin and LY294002 inhibited fluid-phase pinocytosis and Fc receptor-mediated phagocytosis, but they had little effect on the receptor-mediated endocytosis of diI-labeled, acetylated, low density lipoprotein. Large solute probes of endocytosis reported greater inhibition by wortmannin than smaller probes did, indicating that macropinocytosis was affected more than micropinocytosis. Since macropinocytosis and phagocytosis are actin-mediated processes, we expected that their inhibition by wortmannin resulted from deficient signaling from macrophage colony-stimulating factor (M-CSF) receptors or Fc receptors to the actin cytoskeleton. However, video microscopy showed cell surface ruffling in wortmannin-treated cells, and increased ruffling after addition of M-CSF or phorbol myristate acetate. Quantitative measurements of video data reported slightly diminished ruffling in wortmannin-treated cells. Remarkably, the ruffles that formed in wortmannin-treated macrophages all receded into the cytoplasm without closing into macropinosomes. Similarly, wortmannin and LY294002 did not inhibit the extension of actin-rich pseudopodia along IgG- opsonized sheep erythrocytes, but instead prevented them from closing into phagosomes. These findings indicate that PI 3-kinase is not necessary for receptor-mediated stimulation of pseudopod extension, but rather functions in the closure of macropinosomes and phagosomes into intracellular organelles.

Full Text

The Full Text of this article is available as a PDF (1.4 MB).

Selected References

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

  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. Baggiolini M., Dewald B., Schnyder J., Ruch W., Cooper P. H., Payne T. G. Inhibition of the phagocytosis-induced respiratory burst by the fungal metabolite wortmannin and some analogues. Exp Cell Res. 1987 Apr;169(2):408–418. doi: 10.1016/0014-4827(87)90201-1. [DOI] [PubMed] [Google Scholar]
  3. Barker S. A., Caldwell K. K., Hall A., Martinez A. M., Pfeiffer J. R., Oliver J. M., Wilson B. S. Wortmannin blocks lipid and protein kinase activities associated with PI 3-kinase and inhibits a subset of responses induced by Fc epsilon R1 cross-linking. Mol Biol Cell. 1995 Sep;6(9):1145–1158. doi: 10.1091/mbc.6.9.1145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Berthiaume E. P., Medina C., Swanson J. A. Molecular size-fractionation during endocytosis in macrophages. J Cell Biol. 1995 May;129(4):989–998. doi: 10.1083/jcb.129.4.989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brown W. J., DeWald D. B., Emr S. D., Plutner H., Balch W. E. Role for phosphatidylinositol 3-kinase in the sorting and transport of newly synthesized lysosomal enzymes in mammalian cells. J Cell Biol. 1995 Aug;130(4):781–796. doi: 10.1083/jcb.130.4.781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cantley L. C., Auger K. R., Carpenter C., Duckworth B., Graziani A., Kapeller R., Soltoff S. Oncogenes and signal transduction. Cell. 1991 Jan 25;64(2):281–302. doi: 10.1016/0092-8674(91)90639-g. [DOI] [PubMed] [Google Scholar]
  7. Carlberg K., Tapley P., Haystead C., Rohrschneider L. The role of kinase activity and the kinase insert region in ligand-induced internalization and degradation of the c-fms protein. EMBO J. 1991 Apr;10(4):877–883. doi: 10.1002/j.1460-2075.1991.tb08020.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Clague M. J., Thorpe C., Jones A. T. Phosphatidylinositol 3-kinase regulation of fluid phase endocytosis. FEBS Lett. 1995 Jul 3;367(3):272–274. doi: 10.1016/0014-5793(95)00576-u. [DOI] [PubMed] [Google Scholar]
  9. Cooper P. H., Mayer P., Baggiolini M. Stimulation of phagocytosis in bone marrow-derived mouse macrophages by bacterial lipopolysaccharide: correlation with biochemical and functional parameters. J Immunol. 1984 Aug;133(2):913–922. [PubMed] [Google Scholar]
  10. Davidson H. W. Wortmannin causes mistargeting of procathepsin D. evidence for the involvement of a phosphatidylinositol 3-kinase in vesicular transport to lysosomes. J Cell Biol. 1995 Aug;130(4):797–805. doi: 10.1083/jcb.130.4.797. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. De Camilli P., Emr S. D., McPherson P. S., Novick P. Phosphoinositides as regulators in membrane traffic. Science. 1996 Mar 15;271(5255):1533–1539. doi: 10.1126/science.271.5255.1533. [DOI] [PubMed] [Google Scholar]
  12. Fukuda S., Horiuchi S., Tomita K., Murakami M., Morino Y., Takahashi K. Acetylated low-density lipoprotein is endocytosed through coated pits by rat peritoneal macrophages. Virchows Arch B Cell Pathol Incl Mol Pathol. 1986;52(1):1–13. doi: 10.1007/BF02889945. [DOI] [PubMed] [Google Scholar]
  13. Goldstein J. L., Ho Y. K., Basu S. K., Brown M. S. Binding site on macrophages that mediates uptake and degradation of acetylated low density lipoprotein, producing massive cholesterol deposition. Proc Natl Acad Sci U S A. 1979 Jan;76(1):333–337. doi: 10.1073/pnas.76.1.333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gout I., Dhand R., Hiles I. D., Fry M. J., Panayotou G., Das P., Truong O., Totty N. F., Hsuan J., Booker G. W. The GTPase dynamin binds to and is activated by a subset of SH3 domains. Cell. 1993 Oct 8;75(1):25–36. [PubMed] [Google Scholar]
  15. Greenberg S. Signal transduction of phagocytosis. Trends Cell Biol. 1995 Mar;5(3):93–99. doi: 10.1016/s0962-8924(00)88957-6. [DOI] [PubMed] [Google Scholar]
  16. Joly M., Kazlauskas A., Corvera S. Phosphatidylinositol 3-kinase activity is required at a postendocytic step in platelet-derived growth factor receptor trafficking. J Biol Chem. 1995 Jun 2;270(22):13225–13230. doi: 10.1074/jbc.270.22.13225. [DOI] [PubMed] [Google Scholar]
  17. Kodama T., Freeman M., Rohrer L., Zabrecky J., Matsudaira P., Krieger M. Type I macrophage scavenger receptor contains alpha-helical and collagen-like coiled coils. Nature. 1990 Feb 8;343(6258):531–535. doi: 10.1038/343531a0. [DOI] [PubMed] [Google Scholar]
  18. Kotani K., Yonezawa K., Hara K., Ueda H., Kitamura Y., Sakaue H., Ando A., Chavanieu A., Calas B., Grigorescu F. Involvement of phosphoinositide 3-kinase in insulin- or IGF-1-induced membrane ruffling. EMBO J. 1994 May 15;13(10):2313–2321. doi: 10.1002/j.1460-2075.1994.tb06515.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Li G., D'Souza-Schorey C., Barbieri M. A., Roberts R. L., Klippel A., Williams L. T., Stahl P. D. Evidence for phosphatidylinositol 3-kinase as a regulator of endocytosis via activation of Rab5. Proc Natl Acad Sci U S A. 1995 Oct 24;92(22):10207–10211. doi: 10.1073/pnas.92.22.10207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Liscovitch M., Cantley L. C. Signal transduction and membrane traffic: the PITP/phosphoinositide connection. Cell. 1995 Jun 2;81(5):659–662. doi: 10.1016/0092-8674(95)90525-1. [DOI] [PubMed] [Google Scholar]
  21. Nakanishi S., Catt K. J., Balla T. A wortmannin-sensitive phosphatidylinositol 4-kinase that regulates hormone-sensitive pools of inositolphospholipids. Proc Natl Acad Sci U S A. 1995 Jun 6;92(12):5317–5321. doi: 10.1073/pnas.92.12.5317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ninomiya N., Hazeki K., Fukui Y., Seya T., Okada T., Hazeki O., Ui M. Involvement of phosphatidylinositol 3-kinase in Fc gamma receptor signaling. J Biol Chem. 1994 Sep 9;269(36):22732–22737. [PubMed] [Google Scholar]
  23. Racoosin E. L., Swanson J. A. M-CSF-induced macropinocytosis increases solute endocytosis but not receptor-mediated endocytosis in mouse macrophages. J Cell Sci. 1992 Aug;102(Pt 4):867–880. doi: 10.1242/jcs.102.4.867. [DOI] [PubMed] [Google Scholar]
  24. Racoosin E. L., Swanson J. A. Macrophage colony-stimulating factor (rM-CSF) stimulates pinocytosis in bone marrow-derived macrophages. J Exp Med. 1989 Nov 1;170(5):1635–1648. doi: 10.1084/jem.170.5.1635. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Shepherd P. R., Reaves B. J., Davidson H. W. Phosphoinositide 3-kinases and membrane traffic. Trends Cell Biol. 1996 Mar;6(3):92–97. doi: 10.1016/0962-8924(96)80998-6. [DOI] [PubMed] [Google Scholar]
  27. Shepherd P. R., Soos M. A., Siddle K. Inhibitors of phosphoinositide 3-kinase block exocytosis but not endocytosis of transferrin receptors in 3T3-L1 adipocytes. Biochem Biophys Res Commun. 1995 Jun 15;211(2):535–539. doi: 10.1006/bbrc.1995.1846. [DOI] [PubMed] [Google Scholar]
  28. Shpetner H., Joly M., Hartley D., Corvera S. Potential sites of PI-3 kinase function in the endocytic pathway revealed by the PI-3 kinase inhibitor, wortmannin. J Cell Biol. 1996 Feb;132(4):595–605. doi: 10.1083/jcb.132.4.595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Spiro D. J., Boll W., Kirchhausen T., Wessling-Resnick M. Wortmannin alters the transferrin receptor endocytic pathway in vivo and in vitro. Mol Biol Cell. 1996 Mar;7(3):355–367. doi: 10.1091/mbc.7.3.355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Swanson J. A., Baer S. C. Phagocytosis by zippers and triggers. Trends Cell Biol. 1995 Mar;5(3):89–93. doi: 10.1016/s0962-8924(00)88956-4. [DOI] [PubMed] [Google Scholar]
  31. Swanson J. A. Phorbol esters stimulate macropinocytosis and solute flow through macrophages. J Cell Sci. 1989 Sep;94(Pt 1):135–142. doi: 10.1242/jcs.94.1.135. [DOI] [PubMed] [Google Scholar]
  32. Swanson J. A., Watts C. Macropinocytosis. Trends Cell Biol. 1995 Nov;5(11):424–428. doi: 10.1016/s0962-8924(00)89101-1. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. Vlahos C. J., Matter W. F., Hui K. Y., Brown R. F. A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). J Biol Chem. 1994 Feb 18;269(7):5241–5248. [PubMed] [Google Scholar]
  35. Wennström S., Hawkins P., Cooke F., Hara K., Yonezawa K., Kasuga M., Jackson T., Claesson-Welsh L., Stephens L. Activation of phosphoinositide 3-kinase is required for PDGF-stimulated membrane ruffling. Curr Biol. 1994 May 1;4(5):385–393. doi: 10.1016/s0960-9822(00)00087-7. [DOI] [PubMed] [Google Scholar]
  36. Wright S. D., Silverstein S. C. Tumor-promoting phorbol esters stimulate C3b and C3b' receptor-mediated phagocytosis in cultured human monocytes. J Exp Med. 1982 Oct 1;156(4):1149–1164. doi: 10.1084/jem.156.4.1149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Yano H., Agatsuma T., Nakanishi S., Saitoh Y., Fukui Y., Nonomura Y., Matsuda Y. Biochemical and pharmacological studies with KT7692 and LY294002 on the role of phosphatidylinositol 3-kinase in Fc epsilon RI-mediated signal transduction. Biochem J. 1995 Nov 15;312(Pt 1):145–150. doi: 10.1042/bj3120145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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]
  39. van der Bliek A. M., Redelmeier T. E., Damke H., Tisdale E. J., Meyerowitz E. M., Schmid S. L. Mutations in human dynamin block an intermediate stage in coated vesicle formation. J Cell Biol. 1993 Aug;122(3):553–563. doi: 10.1083/jcb.122.3.553. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES