Two Distinct Functions for PI3-kinases in Macropinocytosis

Overview

Journal J Cell Sci

Specialty Cell Biology

Date 2013 Jul 12

PMID 23843627

Citations 65

Authors

Oliver Hoeller

Parvin Bolourani

Jonathan Clark

Len R Stephens

Phillip T Hawkins

Orion D Weiner

Gerald Weeks

Robert R Kay

Affiliations

Soon will be listed here.

Abstract

Class-1 PI3-kinases are major regulators of the actin cytoskeleton, whose precise contributions to chemotaxis, phagocytosis and macropinocytosis remain unresolved. We used systematic genetic ablation to examine this question in growing Dictyostelium cells. Mass spectroscopy shows that a quintuple mutant lacking the entire genomic complement of class-1 PI3-kinases retains only 10% of wild-type PtdIns(3,4,5)P3 levels. Chemotaxis to folate and phagocytosis of bacteria proceed normally in the quintuple mutant but macropinocytosis is abolished. In this context PI3-kinases show specialized functions, only one of which is directly linked to gross PtdIns(3,4,5)P3 levels: macropinosomes originate in patches of PtdIns(3,4,5)P3, with associated F-actin-rich ruffles, both of which depend on PI3-kinase 1/2 (PI3K1/2) but not PI3K4, whereas conversion of ruffles into vesicles requires PI3K4. A biosensor derived from the Ras-binding domain of PI3K1 suggests that Ras is activated throughout vesicle formation. Binding assays show that RasG and RasS interact most strongly with PI3K1/2 and PI3K4, and single mutants of either Ras have severe macropinocytosis defects. Thus, the fundamental function of PI3-kinases in growing Dictyostelium cells is in macropinocytosis where they have two distinct functions, supported by at least two separate Ras proteins.

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References

Rupper A, Lee K, Knecht D, Cardelli J . Sequential activities of phosphoinositide 3-kinase, PKB/Aakt, and Rab7 during macropinosome formation in Dictyostelium. Mol Biol Cell. 2001; 12(9):2813-24. PMC: 59715. DOI: 10.1091/mbc.12.9.2813. View

Cox D, Tseng C, Bjekic G, Greenberg S . A requirement for phosphatidylinositol 3-kinase in pseudopod extension. J Biol Chem. 1999; 274(3):1240-7. DOI: 10.1074/jbc.274.3.1240. View

Cai H, Das S, Kamimura Y, Long Y, Parent C, Devreotes P . Ras-mediated activation of the TORC2-PKB pathway is critical for chemotaxis. J Cell Biol. 2010; 190(2):233-45. PMC: 2930282. DOI: 10.1083/jcb.201001129. View

Peracino B, Balest A, Bozzaro S . Phosphoinositides differentially regulate bacterial uptake and Nramp1-induced resistance to Legionella infection in Dictyostelium. J Cell Sci. 2010; 123(Pt 23):4039-51. DOI: 10.1242/jcs.072124. View

Buczynski G, Grove B, Nomura A, Kleve M, Bush J, Firtel R . Inactivation of two Dictyostelium discoideum genes, DdPIK1 and DdPIK2, encoding proteins related to mammalian phosphatidylinositide 3-kinases, results in defects in endocytosis, lysosome to postlysosome transport, and actin cytoskeleton organization. J Cell Biol. 1997; 136(6):1271-86. PMC: 2132510. DOI: 10.1083/jcb.136.6.1271. View

Fischer M, Haase I, Simmeth E, Gerisch G, Muller-Taubenberger A . A brilliant monomeric red fluorescent protein to visualize cytoskeleton dynamics in Dictyostelium. FEBS Lett. 2004; 577(1-2):227-32. DOI: 10.1016/j.febslet.2004.09.084. View

de Lozanne A, Spudich J . Disruption of the Dictyostelium myosin heavy chain gene by homologous recombination. Science. 1987; 236(4805):1086-91. DOI: 10.1126/science.3576222. View

Eichinger L, Pachebat J, Glockner G, Rajandream M, Sucgang R, Berriman M . The genome of the social amoeba Dictyostelium discoideum. Nature. 2005; 435(7038):43-57. PMC: 1352341. DOI: 10.1038/nature03481. View

Sasaki A, Janetopoulos C, Lee S, Charest P, Takeda K, Sundheimer L . G protein-independent Ras/PI3K/F-actin circuit regulates basic cell motility. J Cell Biol. 2007; 178(2):185-91. PMC: 2064438. DOI: 10.1083/jcb.200611138. View

10.

Bolourani P, Spiegelman G, Weeks G . Delineation of the roles played by RasG and RasC in cAMP-dependent signal transduction during the early development of Dictyostelium discoideum. Mol Biol Cell. 2006; 17(10):4543-50. PMC: 1635367. DOI: 10.1091/mbc.e05-11-1019. View

11.

Bondeva T, Pirola L, Rubio I, Wetzker R, Wymann M . Bifurcation of lipid and protein kinase signals of PI3Kgamma to the protein kinases PKB and MAPK. Science. 1998; 282(5387):293-6. DOI: 10.1126/science.282.5387.293. View

12.

Chubb J, Wilkins A, Thomas G, Insall R . The Dictyostelium RasS protein is required for macropinocytosis, phagocytosis and the control of cell movement. J Cell Sci. 2000; 113 ( Pt 4):709-19. DOI: 10.1242/jcs.113.4.709. View

13.

Pacold M, Suire S, Perisic O, Davis C, WALKER E, Hawkins P . Crystal structure and functional analysis of Ras binding to its effector phosphoinositide 3-kinase gamma. Cell. 2001; 103(6):931-43. DOI: 10.1016/s0092-8674(00)00196-3. View

14.

Willingham M, Haigler H, Fitzgerald D, Gallo M, Rutherford A, Pastan I . The morphologic pathway of binding and internalization of epidermal growth factor in cultured cells. Studies on A431, KB, and 3T3 cells, using multiple methods of labelling. Exp Cell Res. 1983; 146(1):163-75. DOI: 10.1016/0014-4827(83)90334-8. View

15.

Lee E, Knecht D . Visualization of actin dynamics during macropinocytosis and exocytosis. Traffic. 2002; 3(3):186-92. DOI: 10.1034/j.1600-0854.2002.030304.x. View

16.

Suire S, Hawkins P, Stephens L . Activation of phosphoinositide 3-kinase gamma by Ras. Curr Biol. 2002; 12(13):1068-75. DOI: 10.1016/s0960-9822(02)00933-8. View

17.

Tuxworth R, Cheetham J, Machesky L, Spiegelmann G, Weeks G, Insall R . Dictyostelium RasG is required for normal motility and cytokinesis, but not growth. J Cell Biol. 1997; 138(3):605-14. PMC: 2141629. DOI: 10.1083/jcb.138.3.605. View

18.

Zhou K, Takegawa K, Emr S, Firtel R . A phosphatidylinositol (PI) kinase gene family in Dictyostelium discoideum: biological roles of putative mammalian p110 and yeast Vps34p PI 3-kinase homologs during growth and development. Mol Cell Biol. 1995; 15(10):5645-56. PMC: 230815. DOI: 10.1128/MCB.15.10.5645. View

19.

Kortholt A, Kataria R, Keizer-Gunnink I, van Egmond W, Khanna A, van Haastert P . Dictyostelium chemotaxis: essential Ras activation and accessory signalling pathways for amplification. EMBO Rep. 2011; 12(12):1273-9. PMC: 3245697. DOI: 10.1038/embor.2011.210. View

20.

Khosla M, Spiegelman G, Weeks G . The effect of the disruption of a gene encoding a PI4 kinase on the developmental defect exhibited by Dictyostelium rasC(-) cells. Dev Biol. 2005; 284(2):412-20. DOI: 10.1016/j.ydbio.2005.05.037. View