title: Modulation of Rab5 and Rab7 recruitment to phagosomes by phosphatidylinositol 3-kinase.
abstract: Phagosomal biogenesis is central for microbial killing and antigen presentation by leukocytes. However, the molecular mechanisms governing phagosome maturation are poorly understood. We analyzed the role and site of action of phosphatidylinositol 3-kinases (PI3K) and of Rab GTPases in maturation using both professional and engineered phagocytes. Rab5, which is recruited rapidly and transiently to the phagosome, was found to be essential for the recruitment of Rab7 and for progression to phagolysosomes. Similarly, functional PI3K is required for successful maturation. Remarkably, inhibition of PI3K did not preclude Rab5 recruitment to phagosomes but instead enhanced and prolonged it. Moreover, in the presence of PI3K inhibitors Rab5 was found to be active, as deduced from measurements of early endosome antigen 1 binding and by photobleaching recovery determinations. Though their ability to fuse with late endosomes and lysosomes was virtually eliminated by wortmannin, phagosomes nevertheless recruited a sizable amount of Rab7. Moreover, Rab7 recruited to phagosomes in the presence of PI3K antagonists retained the ability to bind its effector, Rab7-interacting lysosomal protein, suggesting that it is functionally active. These findings imply that (i) dissociation of Rab5 from phagosomes requires products of PI3K, (ii) PI3K-dependent effectors of Rab5 are not essential for the recruitment of Rab7 by phagosomes, and (iii) recruitment and activation of Rab7 are insufficient to induce fusion of phagosomes with late endosomes and lysosomes. Accordingly, transfection of constitutively active Rab7 did not bypass the block of phagolysosome formation exerted by wortmannin. We propose that Rab5 activates both PI3K-dependent and PI3K-independent effectors that act in parallel to promote phagosome maturation.
authors.affiliation: Cell Biology Program, Hospital for Sick Children and Department of Biochemistry, University of Toronto, Ontario M5G 1X8, Canada.
authors.forename: Otilia V
authors.initials: OV
authors.lastname: Vieira
authors.forename: Cecilia
authors.initials: C
authors.lastname: Bucci
authors.forename: Rene E
authors.initials: RE
authors.lastname: Harrison
authors.forename: William S
authors.initials: WS
authors.lastname: Trimble
authors.forename: Letizia
authors.initials: L
authors.lastname: Lanzetti
authors.forename: Jean
authors.initials: J
authors.lastname: Gruenberg
authors.forename: Alan D
authors.initials: AD
authors.lastname: Schreiber
authors.forename: Philip D
authors.initials: PD
authors.lastname: Stahl
authors.forename: Sergio
authors.initials: S
authors.lastname: Grinstein
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D016466:CHO Cells / Q000187:drug effects; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D006224:Cricetinae; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D036681:Fluorescence Recovery After Photobleaching; D020690:GTPase-Activating Proteins; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D008264:Macrophages / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008561:Membrane Fusion / Q000187:drug effects; D051379:Mice; D015514:Oncogene Proteins, Fusion / Q000235:genetics; D010053:Ovary / Q000166:cytology / Q000378:metabolism; D010588:Phagosomes / Q000187:drug effects / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D000077191:Wortmannin; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020696:rab5 GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D000090802:rab7 GTP-Binding Proteins
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000730:Androstadienes; D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D020690:GTPase-Activating Proteins; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D015514:Oncogene Proteins, Fusion; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011993:Recombinant Fusion Proteins; D000077191:Wortmannin; D020691:rab GTP-Binding Proteins; D020696:rab5 GTP-Binding Proteins; D000090802:rab7 GTP-Binding Proteins; C498920:Rilp protein, mouse; C100665:USP6NL protein, human; C000716463:rab7 GTP-binding proteins, human; C000716465:rab7 GTP-binding proteins, mouse