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Regulates Phagolysosome Biogenesis the Protein Tyrosine Phosphatase SHP-1 The Protein Tyrosine Phosphatase SHP-1 Regulates Phagolysosome Biogenesis Carolina P. Gómez, Marina Tiemi Shio, Pascale Duplay, Martin Olivier and Albert Descoteaux This information is current as of September 29, 2021. J Immunol 2012; 189:2203-2210; Prepublished online 23 July 2012; doi: 10.4049/jimmunol.1103021 http://www.jimmunol.org/content/189/5/2203 Downloaded from References This article cites 70 articles, 34 of which you can access for free at: http://www.jimmunol.org/content/189/5/2203.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 29, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology The Protein Tyrosine Phosphatase SHP-1 Regulates Phagolysosome Biogenesis Carolina P. Go´mez,*,† Marina Tiemi Shio,†,‡,x Pascale Duplay,* Martin Olivier,†,‡,x and Albert Descoteaux*,† The process of phagocytosis and phagosome maturation involves the recruitment of effector proteins that participate in phagosome formation and in the acidification and/or fusion with various endocytic vesicles. In the current study, we investigated the role of the Src homology region 2 domain-containing phosphatase 1 (SHP-1) in phagolysosome biogenesis. To this end, we used immortalized bone marrow macrophages derived from SHP-1–deficient motheaten mice and their wild-type littermates. We found that SHP-1 is recruited early and remains present on phagosomes for up to 4 h postphagocytosis. Using confocal immunofluorescence micros- copy and Western blot analyses on purified phagosome extracts, we observed an impaired recruitment of lysosomal-associated membrane protein 1 in SHP-1–deficient macrophages. Moreover, Western blot analyses revealed that whereas the 51-kDa pro- Downloaded from cathepsin D is recruited to phagosomes, it is not processed into the 46-kDa cathepsin D in the absence of SHP-1, suggesting a defect in acidification. Using the lysosomotropic agent LysoTracker as an indicator of phagosomal pH, we obtained evidence that in the absence of SHP-1, phagosome acidification was impaired. Taken together, these results are consistent with a role for SHP-1 in the regulation of signaling or membrane fusion events involved in phagolysosome biogenesis. The Journal of Immunology, 2012, 189: 2203–2210. http://www.jimmunol.org/ hagocytosis is the process by which cells internalize large (6). Through these interactions, phagosomes acidify and sequen- particulate materials from their milieu. In contrast to en- tially acquire an array of hydrolases culminating in the generation P docytosis, phagocytosis involves clathrin-independent mecha- of a highly hydrolytic environment (7). This process takes place nisms and requires actin polymerization. Internalization is triggered rapidly, as within a few hours newly formed phagosomes acquire by the interaction of ligands on the target particles with surface phagolysosomal functionality (8). receptors of phagocytic cells. The nascent phagosome is formed Tyrosine (Tyr) phosphorylation is a critical mechanism for the to a large extent by invagination of the plasma membrane (1). control of numerous physiological processes in eukaryotes (9), The particles are surrounded by pseudopods and, following inter- including cytoskeletal function, signal transduction, intracellular by guest on September 29, 2021 nalization, they end up in a vacuole, the phagosome. The newly traffic, and immune defense (9, 10). Phosphotyrosine signaling formed phagosome rapidly matures into a phagolysosome where is regulated by the dynamic interplay of three distinct functional the internalized material is degraded. Phagolysosome biogenesis modules, as follows: protein tyrosine kinases, protein tyrosine is a dynamic process, requiring the coordinated action of multiple phosphatases (PTP), and Src homology 2 domains (11). In met- signaling molecules that regulate the interactions between the azoans, the multiple possible combinations of these modules il- phagosome and the cytoskeleton, and the endosomal/lysosomal lustrate the complexity of phosphotyrosine signaling, which can compartments (2–5). These interactions are transient and are regulate cellular functions either positively or negatively. A recent characterized by the formation of small fusion pores between the large-scale quantitative proteomic and phosphoproteomic analysis two organelles, allowing the exchange of membrane and contents revealed that Tyr phosphorylation sites are overrepresented in phagosomes (12), as compared with global phosphoproteome (13). This finding suggested that Tyr phosphorylation plays an impor- *Institut National de la Recherche Scientifique–Institut Armand-Frappier, Laval, tant role in phagosome signaling. Given their role in controlling † Quebec H7V 1B7, Canada; Center for Host-Parasite Interactions, Laval, Quebec Tyr phosphorylation levels (10, 11), PTPs are expected to partic- H7V 1B7, Canada; ‡Department of Medicine, Centre for the Study of Host Resis- tance, Research Institute of McGill University Health Centre, Montre´al, Quebec H3A ipate in the regulation of phagosome function. Interestingly, x 2B4, Canada; and Department of Microbiology and Immunology, McGill University, characterization of the nonreceptor PTP MEG2 in neutrophils ´ Montreal, Quebec H3A 2B4, Canada revealed that it is present on the cytoplasmic face of secretory Received for publication October 19, 2011. Accepted for publication June 25, 2012. vesicles, and is recruited to nascent phagosomes (14). A physio- This work was supported by the Natural Science and Engineering Research Council logical substrate for PTP-MEG2 is the vesicle fusion protein N- of Canada (to A.D.) and by a New Initiative Fund from the Center for Host-Parasite Interactions (to A.D. and M.O.). A.D. is the holder of a Canada Research Chair, and ethylmaleimide–sensitive factor (NSF), the dephosphorylation of C.P.G. was partly supported by a studentship from the Fondation Armand-Frappier. which stimulates the homotypic fusion of secretory vesicles (15). Address correspondence and reprint requests to Dr. Albert Descoteaux, Institut Na- Additional PTP-MEG2–interacting proteins are associated with tional de la Recherche Scientifique–Institut Armand-Frappier, 531 Boulevard des vesicle traffic, small GTPases, and lipid interaction (16). However, Prairies, Laval, QC H7V 1B7, Canada. E-mail address: [email protected] a role for PTP-MEG2 or any other PTPs in the regulation of phag- Abbreviations used in this article: BMM, bone marrow-derived macrophage; EEA1, early endosome Ag 1; LAMP-1, lysosomal-associated membrane protein 1; LT, osome maturation remains to be demonstrated. LysoTracker; NSF, N-ethylmaleimide–sensitive factor; PTP, protein tyrosine phos- The nonreceptor PTP Src homology region 2 domain-containing phatase; SHP-1, Src homology region 2 domain-containing phosphatase 1; TC-PTP, phosphatase 1 (SHP-1) is expressed at highest levels in hemopoietic T cells–PTP; Tyr, tyrosine; v-ATPase, vacuolar-type H+-ATPase; WT, wild-type. cells. In macrophages, SHP-1 participates in the regulation of Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 various processes, including proinflammatory and innate immune www.jimmunol.org/cgi/doi/10.4049/jimmunol.1103021 2204 SHP-1 REGULATES PHAGOLYSOSOME BIOGENESIS responses (17–20). SHP-1 has previously been shown to associate Phagocytosis assays with phagosomes (21, 22), thereby suggesting a role in the bio- Zymosan (Sigma-Aldrich) and latex beads (3 mm; Polyscience) were genesis of phagolysosomes. In this study, we present evidence that opsonized with mouse serum. For synchronized phagocytosis assays, cells SHP-1 plays a role in phagosome maturation and that it is required were incubated with particles at a particle-to-cell ratio of 5:1 for 10 min at for acidification of this organelle. 4˚C. Excess particles were removed, and phagocytosis was triggered by transferring the cells to 37˚C for the indicated time points before pro- ceeding for microscopy. Materials and Methods Microscopy and immunofluorescence Cell culture Macrophages were fixed and permeabilized using 0.1% Triton X-100, and All animals were handled in strict accordance with good animal practice as nonspecific surface FcgR binding was blocked using 1% BSA, 2% goat defined by the Canadian Council on Animal Care, and all animal work was serum, 6% milk, and 50% FBS (26). Particle internalization was quantified approved by the Comite´ Institutionel de Protection des Animaux of Institut by immunofluorescence microscopy. Quantification of phagosomal re- National de la Recherche Scientifique–Institut Armand-Frappier. Bone cruitment was performed as follows. Slides were prepared with three marrow-derived macrophages (BMM) were obtained by extracting the coverslips per
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