title: FcγRIIB-Independent Mechanisms Controlling Membrane Localization of the Inhibitory Phosphatase SHIP in Human B Cells.
abstract: SHIP is an important regulator of immune cell signaling that functions to dephosphorylate the phosphoinositide phosphatidylinositol 3,4,5-trisphosphate at the plasma membrane and mediate protein-protein interactions. One established paradigm for SHIP activation involves its recruitment to the phospho-ITIM motif of the inhibitory receptor FcγRIIB. Although SHIP is essential for the inhibitory function of FcγRIIB, it also has critical modulating functions in signaling initiated from activating immunoreceptors such as B cell Ag receptor. In this study, we found that SHIP is indistinguishably recruited to the plasma membrane after BCR stimulation with or without FcγRIIB coligation in human cell lines and primary cells. Interestingly, fluorescence recovery after photobleaching analysis reveals differential mobility of SHIP-enhanced GFP depending on the mode of stimulation, suggesting that although BCR and FcγRIIB can both recruit SHIP, this occurs via distinct molecular complexes. Mutagenesis of a SHIP-enhanced GFP fusion protein reveals that the SHIP-Src homology 2 domain is essential in both cases whereas the C terminus is required for recruitment via BCR stimulation, but is less important with FcγRIIB coligation. Experiments with pharmacological inhibitors reveal that Syk activity is required for optimal stimulation-induced membrane localization of SHIP, whereas neither PI3K or Src kinase activity is essential. BCR-induced association of SHIP with binding partner Shc1 is dependent on Syk, as is tyrosine phosphorylation of both partners. Our results indicate that FcγRIIB is not uniquely able to promote membrane recruitment of SHIP, but rather modulates its function via formation of distinct signaling complexes. Membrane recruitment of SHIP via Syk-dependent mechanisms may be an important factor modulating immunoreceptor signaling.
authors.affiliation: Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, Manitoba R3E 0T5, Canada;
authors.forename: Samantha D
authors.identifier: 0000-0003-3866-4649
authors.initials: SD
authors.lastname: Pauls
authors.affiliation: Department of Immunology, University of Manitoba, Winnipeg, Manitoba R3E 0T5, Canada; and.
authors.forename: Arnab
authors.identifier: 0000-0001-6135-373X
authors.initials: A
authors.lastname: Ray
authors.affiliation: Department of Immunology, University of Manitoba, Winnipeg, Manitoba R3E 0T5, Canada; and.
authors.forename: Sen
authors.identifier: 0000-0001-8287-8981
authors.initials: S
authors.lastname: Hou
authors.affiliation: Cancer Sciences Unit, University of Southampton, Southampton SO16 6YD, United Kingdom.
authors.forename: Andrew T
authors.identifier: 0000-0001-6076-3649
authors.initials: AT
authors.lastname: Vaughan
authors.affiliation: Cancer Sciences Unit, University of Southampton, Southampton SO16 6YD, United Kingdom.
authors.forename: Mark S
authors.identifier: 0000-0003-2077-089X
authors.initials: MS
authors.lastname: Cragg
authors.affiliation: Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, Manitoba R3E 0T5, Canada; Department of Immunology, University of Manitoba, Winnipeg, Manitoba R3E 0T5, Canada; and Aaron.Marshall@umanitoba.ca.
authors.forename: Aaron J
authors.identifier: 0000-0002-1175-5498
authors.initials: AJ
authors.lastname: Marshall
mesh_terms: D001402:B-Lymphocytes / Q000201:enzymology* / Q000276:immunology; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000276:immunology / Q000502:physiology*; D002478:Cells, Cultured; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D007191:Indazoles / Q000494:pharmacology; D008213:Lymphocyte Activation / Q000276:immunology; D010078:Oxazines / Q000494:pharmacology; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D000072183:Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D038761:Photobleaching; D011719:Pyrazines / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D011947:Receptors, Antigen, B-Cell / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D017452:Receptors, IgG / Q000276:immunology* / Q000378:metabolism; D015398:Signal Transduction; D000072377:Syk Kinase / Q000276:immunology / Q000378:metabolism; D009025:Morpholines
chemical_list: D019086:Bridged Bicyclo Compounds, Heterocyclic; D049452:Green Fluorescent Proteins; D007191:Indazoles; D010078:Oxazines; D018129:Phosphatidylinositol Phosphates; D000072183:Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases; D011719:Pyrazines; D011725:Pyridines; D011743:Pyrimidines; D011947:Receptors, Antigen, B-Cell; D017452:Receptors, IgG; D000072377:Syk Kinase; C569670:1-(4-((2-(2-aminopyrimidin-5-yl)-7-methyl-4-morpholinothieno(3,2-d)pyrimidin-6-yl)methyl)piperazin-1-yl)-2-hydroxypropan-1-one; C000589391:6-(1H-indazol-6-yl)-N-(4-morpholinophenyl)imidazo(1,2-a)pyrazin-8-amine; D009025:Morpholines; C412373:AG 1879; C510788:N4-(2,2-dimethyl-3-oxo-4H-pyrid(1,4)oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethoxyphenyl)-2,4-pyrimidinediamine; C060974:phosphatidylinositol 3,4,5-triphosphate; C000606896:SYK protein, human; C000606430:INPP5D protein, human; C506918:bafetinib