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FcyRIIB-independent mechanisms controlling membrane localization of the inhibitory phosphatase SHIP in human B cells

FcyRIIB-independent mechanisms controlling membrane localization of the inhibitory phosphatase SHIP in human B cells
FcyRIIB-independent mechanisms controlling membrane localization of the inhibitory phosphatase SHIP in human B cells
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 Fc?RIIB. Although SHIP is essential for the inhibitory function of Fc?RIIB, 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 Fc?RIIB 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 Fc?RIIB 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 Fc?RIIB 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 Fc?RIIB 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.
0022-1767
1587-1596
Pauls, Samantha
f25f9e81-8abd-419b-8b24-7c5fb342295f
Ray, Arnab
09bbf795-f203-48be-befd-86c13a234892
Hou, Sen
8920f60f-f213-40cc-a6bf-a43eb2772a47
Vaughan, Andrew T.
bfb2ceab-a592-457e-89f9-00fcd1dddbdb
Cragg, Mark S.
ec97f80e-f3c8-49b7-a960-20dff648b78c
Marshall, Aaron
4c9335e1-d656-420e-b748-829a38cb59a3
Pauls, Samantha
f25f9e81-8abd-419b-8b24-7c5fb342295f
Ray, Arnab
09bbf795-f203-48be-befd-86c13a234892
Hou, Sen
8920f60f-f213-40cc-a6bf-a43eb2772a47
Vaughan, Andrew T.
bfb2ceab-a592-457e-89f9-00fcd1dddbdb
Cragg, Mark S.
ec97f80e-f3c8-49b7-a960-20dff648b78c
Marshall, Aaron
4c9335e1-d656-420e-b748-829a38cb59a3

Pauls, Samantha, Ray, Arnab, Hou, Sen, Vaughan, Andrew T., Cragg, Mark S. and Marshall, Aaron (2016) FcyRIIB-independent mechanisms controlling membrane localization of the inhibitory phosphatase SHIP in human B cells. Journal of Immunology, 197 (5), 1587-1596. (doi:10.4049/jimmunol.1600105). (PMID:27456487)

Record type: Article

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 Fc?RIIB. Although SHIP is essential for the inhibitory function of Fc?RIIB, 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 Fc?RIIB 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 Fc?RIIB 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 Fc?RIIB 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 Fc?RIIB 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.

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Accepted/In Press date: 21 June 2016
e-pub ahead of print date: 25 July 2016
Published date: 1 September 2016
Organisations: Cancer Sciences

Identifiers

Local EPrints ID: 403221
URI: http://eprints.soton.ac.uk/id/eprint/403221
ISSN: 0022-1767
PURE UUID: 3a72b175-042c-4e16-af15-0dd6331d20ea
ORCID for Andrew T. Vaughan: ORCID iD orcid.org/0000-0001-6076-3649
ORCID for Mark S. Cragg: ORCID iD orcid.org/0000-0003-2077-089X

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Date deposited: 28 Nov 2016 15:04
Last modified: 16 Mar 2024 02:58

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Contributors

Author: Samantha Pauls
Author: Arnab Ray
Author: Sen Hou
Author: Andrew T. Vaughan ORCID iD
Author: Mark S. Cragg ORCID iD
Author: Aaron Marshall

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