Fcγriib-Independent Mechanisms Controlling Membrane Localization

FcγRIIB-Independent Mechanisms Controlling Membrane Localization of the Inhibitory Phosphatase SHIP in Human B Cells This information is current as of September 28, 2021. Samantha D. Pauls, Arnab Ray, Sen Hou, Andrew T. Vaughan, Mark S. Cragg and Aaron J. Marshall J Immunol published online 25 July 2016 http://www.jimmunol.org/content/early/2016/07/23/jimmun ol.1600105 Downloaded from

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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 © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published July 25, 2016, doi:10.4049/jimmunol.1600105 The Journal of Immunology

FcgRIIB-Independent Mechanisms Controlling Membrane Localization of the Inhibitory Phosphatase SHIP in Human B Cells

Samantha D. Pauls,* Arnab Ray,† Sen Hou,† Andrew T. Vaughan,‡ Mark S. Cragg,‡ and Aaron J. Marshall*,†

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 FcgRIIB. Although SHIP is essential for the inhibitory function of FcgRIIB, 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 Downloaded from membrane after BCR stimulation with or without FcgRIIB 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 FcgRIIB 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 FcgRIIB coligation. Exper-

iments with pharmacological inhibitors reveal that Syk activity is required for optimal stimulation-induced membrane localiza- http://www.jimmunol.org/ tion 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 FcgRIIB is not uniquely able to promote membrane recruitment of SHIP, but rather modulates its function via formation of distinct signaling complexes. Mem- brane recruitment of SHIP via Syk-dependent mechanisms may be an important factor modulating immunoreceptor signaling. The Journal of Immunology, 2016, 197: 000–000.

ctivation of immune cells is controlled by an intricate phosphorylated Iga/b ITAMs provide binding sites for Syk, thereby balance of activating and inhibitory signals, which col- initiating its activation (3). These two early signaling kinases A lectively direct effective immune responses while avoiding phosphorylate a variety of target proteins, initiating multiple inde- by guest on September 28, 2021 damage to self tissues. Understanding how activating and inhibitory pendent and overlapping signaling cascades, ultimately resulting in signals are balanced requires a detailed determination of the multiple activation of the B cell (1). One key Syk-dependent pathway is the inputs controlling activity of regulatory signaling molecules. Many of PI3K pathway. Upon BCR ligation, Syk phosphorylates CD19 and the fundamental signaling pathways leading to B cell activation via BCAP, providing binding sites at the membrane for class IA PI3K the BCR have been elucidated; however, the integration of activating adaptor subunits (4, 5). PI3K catalytic subunits then convert the and inhibitory signaling mechanisms is still not well understood. membrane lipid phosphoinositol 4,5-bisphosphate to the second The BCR signaling chains Iga and Igb each contain two ITAMs messenger phosphoinositol 3,4,5-trisphosphate [PI(3,4,5)P3](6), that can be phosphorylated upon receptor cross-linking by the which is of critical importance for cell proliferation, survival, Ag Src family kinase Lyn and, in some contexts, Syk (1, 2). Doubly presentation, class switching, and migration (5). Late in the immune response, both in the context of a protective *Department of Biochemistry and Medical Genetics, University of Manitoba, Win- response to pathogens or a pathological response to self-antigen, nipeg, Manitoba R3E 0T5, Canada; †Department of Immunology, University of Manitoba, Winnipeg, Manitoba R3E 0T5, Canada; and ‡Cancer Sciences Unit, Uni- complexes of Ag and previously generated IgG are present and versity of Southampton, Southampton SO16 6YD, United Kingdom can coligate FcgRIIB along with the BCR (7). This leads to ORCIDs: 0000-0003-3866-4649 (S.D.P.); 0000-0001-6135-373X (A.R.); 0000-0001- phosphorylation of ITIM tyrosines in FcgRIIB and subsequent 8287-8981 (S.H.); 0000-0001-6076-3649 (A.T.V.); 0000-0003-2077-089X (M.S.C.); recruitment of SHIP (8). SHIP controls the accumulation of PI3K 0000-0002-1175-5498 (A.J.M.). products at the plasma membrane by converting PI(3,4,5)P to Received for publication January 19, 2016. Accepted for publication June 21, 2016. 3 phosphatidylinositol 3,4-bisphosphate (9, 10). SHIP was found to This work was supported by Canadian Institutes of Health Research Operating Grant MOP-93771 (to A.J.M.) and by an equipment grant from the Canadian Foundation mediate most of the inhibitory functions of FcgRIIB in B cells for Innovation. S.D.P. is supported by a Canadian Institutes of Health Research (11). Taken together, these findings established the paradigm that doctoral research award. SHIP recruitment to the ITIM of FcgRIIB is a dominant mecha- Address correspondence and reprint requests to Dr. Aaron J. Marshall, University of Manitoba, 471 Apotex Centre, 750 McDermot Avenue, Winnipeg, MB R3E 0T5, nism controlling SHIP activity and thus B cell activation via the Canada. E-mail address: [email protected] PI3K pathway. Because both FcgRIIB and SHIP are implicated in The online version of this article contains supplemental material. control of autoreactive B cells (12, 13), this regulatory circuit is Abbreviations used in this article: EGFP, enhanced GFP; FN, fibronectin; FRAP, critical. However SHIP is also phosphorylated and functional after fluorescence recovery after photobleaching; PI(3,4,5)P3, phosphatidylinositol 3,4,5- triggering activating receptors such as cytokine receptors and trisphosphate; SH2, Src homology 2. the BCR, indicating that it can also function independently of Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 FcgRIIB (9, 10).

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1600105 2 CONTROL OF SHIP LOCALIZATION BY THE BCR AND FcgRIIB

SHIP recruitment to the plasma membrane is required for its function in hydrolyzing PI(3,4,5)P3 substrate, and the present model predicts that inhibitory receptor engagement enhances SHIP membrane recruitment via phoshorylated ITIMs. Unexpectedly, we found in the present study that after BCR stimulation SHIP is indistinguishably recruited to the plasma membrane in the presence or absence of FcgRII coligation; however, FcgRII coligation impacts the mobility of engaged SHIP molecules once at the cell surface based on ﬂuorescence recovery after photobleaching (FRAP) analysis. BCR-induced membrane recruitment requires the Src homology 2 (SH2) domain of SHIP and an intact C terminus containing tyrosine phosphorylation sites and proline-rich region, whereas with FcgRIIB coligation SHIP recruitment becomes less dependent on the C-terminal region. The kinase activity of Syk is strictly required for SHIP recruitment in either stimulation context and for an association of SHIP with another well-characterized binding partner. Taken together, our ﬁndings provide new insight into the regulation of SHIP by BCR and FcgRIIBandidentifyanovelandessential

role for Syk kinase as an upstream regulator of SHIP. Downloaded from

Materials and Methods Cell culture and reagents Ramos clonal cell lines expressing full-length or cytoplasmic truncated

FcgRIIB were generated previously (14). A20 cell culture conditions http://www.jimmunol.org/ are described elsewhere (15). Primary human B cells were isolated from healthy donor blood using the EasySep direct B cell isolation kit (Stemcell Technologies) and were rested 1 h before manipulation. Where indicated, coverglass slides were coated with 5 mg/ml ﬁbro- nectin (FN; Sigma-Aldrich) overnight at 4˚C. Stimulations were performed using goat anti-human IgM or goat anti-mouse IgG Abs (Jackson ImmunoResearch Laboratories and SouthernBiotech) at 10 mg/ml [F(ab9)2] or 20 mg/ml (intact). Inhibitors used were as follows: 3AC (10 mM; Calbiochem), GDC-0980 (1 mM; Selleck Chemicals), bafetinib (1 mM; Selleck Chemicals), PP2 (10 mM; Sigma-Aldrich), R406 (10 mM; Selleck

Chemicals), and GS-9973 (5 mM; Selleck Chemicals). by guest on September 28, 2021 Plasmids and transfections The SHIP–p-enhanced GFP (EGFP)-C1 construct was a gift from Dr. M. Jucker€ (16). The membrane marker used was pmKate2–f-mem (Evrogen), where f-mem is a 20-aa farnesylation signal from c-Ha-Ras (17). Site-directed mutagenesis was performed using the QuikChange II XL kit (Agilent Tech- nologies). Primer sequences are as follows, with codon changes underlined: SH2 domain inactivated SHIP R31G (18): 59-GAGCTTCCTCGTGGGTGC- CAGCGAGTC-39 and 59-GACTCGCTGGCACCCACGAGGAAGCTC-39; phosphatase-deﬁcient SHIP D673G (19, 20): 59-CCTTCCTGGTGTGGC- CGAGTCCTCTGG-39 and 59-CCAGAGGACTCGGCCACACCA GGA- AGG-39; for truncated SHIP 909Trunc, a stop codon was introduced using 39-GTGGCTCCAGCATCACTTAAATCATCAACCCCAAC-59 and 39-GTTG- GGGTTGATGATTTAAGTGATGCTGGAGCCAC-59. Transient expression

was performed by ﬂow cytometry. Percentage increase in mean ﬂuorescence intensity relative to unstimulated cells was determined for triplicate sam-

ples. (D) SHIP-EGFP is phosphorylated on Y1022 after F(ab9)2 and intact anti-IgM stimulation. Twenty-four hours after transfection with SHIP- EGFP vector, FcgRIIB+ Ramos cells were serum starved and stimulated for 5 min with anti-IgM. Lysates were subjected to SDS-PAGE, and Western blotting was performed with the indicated Abs. EGFP-tagged FIGURE 1. SHIP-EGFP is recruited to the plasma membrane with BCR SHIP (upper band, arrow) and endogenous SHIP (lower band) can be ligation alone or with FcgRIIB coligation. (A and B)FcgRIIB/CD32B distinguished by size. (E and F) Transiently transfected FcgRIIB+ (E)or expression in Ramos-derived cell lines. (A) Ramos cell cultures were cell FcgRIIBTrunc (F) Ramos cells expressing SHIP-EGFP and red membrane surface stained with anti-CD32 Ab and analyzed by flow cytometry. (B) marker were stimulated in a cell culture incubator for 2 min, fixed, and Cell lysates were probed with Ab that specifically detects the C terminus of imaged by confocal microscopy at original magnification 363. Repre- anti-CD32B and analyzed by Western blotting. Note that FcgRIIBTrunc is sentative images and quantitation of green/red colocalization by Pearson not detected with this Ab as expected. (C) Stimulation with intact anti-IgM coefficients are shown. Data were analyzed by one-way ANOVA, with a leads to increased SHIP phosphorylation dependent on the FcgRIIB Tukey multiple comparisons test for pairwise comparisons. Data are rep- cytoplasmic tail. The indicated Ramos-derived cell lines were serum resentative of at least three independent experiments for FcgRIIB+ cells or starved for 3 h and then stimulated as indicated. Intracellular staining was two for FcgRIIBTrunc cells. Scale bars, 5 mm. *p . 0.05, **p . 0.01, performed for phospho-Y1022 SHIP or phospho-Y348 Syk and detection ****p . 0.0001. The Journal of Immunology 3 of constructs was achieved using the Neon transfection system (Invitrogen) Statistical analysis to electroporate 5 3 106 cells per 100 mlreactionwith20mg of SHIP vector and/or 10 mg of pmKate2–f-mem. Cells were cultured in antibiotic-free Correlation coefficient data from 10 cells per condition (live cell time courses) medium and used for experiments 18–24 h posttransfection. or 20–30 cells per condition (fixed cell imaging) were analyzed by ANOVA to identify any significant differences in means among multiple condi- Membrane recruitment experiments tions. Posttests for pairwise significance were applied depending on the 3 5 comparison being made. For comparison of three stimulation condi- For live cell imaging experiments, transfected cells were plated at 1 10 / tions, one-way ANOVAwith a Tukey posttest was used; for comparison well on uncoated (A20) or FN-coated (Ramos) coverglass slides, mounted of multiple stimulation time points to unstimulated, nonparametric one- in a stage-top incubator, and stimulated while imaging at original mag- way ANOVA with a Dunn test was used; for multiple time points and nification 363 on a Zeiss AxioObserver spinning disk confocal micro- two stimulation conditions, or for three stimulations conditions and two scope. For fixed-cell experiments, cells were serum starved at 1 3 106/ml drug treatments, two-way ANOVA with a Sidak test was used. All sta- for 3 (Ramos) or 1 h (primary cells) and inhibitors were added during the tistical analysis was performed using GraphPad Prism. For all figures, *p . final hour. Stimulations were performed in a cell culture incubator and 0.05, **p . 0.01, ***p . 0.001, and ****p . 0.0001. stopped with 1–2% ice-cold PFA. Images were then acquired at original magnification 363. Membrane recruitment in transfected cell lines was FRAP experiments measured by colocalization analysis in ImageJ. The Cell Outliner plugin was used to define individual cells, and the Colocalization Indices plugin Ibidi m-slides were coated with 5 mg/ml FN plus 5 mg/ml F(ab9)2 or 10 mg/ml was used to calculate the Pearson coefficient of correlation between green intact anti-IgM where indicated. Cells were serum starved for 2–3 h and then and red signals. The red signal arises from the membrane marker f-mem. plated at 2.5 3 105/well and spread for 30–40 min. Peripheral regions of The green signal arises from either ectopic EGFP or anti-SHIP staining interest were bleached with a 405 nm laser (Rapp OptoElectronic), concur- using anti-SHIP (P1C1; Santa Cruz Biotechnology) as primary Ab at 1:200 rent with high-speed confocal imaging at original magnification 363. In- plus goat anti-mouse Alexa Fluor 488 (Molecular Probes) as secondary tensity values were obtained using the Time Series V3 plugin for ImageJ. To Ab at 1:500. account for initial intensity differences as well as minor whole-cell photo- Downloaded from Membrane recruitment in primary cells was estimated using a modifi- bleaching resulting from the 488-nm imaging laser, intensity values within cation of the previously described maximal method (15). Briefly, for each the bleached regions of interest were normalized to intensity values from an cell the maximal intensity membrane pixel (within 2 pixels of the cell unbleached control region of the same cell. Curves of normalized intensity edge) is divided by the average pixel intensity of the entire cell to calculate (Y) versus time (x)werey-transformed to (0,0) at t = 0 (bleaching event) so a maximal membrane/average pixel intensity ratio. that individual recovery curves begin at the intensity minimum. Each group http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 2. Kinetics of SHIP-EGFP membrane recruitment in live cells. (A)FcgRIIB+ Ramos cells or (B) A20 cells were transiently transfected with plasmids encoding SHIP-EGFP plus a red fluorescent membrane marker and stimulated with anti-Ig while imaging at original magnification 363 in a stage- top incubator. Graphs show change in SHIP membrane localization over time, expressed as average Pearson correlation coefficient of 10 cells per condition

(normalized to time 0). Significance relative to prestimulation, as assessed by a Dunn test, is indicated by * (intact) or t [F(ab9)2]. Two-way ANOVA did not reveal any significant difference between F(ab9)2 and intact anti-Ig. 4 CONTROL OF SHIP LOCALIZATION BY THE BCR AND FcgRIIB of recovery curves was then fit to the following equation, with the constraint IgG BCR and endogenous FcgRIIB and are an established model 2 3 2 2K3x that Yo =0:Y = Yo + [(plateau Yo) (1 e ). The rate constant for inhibitory signaling (23). A20 cells also did not show significant (K) was derived and statistical comparisons between groups were per- differences in the magnitude or kinetics of SHIP recruitment formed using GraphPad Prism software. Two peripheral regions of interest were bleached in at least eight cells per stimulation condition per (Fig. 2B). We further examined membrane association of en- experiment. dogenous SHIP detected by intracellular staining and colocalization with a membrane marker and found results consistent with Coimmunoprecipitation experiments those using SHIP-EGFP (Fig. 3A). Examination of primary human Equal numbers of FcgRIB+ Ramos cells were stimulated as indicated and B cells also revealed that BCR stimulation alone triggers mem- lysed with Nonidet P-40 (Thermo Fisher) lysis buffer containing protease brane recruitment of SHIP that is indistinguishable from that in- and phosphatase inhibitor cocktails (Roche). Three micrograms of specific Ab, either anti-SHIP (P1C1; Santa Cruz Biotechnology) or anti-Shc (PG- duced in the presence of FcgRIIB coligation (Fig. 3B). Taken 797; Santa Cruz Biotechnology), was used for immunoprecipitation with together, these results indicate that neither expression nor en- protein G–Sepharose beads (GE Healthcare). Equal volume of precipitate gagement of FcgRIIB is essential for SHIP accumulation at the was run on mini-protean precast polyacrylamide gels (Bio-Rad) and plasma membrane during B cell activation, counter to the pre- transferred to nitrocellulose membrane (Bio-Rad). Membranes were pro- vailing paradigm. bed with anti-SHIP (P1C1; Santa Cruz Biotechnology), anti-Shc (H-108; Santa Cruz Biotechnology), or anti-pTyr (4G10; Millipore) primary Ab Mobility of SHIP-EGFP at the cell periphery then HRP-linked goat anti-mouse IgG (Jackson ImmunoResearch Labo- ratories) or goat anti-rabbit IgG (Cell Signaling Technology) secondary Although BCR ligation appears sufficient to recruit SHIP to the Ab, then developed by chemiluminescence. plasma membrane, FcgRIIB coligation can impact SHIP phos-

Abs and staining phorylation (Fig. 1) and activity (11, 21). We thus speculated that Downloaded from engagement of this inhibitory receptor may allow SHIP to enter For surface staining, FITC-conjugated anti-FcgRIIB/CD32 (BD Pharmi- into functionally distinct protein complexes at the membrane. One nogen), Cy3-conjugated anti-IgM (Jackson ImmunoResearch Laboratories) and allophycocyanin-conjugated anti-CD19 (BD Pharmingen) were all used measurable parameter that can be influenced by signaling complex at 1:200. For intracellular Phosflow staining, cells were prepared at 1 3 106/ formation is dynamic protein mobility or diffusion rate. We ml in warm serum-free RPMI 1640 and starved for 3 h before stimulation as employed FRAP to determine whether stimulation conditions alter indicated. Cells were centrifuged briefly for harvest, resuspended in Fix/Perm

the mobility of SHIP-EGFP molecules at the cell periphery. We http://www.jimmunol.org/ solution (BD Biosciences), washed, and ﬁnally stained with Alexa Fluor bleached regions of interest and subsequently monitored the 488–conjugated anti–phospho-Syk (BD Biosciences) at 1:5 or rabbit mono- clonal anti–phospho-SHIP (EPR425; Abcam) at 1:200 in combination with Alexa Fluor 488–conjugated donkey anti-rabbit IgG secondary Ab (BD Biosciences) at 1:500. For phospho-SHIP Western blot, anti-pY1020 (Cell Signaling Technologies) was used at 1:1000.

Results FcgRIIB engagement does not signiﬁcantly impact recruitment

of SHIP to the plasma membrane by guest on September 28, 2021

Previous studies report that SHIP-mediated inhibition of PI(3,4,5)P3 and Ca2+ flux is most active when FcgRIIB is coligated with the BCR (11, 21). SHIP is also more highly phosphorylated in its C terminus with FcgRIIB coligation (22). However, the impact of coligation on SHIP recruitment to the plasma membrane has not been directly determined. To assess SHIP recruitment in the presence or absence of FcgRIIB we used human Ramos B cell lines lacking endogenous expression of FcgRIIB but transfected with either wild-type FcgRIIB or a truncation mutant lacking the ITIM motif (Fig. 1A, 1B). As expected, coligation of FcgRIIB using intact anti-IgM led to increased phosphorylation of SHIP on tyrosine 1022, relative to BCR ligation alone using F(ab9)2 anti- IgM, and this increased phosphorylation required the ITIM motif (Fig. 1C, left). In contrast, phosphorylation of Syk was unaffected by FcgRIIB coligation (Fig. 1C, right). SHIP-EGFP fusion protein also showed increased phosphorylation in FcgRIIB-expressing Ramos cells stimulated with intact anti-IgM (Fig. 1D). Unex- pectedly, we found that SHIP-EGFP is significantly recruited to FIGURE 3. Endogenous SHIP is recruited to the plasma membrane after the plasma membrane when the BCR is ligated alone, and there is BCR ligation with or without FcgRIIB coligation. (A)FcgRIIB+ Ramos in fact no measurable difference in the magnitude of recruitment cells transfected with membrane marker only were serum starved for 3 h, induced by F(ab9)2 or intact anti-IgM (Fig. 1E). Additionally, stimulated, fixed, permeabilized, and stained with anti-SHIP Ab to detect SHIP recruitment still occurs in Ramos cells expressing truncated endogenously expressed SHIP. Quantitation of green/red colocalization by FcgRIIB that lacks the ITIM motif (Fig. 1F). Pearson’s coefficient is shown, analyzed by one-way ANOVAwith Tukey’s B We examined whether FcgRIIB coligation impacted the kinetics test for pairwise comparisons. ( ) Primary human B cells were enriched from healthy donor blood and serum starved for 2 h prior to stimulation, of SHIP membrane recruitment. Live cell imaging of FcgRIIB+ fixation, and staining with anti-SHIP Ab. Membrane recruitment of SHIP Ramos coexpressing SHIP-EGFP and a red fluorescent membrane was calculated as the ratio of maximal membrane intensity to average marker indicated that recruitment occurs within the first minute of fluorescence in each cell and statistical differences were calculating by a stimulation with either F(ab9)2 or intact anti-IgM and did not re- Tukey test. Images and analysis are representative of at least three indepen- veal any significant differences between these stimuli (Fig. 2A). A dent experiments. All images were acquired at original magnification 363. similar analysis was conducted in murine A20 cells, which express *p . 0.05, **p . 0.01. The Journal of Immunology 5

fluorescence recovery over time. By curve fitting, rate constants C-terminal region, can play a role together with the SH2 domain in were derived for the recovery, reflective of how quickly the mole- facilitating BCR-induced SHIP recruitment. cules redistribute within the bleached area. Our results in We next examined whether SHIP recruitment is influenced by its FcgRIIB+ Ramos B cells indicate that the mobility of SHIP-EGFP own enzymatic activity or requires activation of Syk, Src, or PI3K is significantly reduced when cells are stimulated with intact anti- kinases (Fig. 5F–I, Supplemental Fig. 1). Neither the SHIP in- IgM relative to F(ab9)2 anti-IgM (Fig. 4B). This difference was not hibitor 3AC (24) (Fig. 5F) nor the pan-PI3K inhibitor apitolisib seen in FcgRIIBTrunc Ramos cells (Fig. 4C). (GDC-0980, Fig. 5G) had any effect, suggesting that binding to phosphoinositides or phosphoinositide-binding proteins is not es- Dependence of SHIP membrane recruitment on upstream sential for SHIP recruitment. Unexpectedly, membrane associa- kinases and protein interaction motifs tion of SHIP was not strongly inhibited by the potent Lyn inhibitor We tested the sensitivity of the membrane recruitment process bafetinib or by the pan-Src kinase inhibitor PP2, even in the to disruption of key elements of SHIP structure (Fig. 5A–D, presence of FcgRIIB coligation (Fig. 5H). However, pretreatment Supplemental Fig. 1). It was found that a functional SH2 domain is with the Syk kinase inhibitor fostamatinib (R406) strongly required for SHIP membrane recruitment irrespective of FcgRIIB inhibited membrane recruitment of SHIP-EGFP in FcgRIIB+ coligation (Fig. 5A). A phosphatase-deficient SHIP mutant was Ramos cells (Fig. 5I). Similar results were obtained using a dis- recruited normally (Fig. 5B). Interestingly, the C-terminal region of tinct Syk inhibitor entospletinib (GS-9973; Supplemental Fig. SHIP is absolutely required for recruitment of SHIP-EGFP upon 1B). Whereas Syk inhibition significantly reduced membrane stimulationwithF(ab9)2 Ab, but it has a less significant role for recruitment with either F(ab9)2 or intact anti-IgM stimulation, we recruitment after intact Ab stimulation (Fig. 5C). This differential noted a trend of decreased percentage inhibition under the latter Downloaded from effect was not observed in cells expressing truncated FcgRIIB condition (Supplemental Fig. 1C). Syk inhibition also blocked lacking the ITIM motif (Fig. 5D). Mutagenesis of Y1022, the best membrane recruitment of endogenous SHIP in both FcgRIIB+ characterized pTyr in the SHIP C-terminal domain, revealed that this Ramos cells (Fig. 6A) and primary human B cells (Fig. 6B). Taken tyrosine is not essential for membrane recruitment (Fig. 5E). These together, these results identify a novel role for Syk kinase activity results suggest that tyrosines other than Y1022, or other motifs in the in recruitment of SHIP to the membrane of human B cells. http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 4. Fluorescence recovery after photobleaching analysis of SHIP-EGFP. FcgRIIB+ or FcgRIIBTrunc Ramos cells transiently transfected to express SHIP-EGFP were plated on either F(ab9)2 or intact anti-IgM for 30–40 min. Photobleaching of peripheral regions of interest was performed concurrently with high-speed confocal imaging at original magniﬁ- cation 363. (A) Representative images showing recovery from bleaching over time. (B)FRAPanalysisofFcgRIIB+ Ramos cells. Left panel shows example of recovery curves, and right panel shows quantiﬁcation of rate constants pooled from four experiments (100– 120 bleaching events total). (C)FRAP analysis of FcgRIIBTrunc cells. Right panel shows data pooled from two experiments (50–60 bleaching events total). Rate constants and p values were obtained by nonlinear regression analysis. **p . 0.01, ****p . 0.0001. 6 CONTROL OF SHIP LOCALIZATION BY THE BCR AND FcgRIIB

FIGURE 5. Structural and signaling requirements for SHIP-EGFP membrane recruitment. FcgRIIB+ Ramos Downloaded from cells expressing red membrane marker plus (A) SH2 mutant (B) phosphatase-deﬁcient (PD) mutant, (C) C-terminal truncation (909Trunc) mutant, or (E) Y1022F mutant SHIP-EGFP were stimulated, ﬁxed, imaged, andanalyzedasinFig.1.(D) 909Trunc mutant expressed in FcgRIIBTrunc Ramos cells. FcgRIIB+ Ramos cells expressing membrane marker plus WT http://www.jimmunol.org/ SHIP-EGFP were preincubated with (F) SHIP phosphatase inhibitor 3AC, (G) PI3K inhibitor apitolisib (GDC-0980), (H) Lyn inhibitor bafetinib or pan-Src kinase inhibitor PP2, (I) Syk inhibitor fostamatinib (R406), or DMSO vehicle control and analyzed as above. Statistical analyses between treated and untreated groups were performed by two-way ANOVA with a Sidak multiple comparisons test. Data are rep-

resentative of at least three independent experiments by guest on September 28, 2021 for FcgRIIB+ cells or two for FcgRIIBTrunc cells, with 20 cells per group per experiment. *p . 0.05, **p . 0.01, ***p . 0.001, ****p . 0.0001.

Syk activity is required for SHIP phosphorylation and teraction detected by reciprocal coimmunoprecipitation (Fig. 7A). association with Shc Interestingly, pretreatment with the Syk inhibitor R406 abrogates the We further assessed whether Syk activity may be required for interaction between SHIP and Shc and substantially reduces the SHIP phosphorylation and interaction with other binding partners. total tyrosine phosphorylation of both proteins (Fig. 7B). A second Stimulation with intact anti-Ig is known to induce a robust inter- Syk inhibitor, GS-9973, had a similar effect (Supplemental Fig. 2). action between SHIP and the adaptor protein Shc1 (25, 26); Phospho flow cytometry confirms that inhibition of Syk signifi- however, it is unknown whether formation of this complex requires cantly reduces SHIP phosphorylation at Y1022; however, this in- coengagement of FcgRIIB or whether Syk activity is required. In hibition appears incomplete in the presence of FcgRIIB coligation this study, we report that F(ab9)2 anti-Ig induces a comparable in- (Fig. 7C). Inhibition of Src family kinases (Fig. 7C), which are directly The Journal of Immunology 7

FIGURE 6. Syk inhibition prevents membrane recruitment of endogenous SHIP. (A)FcgRIIB+ Ramos cells expressing red membrane marker were preincubated with Syk inhibitor R406 then stimulated, fixed, and stained for endogenous SHIP. Example images at original magnification 363 and colocalization analysis are representative of three independent experiments with 20 cells per group. (B) Primary human B cells derived from healthy donor blood were preincubated with Syk inhibitor R406 then stimulated, fixed, and stained for endogenous SHIP. Example images and maximal method

analysis are representative of three donors with 20 cells Downloaded from per group for each donor. Statistical analyses between treated and untreated groups were performed by two-way ANOVA with a Sidak multiple comparisons test. *p . 0.05, **p . 0.01, ***p . 0.001. http://www.jimmunol.org/

responsible for phosphorylating Y1022 (27, 28), abrogated its phos- conditions. Conceptually this result indicates that the activating phorylation (Fig. 7C). Taken together, these results suggest that Syk kinase Syk rather than the inhibitory kinase Lyn can serve as the by guest on September 28, 2021 activity is required for SHIP to form a complex with Shc and further primary driver of SHIP membrane recruitment in human B cells. facilitates SHIP phosphoryation by Src kinases at the plasma mem- Mechanistically, this result could be interpreted in several ways. brane. As Syk is upstream of PI3K (4), the requirement for Syk activity might reflect a requirement for either PI(3,4,5)P3 or phosphoino- Discussion sitol 3,4-bisphosphate as an alternate ligand for plasma membrane Engagement of FcgRIIB provides a critical control mechanism to docking. Indeed. SHIP contains a pleckstrin homology–related attenuate B cell activation in the presence of Ag complexed with domain that is required for localization to the phagocytic cup of pre-existing IgG Ab. This feedback circuit plays essential roles in RAW264.7 macrophages (38) and a C2 domain that can interact preventing inappropriate activation and preventing autoimmunity with its own lipid product to promote enzymatic activity via an and immune complex–mediated inflammatory disease (29–31). allosteric positive feedback mechanism (39). However, we found SHIP is also strongly implicated in control of autoreactive B cells that inhibition of PI3K had no effect on BCR-induced SHIP and inflammation (13), and this is most often attributed to its role membrane recruitment, arguing against this mechanism. Alterna- as mediator of FcgRIIB inhibitory signaling. Our results demon- tively, Syk might be responsible for phosphorylation of a SHIP strate that in the absence of FcgRIIB involvement, BCR ligation binding partner that is required for its recruitment to, or retention alone can trigger robust membrane recruitment of SHIP via pro- at, the plasma membrane. tein–protein interactions driven by Syk. These results are consis- The latter hypothesis led us to test the sensitivity of known tent with a model where SHIP is directly recruited by the BCR and interactions to Syk inhibition. We focused on Shc1 because pre- acts primarily as an intrinsic brake on BCR signaling, and can vious studies report an inducible association with SHIP upon li- secondarily be modulated by FcgRIIB and likely by other re- gation of various receptors (40) and that its phosphorylation is ceptors as well. Indeed, this example of a negative signal initiated impaired in the absence of Syk (41). We found that pretreatment from an activating receptor is one of several recent challenges to with Syk inhibitor abrogates the anti-Ig–induced interaction be- the elegant but simplistic ITAM-ITIM dogma (32, 33). Other tween SHIP and Shc1. Interestingly, immunoprecipitation of SHIP activating receptors have also been found to recruit SHIP (34, 35). by anti-SHIP Ab appeared to be increased when Syk was inhibi- The established model for SHIP membrane recruitment and ted, consistent with an increased amount of uncomplexed SHIP activation predicts the central importance on the Src family kinase accessible to Ab binding. Furthermore, global tyrosine phosphory- Lyn, which can phosphorylate the ITIM motif on FcgRIIB that lation of both SHIP and Shc1 is substantially reduced in the presence binds to the SHIP SH2 domain (36, 37). However, our results of Syk inhibitor. This may to some extent reflect direct phosphor- indicate that activity of this kinase is dispensable for SHIP re- ylation of SHIP and Shc1 by Syk, the latter of which has been cruitment after BCR or BCR/FcgRIIB ligation, whereas inhibi- reported previously (42). We also found that phosphorylation of tion of Syk abrogates membrane recruitment of SHIP under these SHIP at Y1022 is blocked by both Syk and Src kinase inhibitors. 8 CONTROL OF SHIP LOCALIZATION BY THE BCR AND FcgRIIB Downloaded from http://www.jimmunol.org/ FIGURE 7. Syk inhibition prevents SHIP phosphorylation and association with Shc1. (A)FcgRIIB+ Ramos cells were stimulated for 5 min with the indicated anti-IgM Ab, lysed, and subjected to coimmunoprecipitation with specific Ab or isotype control. Eluates were separated by SDS-PAGE and Western blotting was performed with the indicated Abs. (B) Cells were pretreated with Syk inhibitor R406 or DMSO vehicle prior to stimulation and protein extracts were analyzed as above. Blots are representative of three experiments. (C)FcgRIIB+ Ramos cells were pretreated with Syk inhibitor R406 or Src family kinase inhibitor PP2 then stimulated for 5 min, fixed, and stained for intracellular phospho-SHIP (Y1022) and analyzed by flow cytometry. Quantitation is based on triplicate samples. ***p . 0.001, ****p . 0.0001.

Because Lyn or other Src family kinases are directly responsible for Our data indicate that coligation of FcgRIIB modulates BCR- by guest on September 28, 2021 phosphorylating Y1022 (27, 28), this result is consistent with a induced SHIP recruitment such that it becomes less dependent on model where Syk-dependent translocation of SHIP to the C-terminal interactions and immobilized to a greater degree. The membrane is a prerequisite to phosphorylation by membrane- most straightforward interpretation of these results is that SHIP associated Src family kinases. Taken together, our results indi- preferentially binds to phospho-ITIMs when they are available, cate that Syk activity is required not only for SHIP membrane and this promotes SHIP interaction with distinct protein com- recruitment, but also for its phosphorylation and interaction with plexes that promote increased phosphorylation on Y1022 and in- major binding partner Shc1. creased or prolonged enzymatic activity. Our finding that FcgRIIB Our structure–function analysis indicates that BCR-induced SHIP coligation decreases mobility of SHIP-EGFP is consistent with recruitment requires both its SH2 domain and its C-terminal domain. inhibitory receptor driving formation of distinct membrane- The latter contains NPxY motifs at Y917 and Y1022, PxxP motifs, associated protein complexes. Surprisingly, SHIP recruitment, and several other poorly characterized tyrosine phosphorylation phosphorylation, and interaction with Shc1 remain dependent on sites. Because the NPxY motifs can bind to the PTB domain of Shc Syk activity with coligation of FcgRIIB, suggesting an upstream and the SHIP SH2 can bind to Y317 of Shc (43), the requirement of requirement for Syk in generation of both activating and inhibitory these domains is consistent with a role for the SHIP–Shc interaction signaling complexes. in membrane recruitment. Although previous studies have demon- Because modulation of SHIP activity by FcgRIIB cannot be strated that the SHIP–Shc1 interaction is expendable to or even explainedbyanincreaseinaccesstophosphoinositidesubstrate competitive with the SHIP–FcgRIIB interaction (44, 45), it has not at the plasma membrane, more subtle regulatory mechanisms been explored in the context of BCR ligation alone. Both SHIP and must be at play to control the activities of this key enzyme. One Shc have been reported to directly bind to to Iga/b signaling sub- current model of SHIP recruitment to the membrane after BCR/ units of the BCR (46, 47), providing multiple plausible mechanisms FcgRIIB coligation describes the formation of a stable trimolecular for recruitment of this complex. The SHIP SH2 domain was complex containing FcgRIIB, SHIP, and Grb2 and/or GRAP in reported to bind directly to Ig-a (46); however, an intramolecular murine B cells (48). In human B cells, Grb2 and GRAP are dis- interaction between SHIP’s SH2 domain and its C terminus was also pensable but an as-yet unidentified interaction partner that bridges the found to compete for Ig-a binding (46). Thus, it is possible that direct CterminusofFcgRIIB to the C terminus of SHIP is proposed to exist recruitment of SHIP to the BCR complex may require disengagement (11). We were unable to detect SHIP/Grb2 interaction and found that of SHIP’s SH2 domain from its C terminus, and perhaps engagement the C-terminal region of SHIP is less critical for recruitment in the of the C terminus with Shc1 could promote such disengagement. case of BCR/FcgRIIB coligation of human B cells, suggesting that Because the binding of Shc to SHIP’s C terminus involves Shc’s PTB this complex may not have a major role in the human B cell context; domain, the SH2 domain of Shc would potentially remain free to however, we cannot rule out this possibility. Although our data sug- bind Iga/b ITAMs (47). gest that this region is more quantitatively significant for SHIP The Journal of Immunology 9 membrane targeting upon BCR ligation alone, it remains possible that 15. Cheung, S. M., J. C. Kornelson, M. Al-Alwan, and A. J. Marshall. 2007. Reg- ulation of phosphoinositide 3-kinase signaling by oxidants: hydrogen peroxide the C terminus is also engaged in protein interactions within the in- selectively enhances immunoreceptor-induced recruitment of phosphatidylino- hibitory signaling complex that are important for control of enzymatic sitol (3,4) bisphosphate-binding PH domain proteins. Cell. Signal. 19: 902–912. activity. Indeed, previous studies demonstrated reduced functionality 16.Nalaskowski,M.M.,A.Metzner,M.A.Brehm,S.Labiadh,H.Brauer, N. Grabinski, G. W. Mayr, and M. Jucker.€ 2012. The inositol 5-phosphatase SHIP1 of C-terminally truncated SHIP (44). The best characterized tyrosine is a nucleo-cytoplasmic shuttling protein and enzymatically active in cell nuclei. phosphorylation site within the C terminus of SHIP, Y1022, is clearly Cell. Signal. 24: 621–628. phosphorylated to a greater extent with BCR/FcgRIIB coligation, 17. Hancock, J. F., K. Cadwallader, and C. J. Marshall. 1991. Methylation and proteolysis are essential for efficient membrane binding of prenylated p21K-ras perhaps indicating that the inhibitory complex is more accessible to (B). EMBO J. 10: 641–646. Lyn, which phosphorylates this site. However, the functional role of 18. Liu, L., J. E. Damen, M. R. Hughes, I. Babic, F. R. Jirik, and G. Krystal. 1997. Y1022 is unclear, and we find that mutation of this tyrosine has no The Src homology 2 (SH2) domain of SH2-containing inositol phosphatase (SHIP) is essential for tyrosine phosphorylation of SHIP, its association with effect on membrane recruitment. Shc, and its induction of apoptosis. J. Biol. Chem. 272: 8983–8988. Given its established ability to control autoimmune and inflam- 19. Damen, J. E., M. D. Ware, J. Kalesnikoff, M. R. Hughes, and G. Krystal. 2001. matory reactions, SHIP is an attractive drug target (49, 50), high- SHIP’s C-terminus is essential for its hydrolysis of PIP3 and inhibition of mast cell degranulation. Blood 97: 1343–1351. lighting the need to further define mechanisms controlling SHIP 20. Jefferson, A. B., and P. W. Majerus. 1996. Mutation of the conserved domains of activity. Collectively, our results refine the classical paradigm for two inositol polyphosphate 5-phosphatases. Biochemistry 35: 7890–7894. 21. Ono, M., S. Bolland, P. Tempst, and J. V. Ravetch. 1996. Role of the inositol modulation of SHIP activity by inhibitory receptor and suggest that phosphatase SHIP in negative regulation of the immune system by the receptor SHIP might be more accurately viewed as an intrinsic regulator of FcgRIIB. 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