Spatial Coupling of JNK Activation to the B Cell Antigen Receptor by Tyrosine-Phosphorylated Ezrin This information is current as Neetha Parameswaran, Gospel Enyindah-Asonye, Nayer of September 24, 2021. Bagheri, Neilay B. Shah and Neetu Gupta J Immunol 2013; 190:2017-2026; Prepublished online 21 January 2013; doi: 10.4049/jimmunol.1201292 http://www.jimmunol.org/content/190/5/2017 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2013/01/22/jimmunol.120129 Material 2.DC1 http://www.jimmunol.org/ References This article cites 40 articles, 20 of which you can access for free at: http://www.jimmunol.org/content/190/5/2017.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 24, 2021 • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *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 © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Spatial Coupling of JNK Activation to the B Cell Antigen Receptor by Tyrosine-Phosphorylated Ezrin Neetha Parameswaran, Gospel Enyindah-Asonye, Nayer Bagheri,1 Neilay B. Shah,2 and Neetu Gupta The ezrin-radixin-moesin proteins regulate B lymphocyte activation via their effect on BCR diffusion and microclustering. This relies on their ability to dynamically tether the plasma membrane with actin filaments that is in turn facilitated by phosphorylation of the conserved threonine residue in the actin-binding domain. In this study, we describe a novel function of ezrin in regulating JNK activation that is mediated by phosphorylation of a tyrosine (Y353) residue that is unconserved with moesin and radixin. BCR, but not CD40, TLR4, or CXCR5 stimulation, induced phosphorylation of ezrin at Y353 in mouse splenic B cells. Ezrin existed in a pre- formed complex with Syk in unstimulated B cells and underwent Syk-dependent phosphorylation upon anti-IgM stimulation. Y353- phosphorylated ezrin colocalized with the BCR within minutes of stimulation and cotrafficked with the endocytosed BCRs through Downloaded from the early and late endosomes. The T567 residue of ezrin was rephosphorylated in late endosomes and at the plasma membrane at later times of BCR stimulation. Expression of a nonphosphorylatable Y353F mutant of ezrin specifically impaired JNK activation. BCR crosslinking induced the association of Y353-phosphorylated ezrin with JNK and its kinase MAPKK7, as well as spatial co- localization with phosphorylated JNK in the endosomes. The yellow fluorescent protein–tagged Y353F mutant displayed reduced colocalization with the endocytosed BCR as compared with wild-type ezrin-yellow fluorescent protein. Taken together, our data identify a novel role for ezrin as a spatial adaptor that couples JNK signaling components to the BCR signalosome, thus http://www.jimmunol.org/ facilitating JNK activation. The Journal of Immunology, 2013, 190: 2017–2026. ntigen recognition by the BCR in mature B cells triggers influence the assembly of intracellular signaling components with a signaling cascade that culminates in transcriptional the BCR in endosomal signalosomes and play an important role in A activation and proliferation (1, 2). At the outset, BCR regulating BCR signaling. signaling is accompanied by actin cytoskeletal reorganization that The cortical actin filaments are held underneath the plasma facilitates the formation of BCR microclusters, Ag gathering by membrane by adaptor proteins that tether transmembrane proteins the spreading B cell, and the assembly of BCR signalosomes (3, to actin. Ezrin, a plasma membrane–actin cytoskeleton cross- by guest on September 24, 2021 4). This coordination between intracellular signaling molecules linking protein of the ezrin-radixin-moesin (ERM) family, con- and the cytoskeleton modulates the strength of B cell activation (3, tains a conserved threonine residue (T567) in its C-terminal actin- 5, 6). Clustering of the BCR signalosomes is also accompanied binding domain. Phosphorylation of this threonine is critical for by rapid internalization and trafficking of the Ag-bound BCRs to conformational activation and plasma membrane–cytoskeleton the late endosomes for further processing of the Ag and loading crosslinking activity of ERM proteins (9). We previously reported on MHC class II molecules (7). The endocytosed BCRs in turn that ezrin is constitutively phosphorylated at T567 in naive B cells, cosegregate with tyrosine and serine/threonine kinases within the and dephosphorylation of this site upon BCR stimulation results in endosomal compartments and continue to support signal trans- conformational inactivation facilitating lipid raft coalescence (10). duction (8). It is very likely that cytoskeleton-regulating proteins Similarly, chemokine exposure induces T567 dephosphorylation in ezrin in B cells, and the resulting uncoupling of plasma mem- brane from the actin cytoskeleton is required for the morpholog- Department of Immunology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195 ical and cytoskeletal changes essential for B cell migration (11). Ezrin-rich networks confine BCR mobility in the absence of Ag 1Current address: Department of Pathology, Case Western Reserve University, Cleve- land, OH. (5), but they undergo dynamic remodeling upon Ag stimulation 2Current address: Haverford College, Haverford, PA. to facilitate Ag-receptor clustering (12). Therefore, Ag-induced Received for publication May 7, 2012. Accepted for publication December 14, 2012. conformational inactivation of ezrin is an important regulator of membrane dynamics during BCR signal transduction. This work was supported by National Institutes of Health/National Institute of Al- lergy and Infectious Diseases Grant AI081743, as well as by an Investigator Award High structural homology between ezrin and moesin and their from the Cancer Research Institute (to N.G.). well-established role as membrane–cytoskeletal crosslinkers has Address correspondence and reprint requests to Dr. Neetu Gupta, Department of led to the notion that the two proteins have redundant function Immunology, Lerner Research Institute, Cleveland Clinic, 9500 Euclid Avenue, NE40, in lymphocyte activation and migration (9). Indeed, ezrin-deficient Cleveland, OH 44195. E-mail address: [email protected] mature T cells show defect in TCR-dependent IL-2 production, The online version of this article contains supplemental material. which is exacerbated upon additional knockdown of moesin ex- Abbreviations used in this article: EEA1, early endosome Ag 1; ERM, ezrin- radixin-moesin; JIP, JNK-interacting protein; LAMP1, lysosomal-associated membrane pression (13). Interestingly, despite high overall homology between protein 1; MKK7, MAPKK7; pI2E, pIRES-2–enhanced GFP; pThrERM, phos- ezrin and moesin, the amino acid sequence of ezrin contains unique phorylated threonine in ezrin-radixin-moesin; SFK, Src family kinase; YFP, yellow phosphorylation sites (S66, Y353, and Y477), and a polyproline fluorescent protein. stretch at 469–475 (14), suggesting that ezrin may have additional Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 unconserved context-dependent roles. These features in ezrin may www.jimmunol.org/cgi/doi/10.4049/jimmunol.1201292 2018 Y353-PHOSPHORYLATED EZRIN REGULATES JNK ACTIVATION enable protein–protein interactions to facilitate signal transduction were washed and resuspended in DMEM medium supplemented with 1% and/or localization of interacting proteins. S66-phosphorylated BSA and 20 mM HEPES. The cells were maintained in the dark at room ezrin regulates trafficking of H+,K+-ATPase to the apical mem- temperature and a ratio of UV450/525 nm was recorded to establish the baseline fluorescence for unstimulated cells. Cells were stimulated at 30 s brane of gastric parietal cells and is essential for histamine-induced with 10 mg/ml anti-IgM, and the UV450/525 ratio was recorded for 200 s. acid secretion (15). Growth factor–dependent phosphorylation of Samples were acquired using a BD LSRII flow cytometer and data were ezrin at Y353 was shown to regulate survival of nonhematopoietic analyzed using FlowJo (Tree Star). cells (16, 17), whereas Y477 phosphorylation of ezrin regulates Immunofluorescence microscopy growth and invasion of Src-transformed epithelial cells in a three- dimensional environment (18). In human B lymphoma cells, CD81 To visualize the BCR, CH27 cells were stimulated with biotin-conjugated anti-IgM, fixed with 4% PFA, blocked with PBS containing 15% goat serum crosslinking was shown to induce Y353 phosphorylation of ezrin for 30 min, followed by permeabilization and staining with Alexa Fluor 488/ that recruits F-actin and facilitates cytoskeletal reorganization (19). 633–conjugated streptavidin (Molecular Probes) for 45 min at 4˚C. Next, Whether any of these alternate phosphorylation sites contribute the cells were stained with the pY353 Ab overnight at 4˚C and