Cutting Edge: Feed-Forward Activation of γ2 via C2 Domain−Mediated Binding to SLP65

This information is current as Michael Engelke, Thomas Oellerich, Kai Dittmann, of September 29, 2021. He-Hsuan Hsiao, Henning Urlaub, Hubert Serve, Christian Griesinger and Jürgen Wienands J Immunol 2013; 191:5354-5358; Prepublished online 28 October 2013;

doi: 10.4049/jimmunol.1301326 Downloaded from http://www.jimmunol.org/content/191/11/5354

Supplementary http://www.jimmunol.org/content/suppl/2013/10/30/jimmunol.130132 Material 6.DC1 http://www.jimmunol.org/ References This article cites 15 articles, 8 of which you can access for free at: http://www.jimmunol.org/content/191/11/5354.full#ref-list-1

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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 © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Th eJournal of Cutting Edge Immunology

Cutting Edge: Feed-Forward Activation of Phospholipase Cg2 via C2 Domain–Mediated Binding to SLP65 ,1 ,†,1 ‡ Michael Engelke,* x Thomas Oellerich,* Kai Dittmann,*{ He-Hsuan Hsiao, Henning Urlaub,‡, Hubert Serve,† Christian Griesinger, and Ju¨rgen Wienands* Ag-mediated B cell stimulation relies on phospholipase fluxes, suggesting that the activity of PLCg2 or its inducible Cg2(PLCg2) for Ca2+ mobilization. Enzymatic activity association to SLP65 is subject to further regulation. More- of PLCg2 is triggered upon Src homology 2 domain– over, the earliest and most prominent pTyr site of avian mediated binding to the tyrosine-phosphorylated adaptor SLP65, tyrosine 138 (Y138) (8), is not involved in SH2 domain SLP65. However, SLP65 phosphorylation outlasts the binding (4) but is conserved between SLP65 orthologs and elevation of cytosolic Ca2+ concentration suggesting ad- embedded within the consensus sequence EYFDN (single- ditional levels of PLCg2 regulation. We show in this letter code for amino acids with F representing A, V, or I). Downloaded from A similar motif can be found in the T cell paralog SLP76 (3), article that the functionality of the PLCg2/SLP65 com- 173 plex is controlled by the weakly characterized C2 domain albeit at a different position (i.e., Y in murine SLP76) and without a negatively charged residueNterminaltothetyrosine. of PLCg2. Usually C2 domains bind membrane lipids, 173 but that of PLCg2docksinaCa2+-regulated manner Phosphorylation of Y in SLP76 by the Btk-related kinase Itk is pivotal for PLCg1 activation in T lymphocytes and mast

to a distinct phosphotyrosine of SLP65. Hence, early http://www.jimmunol.org/ 2+ cells in vitro and in vivo (9). The mechanism of phospho-Y173 Ca fluxing provides feed-forward signal amplifica- signaling remained elusive. In this article, we show that the tion by promoting anchoring of the PLCg2C2do- corresponding EYFDN motif of SLP65 critically contributes main to phospho-SLP65. As the cellular Ca2+ resources 2+ to B cell activation. The underlying mechanism is unusual. become exhausted, the concomitant decline of Ca Unlike canonical pTyr–SH2 interactions, the phosphory- dampens the C2-phosphotyrosine interaction so that lated EYFDN motif provides a selective and Ca2+-regulated PLCg2 activation terminates despite sustained SLP65 docking site for the C2 domain of PLCg2. This finding phosphorylation. The Journal of Immunology, 2013, uncovers a feed-forward activation loop in that binding to

191: 5354–5358. phosphorylated SLP65 is primed by the PLCg2 SH2 do- by guest on September 29, 2021 mains and further stabilized by the C2 domain upon initial release of Ca2+. The transient nature of Ca2+ fluxes even- ngagement of the BCR and activation of spleen tyrosine tually limits the C2–pTyr interaction, which provides neg- kinase Syk triggers phosphorylation of the adaptor ative feedback regulation to PLCg2 during later phases of SLP65 (1) (or BLNK) (2). Phospho-SLP65 E B cell stimulation. nucleates the assembly of downstream effectors into larger complexes to control a plethora of signaling pathways (3). For mobilization of the Ca2+ second messenger, SLP65 simul- Materials and Methods Cell culture and signal transduction analyses taneously recruits phospholipase Cg2 (PLCg2) and Bruton’s tyrosine kinase (Btk) to distinct phosphotyrosine (pTyr) res- DT40 and Ramos B cells have been described previously (8, 10). Primary human B cells purified via the Isolation Kit II (Miltenyi) were stimulated idues by virtue of the ’ Src homology (SH) 2 domains with 10 mg/ml goat anti-human IgM (Southern Biotechnology). Abs recog- (4–6). This structural organization allows Btk to phosphorylate nizing the phospho-Y119 motif were raised against the peptide 113PFARGE and activate PLCg2. The model and its role for B cell acti- (pY)IDNRS124 (Perbio Science) and immobilized with AminoPlus Kit (Perbio vation is supported by mutational analyses and biochemical Science). Abs against GFP or pTyr (4G10), and recombinant PLCg were purchased from Roche and Millipore, respectively. Ratiometric Ca2+ con- approaches in various experimental systems (7). However, centrations were measured by flow cytometry (8, 10). Confocal laser-scanning 2+ SLP65 phosphorylation lasts longer than intracellular Ca microscopy was conducted on a Leica SP2 system.

*Institute of Cellular and Molecular Immunology, Georg August University of Go¨ttingen, Address correspondence and reprint requests to Dr. Ju¨rgen Wienands, Institute of Cellular 37073 Go¨ttingen, Germany; †Department of Hematology and Oncology, Johann and Molecular Immunology, Georg August University of Go¨ttingen, Humboldtallee 34, Wolfgang Goethe University of Frankfurt, 60590 Frankfurt, Germany; ‡Core Facility of 37073 Go¨ttingen, Germany. E-mail address: [email protected] Bioanalytical Mass Spectrometry, Max Planck Institute for Biophysical Chemistry, x The online version of this article contains supplemental material. 37077 Go¨ttingen, Germany; Bioanalytics Group, Department of Clinical Chemistry, { University Medical Center Go¨ttingen, 37075 Go¨ttingen, Germany; and Department Abbreviations used in this article: Btk, Bruton’s tyrosine kinase; LC-MS/MS, liquid for Nuclear Magnetic Resonance–Based Structural Biology, Max Planck Institute for chromatography–coupled tandem mass spectrometry; PLC, phospholipase C; pTyr, Biophysical Chemistry, 37077 Go¨ttingen, Germany phosphotyrosine; SH, Src homology; SILAC, stable isotope labeling with amino acids in cell culture. 1M.E. and T.O. contributed equally to this work. Received for publication June 18, 2013. Accepted for publication October 6, 2013. Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 This work was supported by the Deutsche Forschungsgemeinschaft through Grant SFB 860, Project B5.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1301326 The Journal of Immunology 5355

Expression constructs and transfections mary murine B cells from wild-type or SLP65-deficient mice Vectors encoding citrine- or STrEP-tagged SLP65 have been described previously that were retrovirally transduced to express wild-type human 119 (8, 10). The cDNA of rat PLCg2 was inserted into pEGFP-N1 (BD Biosciences), SLP65 responded more effectively than Y F-expressing trans- and the PLCg2-EGFP coding fragment was ligated into pcDNA3 (Invitrogen) ductants (Fig. 1D, black and gray curves, respectively). The containing a puromycin resistance cassette. The C2 mutant of PLCg2 generated 2+ 2/2 by overlap extension PCR lacks aa M1043 to V1237. Indicated point mutations Ca profile of the latter B cells was similar to that of slp65 were introduced by site-directed mutagenesis (Quickchange, Stratagene). Trans- control cells (red curve). Likewise, SLP65-deficient DT40 B cells duced or transfected cells were selected with puromycin at 1 mg/ml. that were reconstituted with wild-type versions of human or avian SLP65 showed markedly increased Ca2+ fluxes com- Proteome analyses of stable isotope labeling with amino acids in cell pared with those B cells that expressed the species-matched culture–labeled signaling complexes Y-to-F variant (Supplemental Fig. 1B). Hence, SLP65 ortho- Stable isotope labeling with amino acids in cell culture (SILAC) as well as logs that cannot be phosphorylated at the EYFDN motif fail mass spectrometric identification and quantification of phospho–acceptor 2+ sites or SLP65 ligands have been described (8, 10). In brief, “heavy” SILAC to support a proper Ca response in B cells from different 2 12 14 13 14 medium contained D4, C6, N2-lysine and [ C]6, N4-arginine, whereas species. This deficit translated into compromised activation of 12 14 12 14 “light” medium contained C6, N2-lysine and C6, N4-arginine. JNK and ERK as determined by phospho–site-specific immu- 113 124 bound to immobilized peptides [ PFARGE(p)YIDNRS ]orSTrEP-tagged noblottings (Supplemental Fig. 1C, 1D). Next, we expressed SLP65 were identified on an Orbitrap Velos instrument. Specific interactors were represented by $3 unique peptides at a “heavy-to-light” ratio .5. SLP65 variants as citrine fusion proteins to directly monitor the impact of Y119 phosphorylation on SLP65 recruitment from

Results and Discussion the cytosol to the activated BCR by confocal laser-scanning Downloaded from The EYFDN motif of SLP65 serves as Syk substrate in primary microscopy. This process is a critical step for SLP65 phos- human B cells phorylation and signaling (10). The images of Fig. 1E show 119 We previously determined the phosphorylation dynamics of that both wild-type and Y F mutant SLP65 translocated avian SLP65 in DT40 chicken B cells and identified Y138 within equally well to the plasma membrane on BCR ligation. Fur- thermore, Syk became activated with the same efficacy in both a conserved EYFDN sequence (see Supplemental Fig. 1A) as 2/2 major and immediate early pTyr site (8). We now repeated types of B cells as well as in slp65 mutants (Supplemental http://www.jimmunol.org/ these studies with primary human B cells. The mass spectro- Fig. 1C, 1D). Our results reveal that the major pTyr site of metric profiles in Fig. 1A show that the corresponding site of SLP65 in avian and human orthologs is dispensable for the human SLP65, Y119, was likewise robustly phosphorylated early subcellular navigation of SLP65, but mandatory for proper after BCR stimulation. To determine the upstream kinase, we activation of BCR signal cascades. generated phospho–site-specific Abs (anti-pY119) and expressed PLCg2 is the unique in vivo effector of phospho-Y119 human SLP65 or, as control, a Y119Fvariantinwild-type 119 DT40 B cells or mutant cells that lacked Lyn, Syk, or Btk. To decipher the mechanism of how Y phosphorylation boosts 119 Anti-pY immunoblotting detected human SLP65 from ac- SLP65 signaling, we used a 2-fold mass spectrometric ap- by guest on September 29, 2021 tivated but not from resting wild-type B cells (Fig. 1B, lanes 1, proach. First, we screened for putative ligands of phospho- 119 2). The Y119F variant was not detected at all (lanes 3, 4). Y –encompassing peptides in vitro. Second, we conducted a 119 Inducible phosphorylation of Y119 was also observed in Lyn- differential interactome analysis of wild-type versus Y F deficient B cells (lanes 5–8). Notably, no other Src family mutant SLP65 in vivo (“reverse proteomics”) (10). For peptide- kinase is expressed in these cells. The absence of Syk abrogated based screening of ligands, Ramos B cells were metabolically Y119 phosphorylation (lanes 9–12), suggesting that Syk phos- labeled in “heavy” or “light” SILAC culture medium. Sub- phorylates Y119 either directly or through the activation of the sequently, lysates of the heavily labeled cells were subjected downstream kinase Btk. In support of the former possibility, to affinity purification with the immobilized SLP65 phospho- 113 124 inducible phosphorylation of wild-type SLP65 was still ob- peptide PFARGE(pY)IDNRS , whereas the unphosphory- 2 2 served in btk / Bcells(lanes 13–16). Furthermore, Y119 was lated counterpart was incubated with lysates of lightly labeled also phosphorylated in SLP65 variants that lacked the Btk- cells. Purified proteins were identified and relatively quantified binding site (Y96F) or that harbor Y119 as the only tyrosine by liquid chromatography–coupled tandem mass spectrometry residue (Fig. 1C). These results strongly suggest that Y119 is a (LC-MS/MS). For evaluation of the results, we determined the direct substrate of Syk in cultured and primary human B cells. enrichment factor with which a given protein was selectively The phosphorylation reaction is independent of other pTyr. By purified via the phosphorylated SLP65 peptide by calculating contrast, the corresponding site of SLP76 becomes phosphory- the ratio of the heavily versus the lightly labeled peptide spe- lated by Btk-related Itk in a hierarchical manner only upon cies from that ligand. Then, the individual ratios were plotted preceding phosphorylations on other sites by Syk-related against the ligand’s identification intensity that is represented ZAP70 (9). The independence of Y119 phosphorylation of a by the number of different peptides derived from an individual “primer kinase” is most likely caused by the N-terminal gluta- protein (Fig. 2A). Except for one ligand, all of the obtained mate, which is absent in SLP76. Negatively charged amino acids proteins showed a “heavy-to-light” ratio 1, demonstrating guide tyrosine kinases to a C-terminally located substrate site that they bound the unphosphorylated and phosphorylated (11). The 4G10 anti-pTyr Ab weakly recognizes phospho-Y119 SLP65 peptide with the same intensity irrespectively of how (data not shown) so that it was missed in an earlier study (4). many peptides from those ligands were identified. The excep- tion was PLCg2, which was up to 20-fold enriched by the Phosphorylation of EYFDN controls SLP65-mediated B cell phosphorylated form of the Y119 peptide. This strongly sug- activation gested PLCg2 to be an exquisite ligand of phospho-Y119.To The signaling function of the phospho-Y119 was assessed by explore that possibility in vivo, and moreover in an unbiased flow cytometric recording of BCR-induced Ca2+ fluxes. Pri- manner, we asked whether the loss of Y119 phosphorylation 5356 CUTTING EDGE: FEED-FORWARD ACTIVATION OF PLCg2 Downloaded from http://www.jimmunol.org/

FIGURE 1. (A) LC-MS/MS and MS spectra (left and right, respectively) with the indicated fragment ions identify a singly phosphorylated peptide with m/z= 473,6866 as 117GE(pY)IDNR123 of SLP65 from primary human B cells that were BCR-stimulated for 1 min. (B) Wild-type DT40 B cells (lanes 1–4) or mutants that lack Lyn, Syk, or Btk (lanes 5–8, 9–12, and 13–16, respectively) were reconstituted with tagged versions of human SLP65 or the Y119F variant. Transfectants were left untreated (0) or BCR-stimulated for 3 min (3), and subjected to immunoblotting with Abs recognizing the phosphorylated EYI/VDNR motif (upper by guest on September 29, 2021 panels) or SLP65 itself via anti-tag Abs (lower panels, respectively). (C) SLP65-deficient DT40 B cells were transfected with an empty control vector (crtl, lanes 1, 2) or expression constructs encoding either wild-type SLP65 (lanes 3, 4), Y96F SLP65 that is unable to bind Btk (lanes 5, 6), Y119F SLP65 (lanes 7, 8), or a SLP65 variant that accommodates Y119 as the only tyrosine phosphorylation site (Y119only, lanes 9, 10). Cells were treated and analyzed as in (B). (D) BCR-induced flow 2 2 cytometric Ca2+ profiles of wild-type primary B cells (green line) and slp65 / mouse B cells (red line) and retroviral transductants expressing citrine-tagged human SLP65 (black line) or the Y119F mutant (gray line). (E) DT40 B cells expressing citrine-tagged human wild-type SLP65 or the Y119F variant were analyzed by confocal microscopy in the absence or presence of 3-min BCR stimulation (left and right image, respectively). Each image represents a 25 mm 3 25 mm section. affects the overall composition of the SLP65 interactome in in BCR-induced Ca2+ fluxes (12). C2 domains were described stimulated B cells. We therefore quantitatively compared the first as conserved region number two in classical protein kinase C interactomes of wild-type and Y119F mutant SLP65 by SILAC- isoforms, and are classical lipid-binding moieties. However, the based mass spectrometry (“reverse proteomics”) (10). The C2 domains of protein kinase Cd and u can bind pTyr-based amount of a given ligand that was purified with the Y119F peptides (13, 14). We used the immobilized SLP65 peptides variant was normalized to that obtained with wild-type SLP65 to purify proteins from DT40 transfectants expressing either (Fig. 2B). Consistent with our peptide screening approach, wild-type PLCg2 or a truncated version that lacks the C2 do- 2 2 only PLCg2 showed an altered SLP65 association ratio that main (PLCg2DC2). As control, we used / parental cells. was decreased by ∼50% for the Y119F mutant. Fig. 2C shows Fig. 3A shows that wild-type PLCg2, but not PLCg2DC2, the functional consequence of the Y119FexchangeforBCR- complexed with the phosphorylated SLP65 peptide (upper induced PLCg2 phosphorylation. It was barely detectable in panel, lanes 3, 4 and lanes 5, 6, respectively). Binding strongly B cells expressing the Y119F variant. In summary, the major increased in the presence of Ca2+ (upper panel, lanes 3, 4), which pTyr site of SLP65 controls the recruitment and subsequent may induce conformational changes that tighten the inter- activation of a single effector protein, namely, PLCg2. action as reported for the homologous C2 domain of PLCd g 119 (15). No PLC 2 signal was detected in the negative control The C2 domain of PLCg2 interacts with phopho-Y (upper panel, lanes 1, 2) or in the material that was purified Putative PLCg2 docking modules for phospho-Y119 are the with the unphosphorylated peptide (middle panel). Incubation lipase’s two SH2 domains. However, their consensus binding of the peptides with different concentrations of recombinantly motif (11) markedly differs from EYFDN. In fact, they rec- expressed PLCg2 showed that the binding is direct (Fig. 3B, ognize three YXXP type phosphorylation sites in SLP65 (4). upper panel). In this approach, the positive influence of Ca2+ We therefore asked whether phospho-Y119 binds other PLCg2 ions was obvious at PLCg2concentrations,100 pg/ml, which modules such as the C2 domain, which has been implicated is in accordance with physiological conditions. Interestingly, The Journal of Immunology 5357 Downloaded from

FIGURE 2. (A) Ramos B cells were metabolically labeled in either “light” (L) or “heavy” (H) SILAC medium. Upon BCR stimulation, proteins of H- and L-labeled cells were purified with immobilized versions of the phosphorylated or unphosphorylated Y119 peptide, respectively. Obtained proteins were pooled at http://www.jimmunol.org/ a 1:1 ratio, digested with trypsin, and subjected to quantitative LC-MS/MS analysis. H/L ratios of identified proteins were plotted against the number of peptides (data point for PLCg2 in red). The complete proteomic data sets and statistics are listed in Supplemental Table 1. (B) Differential interactome analysis of wild-type versus Y119F mutant SLP65 (“reverse proteomics”). SLP65 assembled signalosomes were affinity-purified via the STrEP tag from SILAC-labeled DT40 B cells that were BCR-stimulated for 3 min. Signalosome components were identified and quantified by LC-MS/MS analysis. The amount of a given ligand that was purified with wild-type SLP65 was set to 1 (indicated by the red line). The amounts of Y119F ligands were normalized accordingly and expressed in the bar diagram as the fold change to 1 including SDs measured in three independent experiments. For complete data sets, see Supplemental Table 1. (C) SLP65-deficient cells (lanes 1–4) and reconstituted transfectants expressing wild-type SLP65 (lanes 5–8) or the Y119F mutant (lanes 9–12) were left untreated or BCR-stimulated for the indicated times (min). PLCg2 was immunopurified from cellular lysates and analyzed by anti-pTyr or anti-PLCg2 immunoblotting (upper and lower panels, respectively). by guest on September 29, 2021 no binding was observed between SLP65 peptides and the restricted function of the phospho-Y119, namely, to promote g1 isoform of PLC (Fig. 3B, lower panel). Not only does the the recruitment of PLCg2. Furthermore, B cells that express latter result demonstrate the specificity of the experimental assay, PLCg2DChardlymobilizedCa2+ upon BCR ligation (Fig. it also confirms our proteomic results that indicated a highly 3C), which is consistent with the almost blunted Ca2+ response

FIGURE 3. (A) PLCg2-deficient DT40 B cells were transfected with an empty control vector (lanes 1, 2), or expression constructs encoding GFP-tagged PLCg (lanes 3, 4) or PLCg2DC2 (lanes 5, 6). On BCR activation (3 min), lysates were subjected to affinity purifications using the phosphorylated or unphosphorylated forms of the immobilized peptide 113PFARGEYINRS124 (upper and middle panels, respectively) in the absence (2) or presence (+) of 0.5 mM Ca2+. Obtained proteins and cellular lysates (lower panel) were analyzed by anti-GFP immunoblotting. (B) Indicated amounts of recombinantly expressed PLCg2(upper panel) or PLCg1(lower panel) were incubated with the unphosphorylated or phosphorylated Y119 peptides (lanes 1–6 and 7–12, respectively) in the absence (2)or presence (+) of 0.2 mM Ca2+. Bound proteins were detected by immunoblotting with anti-PLCg2 or anti-PLCg1 Abs (upper and lower panels, respectively). Signals of lanes 7–12 (upper panel) were quantified, and the relative signal increase for Ca2+-containing samples was plotted. (C) BCR-induced Ca2+ mobilization profiles of DT40 B cell transfectants expressing wild-type or C2 domain–truncated PLCg2 (black and gray curves, respectively). 5358 CUTTING EDGE: FEED-FORWARD ACTIVATION OF PLCg2 of Y119F-expressing cells (see earlier). Collectively, the data 4.Chiu,C.W.,M.Dalton,M.Ishiai,T.Kurosaki,andA.C.Chan.2002.BLNK: molecular scaffolding through ‘cis’-mediated organization of signaling proteins. reported in this article uncover a specific and unusual inter- EMBO J. 21: 6461–6472. 119 action between phospho-Y and the C2 domain of PLCg2. 5. Hashimoto, S., A. Iwamatsu, M. Ishiai, K. Okawa, T. Yamadori, M. Matsushita, This is mandatory to stabilize the SH2-initiated complex for- Y. Baba, T. Kishimoto, T. Kurosaki, and S. Tsukada. 1999. Identification of the 2+ 2+ SH2 domain binding protein of Bruton’s tyrosine kinase as BLNK—functional mation with SLP65 for proper Ca flux. The Ca sensitivity significance of Btk-SH2 domain in B-cell antigen receptor-coupled signaling. of the C2 interaction has two consequences. Early after BCR Blood 94: 2357–2364. 2+ 6. Su, Y. W., Y. Zhang, J. Schweikert, G. A. Koretzky, M. Reth, and J. Wienands. ligation, an increase of cytosolic Ca boosts SLP65/PLCg2 1999. Interaction of SLP adaptors with the SH2 domain of Tec family kinases. Eur. complex formation, whereas the later adjustment of Ca2+ to J. Immunol. 29: 3702–3711. 7. Engelke, M., N. Engels, K. Dittmann, B. Stork, and J. Wienands. 2007. Ca(2+) baseline concentrations weakens that interaction, which may signaling in antigen receptor-activated B lymphocytes. Immunol. Rev. 218: 235– contribute signal termination independently of . 246. 8. Oellerich, T., M. Grønborg, K. Neumann, H. H. Hsiao, H. Urlaub, and Hence balanced PLCg2 activation is controlled by a single J. Wienands. 2009. SLP-65 phosphorylation dynamics reveals a functional basis phosphorylation event in SLP65. This ensures a rapid B cell for signal integration by receptor-proximal adaptor proteins. Mol. Cell. Proteomics g 8: 1738–1750. response on the one hand, and a transient action of PLC 2 9.Sela,M.,Y.Bogin,D.Beach,T.Oellerich,J.Lehne,J.E.Smith-Garvin,M.Okumura, for limited B cell stimulation on the other hand. E. Starosvetsky, R. Kosoff, E. Libman, et al. 2011. Sequential phosphorylation of SLP-76 at tyrosine 173 is required for activation of T and mast cells. EMBO J. 30: 3160–3172. Acknowledgments 10. Oellerich, T., V. Bremes, K. Neumann, H. Bohnenberger, K. Dittmann, H. H. Hsiao, We thank Ines Heine and Tabea Testa for excellent assistance and Dr. Michael M. Engelke, T. Schnyder, F. D. Batista, H. Urlaub, and J. Wienands. 2011. The B-cell 2/2 antigen receptor signals through a preformed transducer module of SLP65 and Reth for providing slp65 mice. CIN85. EMBO J. 30: 3620–3634. Downloaded from 11. Songyang, Z., and L. C. Cantley. 1995. Recognition and specificity in protein tyrosine kinase-mediated signalling. Trends Biochem. Sci. 20: 470–475. Disclosures 12. Nishida, M., K. Sugimoto, Y. Hara, E. Mori, T. Morii, T. Kurosaki, and Y. Mori. The authors have no financial conflicts of interest. 2003. Amplification of receptor signalling by Ca2+ entry-mediated translocation and activation of PLCgamma2 in B lymphocytes. EMBO J. 22: 4677–4688. 13.Benes,C.H.,N.Wu,A.E.Elia,T.Dharia,L.C.Cantley,andS.P.Soltoff. 2005. The C2 domain of PKCdelta is a phosphotyrosine binding domain. Cell

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