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• W3043290063 abstract "Although signal transduction by immunoreceptors such as the T cell antigen receptor (TCR), B cell antigen receptor (BCR), and Fc receptors uses the same schematic and similar molecules, the threshold and the fine-tuning are set differently for each receptor. One manifestation of these differences is that inhibition of Src family kinases (SFK) blocks TCR but not BCR signaling. SFKs are key kinases phosphorylating immunoreceptor tyrosine-based activation motifs (ITAM) in both these receptors. However, it has been proposed that in B cells, downstream kinase SYK can phosphorylate ITAM sequences independently of SFK, allowing it to compensate for the loss of SFK activity, whereas its T cell paralog ZAP-70 is not capable of this compensation. To test this proposal, we examined signaling in SYK- and ZAP-70–deficient B and T cell lines expressing SYK or ZAP-70. We also analyzed signal transduction in T cells expressing BCR or B cells expressing part of the TCR complex. We show that when compared with ZAP-70, SYK lowered the threshold for SFK activity necessary to initiate antigen receptor signaling in both T and B cells. However, neither SYK nor ZAP-70 were able to initiate signaling independently of SFK. We further found that additional important factors are involved in setting this threshold. These include differences between the antigen receptor complexes themselves and the spatial separation of the key transmembrane adaptor protein LAT from the TCR. Thus, immunoreceptor sensing of SFK activity is a complex process regulated at multiple levels. Although signal transduction by immunoreceptors such as the T cell antigen receptor (TCR), B cell antigen receptor (BCR), and Fc receptors uses the same schematic and similar molecules, the threshold and the fine-tuning are set differently for each receptor. One manifestation of these differences is that inhibition of Src family kinases (SFK) blocks TCR but not BCR signaling. SFKs are key kinases phosphorylating immunoreceptor tyrosine-based activation motifs (ITAM) in both these receptors. However, it has been proposed that in B cells, downstream kinase SYK can phosphorylate ITAM sequences independently of SFK, allowing it to compensate for the loss of SFK activity, whereas its T cell paralog ZAP-70 is not capable of this compensation. To test this proposal, we examined signaling in SYK- and ZAP-70–deficient B and T cell lines expressing SYK or ZAP-70. We also analyzed signal transduction in T cells expressing BCR or B cells expressing part of the TCR complex. We show that when compared with ZAP-70, SYK lowered the threshold for SFK activity necessary to initiate antigen receptor signaling in both T and B cells. However, neither SYK nor ZAP-70 were able to initiate signaling independently of SFK. We further found that additional important factors are involved in setting this threshold. These include differences between the antigen receptor complexes themselves and the spatial separation of the key transmembrane adaptor protein LAT from the TCR. Thus, immunoreceptor sensing of SFK activity is a complex process regulated at multiple levels. Evolution of receptors and associated signaling pathways has generated a range of different signal transduction systems. However, among this variety, groups of receptors can be distinguished which utilize common schemes of signal transmission. The multiple usage of similar molecules in similar arrangements is typical for immunoreceptor signaling, including signaling by TCR, BCR, Fc receptors, NK cell receptors, and several others (1Irving B. Weiss A. A clue to antigen receptor tails.J. Immunol. 2014; 192 (24748634): 4013-401410.4049/jimmunol.1400660Crossref PubMed Scopus (3) Google Scholar, 2Getahun A. Cambier J.C. Of ITIMs, ITAMs, and ITAMis: Revisiting immunoglobulin Fc receptor signaling.Immunol. Rev. 2015; 268 (26497513): 66-7310.1111/imr.12336Crossref PubMed Scopus (91) Google Scholar, 3Packard T.A. Cambier J.C. B lymphocyte antigen receptor signaling: Initiation, amplification, and regulation.F1000Prime Rep. 2013; 5 (24167721): 4010.12703/P5-40Crossref PubMed Scopus (67) Google Scholar, 4Lanier L.L. Up on the tightrope: Natural killer cell activation and inhibition.Nat. Immunol. 2008; 9 (18425106): 495-50210.1038/ni1581Crossref PubMed Scopus (1181) Google Scholar). The signaling is initiated by Src family kinases (SFK), which phosphorylate immunoreceptor tyrosine-based activation motifs (ITAM), located within intracellular domains of receptor-associated transmembrane adaptor proteins. Phosphorylated ITAM motifs serve as docking sites for SYK family kinases. Their activation is a crucial step in subsequent signal transduction. It results in the recruitment and assembly of a signalosome activating Ca2+ response (increase of intracellular concertation of free Ca2+ ions) (1Irving B. Weiss A. A clue to antigen receptor tails.J. Immunol. 2014; 192 (24748634): 4013-401410.4049/jimmunol.1400660Crossref PubMed Scopus (3) Google Scholar, 2Getahun A. Cambier J.C. Of ITIMs, ITAMs, and ITAMis: Revisiting immunoglobulin Fc receptor signaling.Immunol. Rev. 2015; 268 (26497513): 66-7310.1111/imr.12336Crossref PubMed Scopus (91) Google Scholar, 3Packard T.A. Cambier J.C. B lymphocyte antigen receptor signaling: Initiation, amplification, and regulation.F1000Prime Rep. 2013; 5 (24167721): 4010.12703/P5-40Crossref PubMed Scopus (67) Google Scholar, 4Lanier L.L. Up on the tightrope: Natural killer cell activation and inhibition.Nat. Immunol. 2008; 9 (18425106): 495-50210.1038/ni1581Crossref PubMed Scopus (1181) Google Scholar). Several studies have questioned this basic scheme of immunoreceptor signaling by suggesting that under some circumstances BCR signaling can be triggered independently of SFKs (5Rolli V. Gallwitz M. Wossning T. Flemming A. Schamel W.W. Zürn C. Reth M. Amplification of B cell antigen receptor signaling by a Syk/ITAM positive feedback loop.Mol. Cell. 2002; 10 (12453414): 1057-106910.1016/S1097-2765(02)00739-6Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar, 6Saijo K. Schmedt C. Su I.H. Karasuyama H. Lowell C.A. Reth M. Adachi T. Patke A. Santana A. Tarakhovsky A. Essential role of Src-family protein tyrosine kinases in NF-κB activation during B cell development.Nat. Immunol. 2003; 4 (12563261): 274-27910.1038/ni893Crossref PubMed Scopus (234) Google Scholar, 7Takata M. Sabe H. Hata A. Inazu T. Homma Y. Nukada T. Yamamura H. Kurosaki T. Tyrosine kinases Lyn and Syk regulate B cell receptor-coupled Ca2+ mobilization through distinct pathways.EMBO J. 1994; 13 (8137818): 1341-134910.1002/j.1460-2075.1994.tb06387.xCrossref PubMed Scopus (587) Google Scholar, 8Mukherjee S. Zhu J. Zikherman J. Parameswaran R. Kadlecek T.A. Wang Q. Au-Yeung B. Ploegh H. Kuriyan J. Das J. Weiss A. Monovalent and multivalent ligation of the B cell receptor exhibit differential dependence upon Syk and Src family kinases.Sci. Signal. 2013; 6 (23281368): ra110.1126/scisignal.2003220Crossref PubMed Scopus (57) Google Scholar). We and others have demonstrated that SFK inhibition by widely used inhibitor PP2 (9Hanke J.H. Gardner J.P. Dow R.L. Changelian P.S. Brissette W.H. Weringer E.J. Pollok B.A. Connelly P.A. Discovery of a novel, potent, and Src family-selective tyrosine kinase inhibitor. Study of Lck- and FynT-dependent T cell activation.J. Biol. Chem. 1996; 271 (8557675): 695-70110.1074/jbc.271.2.695Abstract Full Text Full Text PDF PubMed Scopus (1784) Google Scholar, 10Zhu X. Kim J.L. Newcomb J.R. Rose P.E. Stover D.R. Toledo L.M. Zhao H. Morgenstern K.A. Structural analysis of the lymphocyte-specific kinase Lck in complex with non-selective and Src family selective kinase inhibitors.Structure. 1999; 7 (10404594): 651-66110.1016/S0969-2126(99)80086-0Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar) abolished TCR signaling in T cells but was unable to block signaling by BCR in B lymphocytes (8Mukherjee S. Zhu J. Zikherman J. Parameswaran R. Kadlecek T.A. Wang Q. Au-Yeung B. Ploegh H. Kuriyan J. Das J. Weiss A. Monovalent and multivalent ligation of the B cell receptor exhibit differential dependence upon Syk and Src family kinases.Sci. Signal. 2013; 6 (23281368): ra110.1126/scisignal.2003220Crossref PubMed Scopus (57) Google Scholar, 11Stepanek O. Draber P. Drobek A. Horejsi V. Brdicka T. Nonredundant roles of Src-family kinases and Syk in the initiation of B-cell antigen receptor signaling.J. Immunol. 2013; 190 (23335753): 1807-181810.4049/jimmunol.1202401Crossref PubMed Scopus (24) Google Scholar). Similar observation was later also reported by another group, showing the same effect at the level of SLP65 and SLP76 adaptor protein phosphorylation (12Fasbender F. Claus M. Wingert S. Sandusky M. Watzl C. Differential requirements for Src-family kinases in SYK or ZAP70-mediated SLP-76 phosphorylation in lymphocytes.Front. Immunol. 2017; 8 (28736554): 78910.3389/fimmu.2017.00789Crossref PubMed Scopus (15) Google Scholar). It has been proposed that SFK-independent signaling in B cells can be explained by unique features of SYK. SYK family of kinases contains only two members, SYK and ZAP-70. They show different expression patterns. B cells and myeloid cells express mainly SYK and T cells mainly ZAP-70 (13Mócsai A. Ruland J. Tybulewicz V.L. The SYK tyrosine kinase: A crucial player in diverse biological functions.Nat. Rev. Immunol. 2010; 10 (20467426): 387-40210.1038/nri2765Crossref PubMed Scopus (861) Google Scholar, 14Au-Yeung B.B. Deindl S. Hsu L.Y. Palacios E.H. Levin S.E. Kuriyan J. Weiss A. The structure, regulation, and function of ZAP-70.Immunol. Rev. 2009; 228 (19290920): 41-5710.1111/j.1600-065X.2008.00753.xCrossref PubMed Scopus (149) Google Scholar). Both kinases are in the steady state in the cytoplasm in a closed inactive conformation and upon receptor activation, they are recruited to the phosphorylated ITAM motifs. Binding to ITAMs via their tandem SH2 domains leads to their transition to active conformation (15Klammt C. Novotná L. Li D.T. Wolf M. Blount A. Zhang K. Fitchett J.R. Lillemeier B.F. T cell receptor dwell times control the kinase activity of Zap70.Nat. Immunol. 2015; 16 (26237552): 961-96910.1038/ni.3231Crossref PubMed Scopus (49) Google Scholar, 16Grädler U. Schwarz D. Dresing V. Musil D. Bomke J. Frech M. Greiner H. Jäkel S. Rysiok T. Müller-Pompalla D. Wegener A. Structural and biophysical characterization of the Syk activation switch.J. Mol. Biol. 2013; 425 (23154170): 309-33310.1016/j.jmb.2012.11.007Crossref PubMed Scopus (56) Google Scholar, 17Deindl S. Kadlecek T.A. Brdicka T. Cao X. Weiss A. Kuriyan J. Structural basis for the inhibition of tyrosine kinase activity of ZAP-70.Cell. 2007; 129 (17512407): 735-74610.1016/j.cell.2007.03.039Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 18Yan Q. Barros T. Visperas P.R. Deindl S. Kadlecek T.A. Weiss A. Kuriyan J. Structural basis for activation of ZAP-70 by phosphorylation of the SH2-kinase linker.Mol. Cell Biol. 2013; 33 (23530057): 2188-220110.1128/MCB.01637-12Crossref PubMed Scopus (65) Google Scholar). However, to become fully active and/or to stabilize interaction with ITAMs, SYK family kinases must be further phosphorylated. For ZAP-70 this phosphorylation seems to be more dependent on SFKs than for SYK, which shows higher propensity to autophosphorylate during this step (13Mócsai A. Ruland J. Tybulewicz V.L. The SYK tyrosine kinase: A crucial player in diverse biological functions.Nat. Rev. Immunol. 2010; 10 (20467426): 387-40210.1038/nri2765Crossref PubMed Scopus (861) Google Scholar, 17Deindl S. Kadlecek T.A. Brdicka T. Cao X. Weiss A. Kuriyan J. Structural basis for the inhibition of tyrosine kinase activity of ZAP-70.Cell. 2007; 129 (17512407): 735-74610.1016/j.cell.2007.03.039Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 18Yan Q. Barros T. Visperas P.R. Deindl S. Kadlecek T.A. Weiss A. Kuriyan J. Structural basis for activation of ZAP-70 by phosphorylation of the SH2-kinase linker.Mol. Cell Biol. 2013; 33 (23530057): 2188-220110.1128/MCB.01637-12Crossref PubMed Scopus (65) Google Scholar, 19Chan A.C. Dalton M. Johnson R. Kong G.H. Wang T. Thoma R. Kurosaki T. Activation of ZAP-70 kinase activity by phosphorylation of tyrosine 493 is required for lymphocyte antigen receptor function.EMBO J. 1995; 14 (7781602): 2499-250810.1002/j.1460-2075.1995.tb07247.xCrossref PubMed Scopus (325) Google Scholar, 20Brdicka T. Kadlecek T.A. Roose J.P. Pastuszak A.W. Weiss A. Intramolecular regulatory switch in ZAP-70: analogy with receptor tyrosine kinases.Mol. Cell Biol. 2005; 25 (15923611): 4924-493310.1128/MCB.25.12.4924-4933.2005Crossref PubMed Scopus (108) Google Scholar, 21Rowley R.B. Burkhardt A.L. Chao H.G. Matsueda G.R. Bolen J.B. Syk protein-tyrosine kinase is regulated by tyrosine-phosphorylated Igα/Igβ immunoreceptor tyrosine activation motif binding and autophosphorylation.J. Biol. Chem. 1995; 270 (7538118): 11590-1159410.1074/jbc.270.19.11590Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar, 22El-Hillal O. Kurosaki T. Yamamura H. Kinet J.P. Scharenberg A.M. Syk kinase activation by a Src kinase-initiated activation loop phosphorylation chain reaction.Proc. Natl. Acad. Sci. U. S. A. 1997; 94 (9050880): 1919-192410.1073/pnas.94.5.1919Crossref PubMed Scopus (108) Google Scholar, 23Zoller K.E. MacNeil I.A. Brugge J.S. Protein tyrosine kinases Syk and ZAP-70 display distinct requirements for Src family kinases in immune response receptor signal transduction.J. Immunol. 1997; 158 (9029101): 1650-1659PubMed Google Scholar). Furthermore, it has been proposed that intense stimulation of B cells with multivalent ligands can trigger SYK to phosphorylate ITAM motifs independently of SFKs; ZAP-70 has been thought to lack this ability. However, the data directly demonstrating the SYK-mediated phosphorylation of ITAM motifs were all generated using either recombinant proteins and peptides in vitro or overexpression in nonhematopoietic cells, such as S2, 293T, or COS cells (5Rolli V. Gallwitz M. Wossning T. Flemming A. Schamel W.W. Zürn C. Reth M. Amplification of B cell antigen receptor signaling by a Syk/ITAM positive feedback loop.Mol. Cell. 2002; 10 (12453414): 1057-106910.1016/S1097-2765(02)00739-6Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar, 8Mukherjee S. Zhu J. Zikherman J. Parameswaran R. Kadlecek T.A. Wang Q. Au-Yeung B. Ploegh H. Kuriyan J. Das J. Weiss A. Monovalent and multivalent ligation of the B cell receptor exhibit differential dependence upon Syk and Src family kinases.Sci. Signal. 2013; 6 (23281368): ra110.1126/scisignal.2003220Crossref PubMed Scopus (57) Google Scholar, 23Zoller K.E. MacNeil I.A. Brugge J.S. Protein tyrosine kinases Syk and ZAP-70 display distinct requirements for Src family kinases in immune response receptor signal transduction.J. Immunol. 1997; 158 (9029101): 1650-1659PubMed Google Scholar, 24Tsang E. Giannetti A.M. Shaw D. Dinh M. Tse J.K. Gandhi S. Ho H. Wang S. Papp E. Bradshaw J.M. Molecular mechanism of the Syk activation switch.J. Biol. Chem. 2008; 283 (18818202): 32650-3265910.1074/jbc.M806340200Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). In this study, we have re-evaluated these data using human B and T cell lines. We show that SYK-mediated ITAM phosphorylation is rather limited and that what may appear as SFK-independent signaling by SYK in studies employing SFK inhibitors can be largely explained by incomplete inhibition of SFKs, coupled to a low threshold for signaling initiation in B cells. Importantly, multiple components of TCR and BCR signaling apparatuses, including antigen receptors themselves, Src and Syk family kinases, and LAT, appear to be parts of a complex mechanism differentially setting this threshold for signaling by BCR and TCR. First, we established our experimental system using model T cell line Jurkat and B cell line Ramos. In Jurkat T cells, 2 μm concentration of PP2 was sufficient to completely inhibit Ca2+ response initiated by TCR crosslinking with anti–TCR antibody C305 (Fig. 1A). Consistent with previously published data (8Mukherjee S. Zhu J. Zikherman J. Parameswaran R. Kadlecek T.A. Wang Q. Au-Yeung B. Ploegh H. Kuriyan J. Das J. Weiss A. Monovalent and multivalent ligation of the B cell receptor exhibit differential dependence upon Syk and Src family kinases.Sci. Signal. 2013; 6 (23281368): ra110.1126/scisignal.2003220Crossref PubMed Scopus (57) Google Scholar, 11Stepanek O. Draber P. Drobek A. Horejsi V. Brdicka T. Nonredundant roles of Src-family kinases and Syk in the initiation of B-cell antigen receptor signaling.J. Immunol. 2013; 190 (23335753): 1807-181810.4049/jimmunol.1202401Crossref PubMed Scopus (24) Google Scholar), Ca2+ response in B cell line Ramos stimulated with anti-BCR antibody was resistant to at least 20 μm PP2, although it exhibited a delay dependent on the PP2 dose (Fig. 1B). The difference can be explained by two alternative hypotheses. First, that downstream B cell kinase SYK but not its T cell paralogue ZAP-70 can initiate receptor signaling by phosphorylating ITAMs independently of SFKs. Second, it is also possible that SFKs are incompletely inhibited by PP2 and their residual activity is sufficient to initiate signaling by BCR but not by TCR. To start addressing these questions we employed Jurkat-derived T cell line P116, which lacks expression of SYK and ZAP-70 (25Williams B.L. Schreiber K.L. Zhang W. Wange R.L. Samelson L.E. Leibson P.J. Abraham R.T. Genetic evidence for differential coupling of Syk family kinases to the T-cell receptor: reconstitution studies in a ZAP-70-deficient Jurkat T-cell line.Mol. Cell Biol. 1998; 18 (9488454): 1388-139910.1128/mcb.18.3.1388Crossref PubMed Scopus (224) Google Scholar). We reconstituted these cells with SYK, ZAP-70, or empty vector (all containing IRES-CD90.1 reporter) and stimulated these cells with anti–TCR antibody C305. To select the cells with expression of SYK and ZAP-70 similar to their native expression in Jurkat and Ramos cells, we have determined the level of reporter gene CD90.1, at which the amount of transduced SYK or ZAP-70 was similar to the endogenous levels and then we gated on these cells using only the reporter (Fig. 1C and Fig. S1). Interestingly, SYK expression somewhat increased TCR signaling resistance to SFK inhibition and these cells responded even in the presence of 5 μm PP2. However, 10 μm concentration, a standard dose widely used to inhibit SFK activity, completely abolished Ca2+ response in SYK expressing P116 cells (Fig. 1, D and E). This result suggested that although it contributes to the resistance of the signaling to SFK inhibition, SYK activity alone cannot explain it. To further dissect the role of SYK, we complemented the experiment in T cells with a similar experiment in B cell line Ramos. Using CRISPR-Cas9 technology, we have obtained several clones of Ramos B cells, which lack expression of SYK (Fig. 1F). None of the tested clones was responsive to BCR stimulation and all the clones had comparable expression of BCR (Fig. 1G and data not shown). For further experiments, we have used one of these clones, hereafter termed R.SYKKO. To avoid clonal bias, we always used SYK reconstituted cells as a control. We transduced R.SYKKO cells with SYK, ZAP-70, and empty vector (Fig. 1H) and stimulated the cells with anti BCR antibody in the presence of different doses of PP2. SYK-expressing cells were similarly resistant to PP2 inhibition as original Ramos cells. Importantly, ZAP-70 expressing cells were also resistant to 10 μm PP2 and the signaling was only inhibited by 20 μm concentration (Fig. 1, I and J). Such a high resistance of ZAP-70–expressing cells suggests that the differences between ZAP-70 and SYK account only for part of the resistance of BCR signaling to SFK inhibition and additional mechanisms substantially contribute to this phenomenon. To test if SYK ability to phosphorylate ITAMs contributes to the BCR resistance to SFK inhibition, we have analyzed tyrosine phosphorylation of CD79a immunoprecipitated from BCR-stimulated R.SYKKO cells reconstituted (or not) with SYK or ZAP-70. We also probed the phosphorylation of tyrosine 182 of CD79a in the whole cell lysates of the same cells to complement immunoprecipitation with an independent method (Fig. 2A). Expression of both SYK and ZAP-70 increased ITAM phosphorylation in activated cells detected by CD79a immunoprecipitation followed by phosphotyrosine staining. However, only in the case of ZAP-70 the difference was statistically significant (Fig. 2, A and B). Staining for phosphotyrosine 182 of CD79a in the whole cell lysates did not reveal any significant effects, although the results in general followed a similar trend with slight increase of tyrosine 182 phosphorylation in the presence of SYK or ZAP-70 (Fig. 2, A and C). These data do not support the hypothesis that SYK can potentiate ITAM phosphorylation more efficiently than ZAP-70. They rather suggest that ZAP-70 is at least as efficient as SYK if not better. Despite this, SYK was better able to restore the resistance of BCR signaling to SFK inhibition (Fig. 1, I and J). These observations led to the conclusion that any potentially unique ability of SYK to phosphorylate ITAM motifs is not the reason why BCR better tolerates SFK inhibition. Importantly, increase in ITAM phosphorylation induced by BCR stimulation could be observed even after treatment with 20 μm PP2 in the absence of SYK and presence of ZAP-70 or in the complete absence of SYK or ZAP-70 (Fig. 2, A–C), demonstrating that even high dose of PP2 is not able to completely inhibit ITAM phosphorylation by SFKs. Collectively, these data show that SYK ability to phosphorylate ITAMs does not explain increased resistance of SYK-expressing cells to SFK inhibition. In addition, they also show that there still is a residual SFK activity in PP2-treated cells, which is probably sufficient to trigger BCR-mediated signaling. To address the question whether TCR per se is inherently less sensitive than BCR to SFK activity, we transduced our T and B cell lines with chimeric protein composed of extracellular domain of CD16 and full-length TCRζ (Fig. 3, A and B). Importantly, CD16 is not expressed in any of the cell lines used in our experiments (Fig. 3C). Crosslinking of CD16-ζ on T cells induced strong Ca2+ response similar to the one elicited by TCR stimulation (Fig. 3D). Crosslinking of CD16-ζ on B cells induced relatively strong but already delayed Ca2+ response, which was inhibited by 10 μm concentration of PP2 (Fig. 3, E and F), indicating that antigen receptor composition is important for the resistance to SFK inhibition. To further explore this possibility, we transduced Jurkat T cells with the BCR complex, including IgH, Igα, and Igβ (Fig. 4A). We stimulated these cells with anti-TCR or anti-BCR antibody and treated with different doses of PP2. Interestingly, and analogously to CD16-ζ–induced signaling in B cells, BCR signaling in T cells was delayed compared with TCR signaling even in PP2 nontreated cells (Fig. 4B). However, despite this, BCR signaling was still more resistant to PP2 inhibition than TCR signaling in the same cells (Fig. 4, B–D), demonstrating that structural features of BCR contribute to the higher resistance of BCR signaling to SFK inhibition.Figure 4Ectopic expression of BCR in T cells increases the resistance to SFK inhibition. A, flow cytometry analysis of CD79b and surface IgM expression in Jurkat BCR cells. B and C, Ca2+ response in Jurkat BCR cells stimulated with antibodies to TCR (B) or BCR (C) in the presence or absence of various concentrations of PP2. Ca2+ response measurements were performed as in Fig. 1A (n = 4). Black arrows indicate the time of the stimulation. D, quantification of Jurkat BCR Ca2+ response from (B) and (C). Data are plotted and analyzed similarly as in Fig. 1E.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Even though BCR signaling in Jurkat T cells was more resistant to PP2 inhibition than TCR signaling in the same cells, it still could be completely inhibited by a relatively low dose of PP2, suggesting that other factors are contributing too. We have shown above that SYK activity is one of these factors. Next we wanted to test whether differences between SFK members in T cells and B cells can also be responsible. Therefore, we transduced the Jurkat BCR cells with Lyn, a major B cell member of Src family (Fig. 5A). Interestingly, in Jurkat BCR cells, Lyn expression accelerated Ca2+ response both in the absence and in the presence of PP2, which demonstrates certain level of nonredundancy between the T cell and B cell SFK and suggests that Lyn expression also slightly increases the resistance of BCR signaling to PP2. Moreover, the fact that Lyn was able to alter signaling output significantly more in the presence of PP2 further supports the argument that signaling in PP2-treated cells is still initiated by SFKs (Fig. 5, B and C). The data described above suggested that multiple members of antigen receptor signaling apparatus cooperate to set the threshold for the amount of SFK activity required for receptor activation. This threshold appears to be lower in B cells than in T cells, allowing B cells to respond even to the residual SFK activity surviving in the presence of PP2. When considering the differences between BCR and TCR signaling at the level of organization of the molecules potentially involved in regulating this threshold, we realized that one of the most apparent differences is in the usage of receptor-independent transmembrane adaptor LAT by TCR signaling machinery, which serves as a docking site for assembly of PLCγ containing signalosome. It was recently published that bridging the TCR to LAT requires adaptor function of Lck, which depends on Lck-mediated phosphorylation of ZAP-70 (18Yan Q. Barros T. Visperas P.R. Deindl S. Kadlecek T.A. Weiss A. Kuriyan J. Structural basis for activation of ZAP-70 by phosphorylation of the SH2-kinase linker.Mol. Cell Biol. 2013; 33 (23530057): 2188-220110.1128/MCB.01637-12Crossref PubMed Scopus (65) Google Scholar, 20Brdicka T. Kadlecek T.A. Roose J.P. Pastuszak A.W. Weiss A. Intramolecular regulatory switch in ZAP-70: analogy with receptor tyrosine kinases.Mol. Cell Biol. 2005; 25 (15923611): 4924-493310.1128/MCB.25.12.4924-4933.2005Crossref PubMed Scopus (108) Google Scholar, 26Lo W.L. Shah N.H. Ahsan N. Horkova V. Stepanek O. Salomon A.R. Kuriyan J. Weiss A. Lck promotes Zap70-dependent LAT phosphorylation by bridging Zap70 to LAT.Nat. Immunol. 2018; 19 (29915297): 733-74110.1038/s41590-018-0131-1Crossref PubMed Scopus (77) Google Scholar). We speculated that because of its relative complexity, maintaining this bridge may require comparatively high SFK activity. Moreover, when functioning as a mere adaptor, Lck cannot employ enzymatic amplification and, thus, more Lck molecules in open active conformation are required to transduce the signal. To test this possibility, we wanted to bring LAT to the proximity of TCR independently of Lck-mediated bridging mechanism. To achieve this, we prepared chimeric protein composed of extracellular domain of CD16 and full-length LAT (Fig. 6A). It enabled us to co-crosslink TCR with LAT and thus probe the resistance of TCR signaling to SFK inhibition independently of LAT-bridging step. We transduced ZAP-70 or SYK expressing P116 T cells with chimeric construct coding for CD16-LAT (Fig. 6B). Strikingly, co-crosslinking of LAT to TCR resulted in resistance of the TCR signaling to 10 μm concentration of PP2, which is the highest level of resistance we were so far able to achieve in T cells. As expected, the resistance was more profound in case of SYK-expressing cells (Fig. 6, C–F). Although the resistance was still not as strong as in the case of B cells, the results support the conclusion that LAT bridging step is regulated by SFKs and that it is an important part of the mechanism by which TCR signaling apparatus sets the threshold for the amount of SFK activity required for signal propagation. In our work, we analyzed the mechanisms that make TCR signaling sensitive and BCR signaling resistant to the inhibition of SFK. This phenomenon has been observed in previous works. Similar difference in the sensitivity of antigen receptor signaling to PP2-mediated inhibition has been observed in primary mouse T cells and B cells (8Mukherjee S. Zhu J. Zikherman J. Parameswaran R. Kadlecek T.A. Wang Q. Au-Yeung B. Ploegh H. Kuriyan J. Das J. Weiss A. Monovalent and multivalent ligation of the B cell receptor exhibit differential dependence upon Syk and Src family kinases.Sci. Signal. 2013; 6 (23281368): ra110.1126/scisignal.2003220Crossref PubMed Scopus (57) Google Scholar, 11Stepanek O. Draber P. Drobek A. Horejsi V. Brdicka T. Nonredundant roles of Src-family kinases and Syk in the initiation of B-cell antigen receptor signaling.J. Immunol. 2013; 190 (23335753): 1807-181810.4049/jimmunol.1202401Crossref PubMed Scopus (24) Google Scholar). It was also described in cell lines where SFKs were inhibited genetically by membrane-targeted Csk (11Stepanek O. Draber P. Drobek A. Horejsi V. Brdicka T. Nonredundant roles of Src-family kinases and Syk in the initiation of B-cell antigen receptor signaling.J. Immunol. 2013; 190 (23335753): 1807-181810.4049/jimmunol.1202401Crossref PubMed Scopus (24) Google Scholar). Delayed but still robust Ca2+ response has also been observed in LYN-deficient DT-40 cell line (7Takata M. Sabe H. Hata A. Inazu T. Homma Y. Nukada T. Yamamura H. Kurosaki T. Tyrosine kinases Lyn and Syk regulate B cell receptor-coupled Ca2+ mobilization through distinct pathways.EMBO J. 1994; 13 (8137818)" @default.
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• W3043290063 date "2020-09-01" @default.
• W3043290063 modified "2023-10-06" @default.
• W3043290063 title "Mechanisms determining a differential threshold for sensing Src family kinase activity by B and T cell antigen receptors" @default.
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