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CD28 Costimulation in Jurkat Cells Signaling Networks Triggered By Quantitative Analysis of Phosphotyrosine Signaling Networks Triggered by CD3 and CD28 Costimulation in Jurkat Cells This information is current as Ji-Eun Kim and Forest M. White of September 26, 2021. J Immunol 2006; 176:2833-2843; ; doi: 10.4049/jimmunol.176.5.2833 http://www.jimmunol.org/content/176/5/2833 Downloaded from References This article cites 47 articles, 22 of which you can access for free at: http://www.jimmunol.org/content/176/5/2833.full#ref-list-1 Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on September 26, 2021 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 © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Quantitative Analysis of Phosphotyrosine Signaling Networks Triggered by CD3 and CD28 Costimulation in Jurkat Cells1 Ji-Eun Kim and Forest M. White2 The mechanism by which stimulation of coreceptors such as CD28 contributes to full activation of TCR signaling pathways has been intensively studied, yet quantitative measurement of costimulation effects on functional TCR signaling networks has been lacking. In this study, phosphotyrosine networks triggered by CD3, CD28, or CD3 and CD28 costimulation were analyzed by site-specific quantitative phosphoproteomics, resulting in identification of 101 tyrosine and 3 threonine phosphorylation sites and quantification of 87 sites across four cell states. As expected, CD3 stimulation induced phosphorylation of CD3 chains and upstream components of TCR pathways such as Zap70, while CD28 stimulation induced phosphorylation of CD28, Vav-1, and other adaptor proteins including downstream of tyrosine kinase 1, Grb2-associated protein 2 (Grap2), and Wiskott-Aldrich syndrome protein. CD3 and CD28 costimulation induced a complex response including decreased threonine phosphorylation in Downloaded from the ERK1 and ERK2 activation loops and increased phosphorylation of selected tyrosine sites on ERK1/2, p38, phospholipase C-␥, Src homology 2 domain-containing transforming protein 1, Grap2, and Vav-1, potentiating T cell activation. Hierarchical clus- tering and self-organizing maps were used to identify modules of coregulated phosphorylation sites within the network. Quanti- tative information in our study suggests quantitative and qualitative contribution by costimulation of CD28 on CD3-stimulated TCR signaling networks via enhanced phosphorylation of phospholipase C-␥/Src homology 2 domain-containing transforming protein 1/Grap2/Vav-1 and their effects on downstream components including MAPKs. The Journal of Immunology, 2006, 176: http://www.jimmunol.org/ 2833–2843. tudies on T cell activation leading to diverse cellular out- Zap70, in turn, phosphorylates phospholipase C-␥ (PLC-␥), which comes such as cytokine production have resulted in iden- is promoted after its binding to an adaptor protein, linker for ac- S tification of a number of cellular components involved in tivation of T cells (LAT), and Src homology 2 domain-containing T cell signaling pathways (reviewed in Refs. 1–4). Briefly, once leukocyte protein of 76 kDa (SLP-76). PLC-␥ can further activate the MHC molecule presents processed Ag to TCR ␣- and ␤-chains, protein kinase C (PKC), leading to NF-␬B transactivation, and CD4 binds to the MHC molecule, resulting in the proximity of calcium-dependent pathways, resulting in the transport of NF-AT lymphocyte-specific protein kinase (Lck),3 which exists in a com- into the nucleus for its transcription activation. During early phos- by guest on September 26, 2021 plex with CD4, to CD3-␨ and thereby phosphorylation of CD3 phorylation events in lipid raft clusters, Src homology 2 domain- chains in the preexisting molecular cluster within plasma mem- containing transforming protein 1 (Shc) also interacts with phos- brane microdomains. Each CD3 chain (CD3-␦,-⑀,-␥) contains two phorylated ITAM in the ␨-chain and LAT, which interacts with phosphotyrosine sites in the ITAM motif (ITAM; YxxI/L) with the other adaptor molecules such as Vav-1 or Ras via Grb2 and Grap2/ exception of the ␨-chain, which possesses six phosphotyrosine Grb2-associated protein downstream of Shc (Gads), leading to the sites. TCR Zap70 is then recruited, binds to phosphorylated CD3 activation of MAPKs such as JNK, p38, and ERK 1/2 for subse- chains, and becomes phosphorylated by Lck. Phosphorylated quent AP-1 transactivation. The two-signal theory suggests that T cell activation requires both Ag recognition via the TCR-CD3 complex and additional Biological Engineering Division, Massachusetts Institute of Technology, Cambridge, costimulatory signals derived from CD28 and other receptors (5). MA 02139 Correspondingly, CD28-negative mice require repeated stimula- Received for publication September 26, 2005. Accepted for publication December tion to elicit T cell response and respond poorly to immunological 8, 2005. challenge (reviewed in Ref. 6). Studies on the mechanism by The costs of publication of this article were defrayed in part by the payment of page which costimulation of CD3 and other coreceptors, particularly charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. CD28, leads to complete activation of T cells have generated two hypotheses of T cell activation by CD3/CD28 costimulation, re- 1 This work was supported by National Institutes of Health Grants R21-AI065354-2, P50-GM68762, R01-AI065824, and P30-ES002109. ferred to as either the quantitative or qualitative mechanisms (6–8) 2 Address correspondence and reprint requests to Dr. Forest M. White, Biological The quantitative mechanism proposes cis-activation of the TCR Engineering Division, Massachusetts Institute of Technology, 77 Massachusetts Av- signaling network by CD28 stimulation, affecting the phosphory- enue, Room 56-787A, Cambridge, MA 02139. E-mail address: [email protected] lation level and activation of components downstream of the TCR 3 Abbreviations used in this paper: Lck, lymphocyte-specific protein kinase; IMAC, to overcome a threshold necessary for T cell activation. Promotion immobilized metal-affinity chromatography; MS, mass spectrometry; LC, liquid chro- matography; PLC-␥, phospholipase C-␥; LAT, linker for activation of T cell; SLP-76, of TCR localization with lipid rafts and reorganization of mem- Src homology 2 domain-containing leukocyte protein of 76 kDa; PKC, protein kinase brane microdomains have supported these quantitative effects of C; Grap2, Grb2-associated protein 2; Shc, Src homology 2 domain-containing trans- CD28 costimulation (6, 8–10). Additionally, the quantitative forming protein 1; Gads, Grb2-associated protein downstream of Shc; o.d., outside diameter; i.d., inside diameter; GEM, glycolipid-enriched membrane microdomain; mechanism emphasizes the role of Vav-1 as an important effector WASP, Wiskott-Aldrich syndrome protein; SOM, self-organizing map; PAG, phos- in the CD28 signaling pathway and proposes that the activation of phoprotein associated with GEM; Gsk, glycogen synthase kinase 3; Itk, IL-2-induc- ible T cell kinase; SLAP-130, SLP-76-associated protein 130; Dok-1, downstream of Vav-1 might differ from the CD3 signaling pathway, possibly tyrosine kinase 1. through prolonged Lck activation (8). The qualitative mechanism Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 2834 SIGNALING NETWORKS IN T CELL COSTIMULATION focuses more on trans-activation of the TCR signaling network, 2.5) for further immobilized metal affinity chromatography (IMAC)-based with CD28 stimulation resulting in distinct signaling pathways phosphopeptide enrichment. leading to cytoskeleton rearrangement and mediation by key pro- Phosphopeptide enrichment using IMAC teins such as PI3K, Vav-1, and SLP-76 (7). In fact, cytoskeletal scaffolding following T cell stimulation is indispensable for T cell IMAC columns were prepared as described elsewhere (13). Briefly, a 15-cm long (360 ␮m outside diameter (o.d.) ϫ 200 ␮m inside diameter activation (11). (i.d.)) microcapillary fused-silica column (Polymicro Technologies) was Studies to elucidate signaling pathways triggered by TCR stim- packed with POROS 20MC in water (Applied Biosystems) and washed ulation or TCR and coreceptor stimulation have identified many of with 100 mM EDTA (10 min at 10 ␮l/min). After rinsing with water to remove EDTA, the column was charged with 100 mM FeCl3 for 10 min at the components, their interactions, and protein phosphorylation ␮ 10 l/min. Excess FeCl3 was removed by rinsing with 0.1 M acetic acid, sites in the TCR signaling network under a variety of conditions. and the column was equilibrated with 0.1% trifluoroacetic acid. Immuno- These efforts have generated a fairly comprehensive map of the T precipitated peptides in 30% acetonitrile and 0.1% trifluoroacetic acid were cell signaling
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