Combinatorial proteomic analysis of intercellularsignaling applied to the CD28 T-cellcostimulatory receptorRuijun Tiana,b,c,1,2, Haopeng Wangd,1, Gerald D. Gisha, Evangelia Petsalakia, Adrian Pasculescua, Yu Shie,Marianne Mollenauerd, Richard D. Bagshawa, Nir Yoseff, Tony Huntere, Anne-Claude Gingrasa,g, Arthur Weissd,h,2,and Tony Pawsona,g,3

aLunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada M5G 1X5; bDepartment of Chemistry and cShenzhen Key Laboratoryof Cell Microenvironment, South University of Science and Technology of China, Shenzhen 518055, China; dDivision of Rheumatology, Department ofMedicine, Rosalind Russell and Ephraim P. Engleman Rheumatology Research Center, and hHoward Hughes Medical Institute, University of California,San Francisco, CA 94143; eMolecular and Cell Biology Laboratory, Salk Institute for Biological Studies, La Jolla, CA 92037; fDepartment of ElectricalEngineering and Computer Sciences, University of California, Berkeley, CA 94720; and gDepartment of Molecular Genetics, University of Toronto, Toronto,ON, Canada M5S 1A8

Contributed by Arthur Weiss, February 20, 2015 (sent for review October 15, 2014)

Systematic characterization of intercellular signaling approxi-mating the physiological conditions of stimulation that involvedirect cell–cell contact is challenging. We describe a proteomicstrategy to analyze physiological signaling mediated by the T-cellcostimulatory receptor CD28. We identified signaling pathwaysactivated by CD28 during direct cell–cell contact by global anal-ysis of protein phosphorylation. To define immediate CD28 tar-gets, we used phosphorylated forms of the CD28 cytoplasmicregion to obtain the CD28 interactome. The interaction profilesof selected CD28-interacting proteins were further characterizedin vivo for amplifying the CD28 interactome. The combination ofthe global phosphorylation and interactome analyses revealedbroad regulation of CD28 and its interactome by phosphoryla-tion. Among the cellular phosphoproteins influenced by CD28signaling, CapZ-interacting protein (CapZIP), a regulator of theactin cytoskeleton, was implicated by functional studies. Thecombinatorial approach applied herein is widely applicable forcharacterizing signaling networks associated with membranereceptors with short cytoplasmic tails.

intercellular signaling | proteomics | T cells | phosphorylation |signal transduction

During some forms of intercellular communication, solublegrowth factors are secreted by one cell type and bind specific

transmembrane receptor tyrosine kinases (RTKs) present onneighboring cells. Activated RTKs then undergo autophos-phorylation and also phosphorylate associated scaffold pro-teins, thereby creating docking sites for effector proteins withphosphotyrosine (pTyr)-recognition modules such as Src ho-mology 2 (SH2) and pTyr-binding (PTB) domains (1). Theseimmediate targets of growth-factor receptors control a varietyof intracellular responses, typified by the activation of serine/threonine protein kinases such as those involved in the ERKMAP kinase pathway. Growth-factor signaling therefore leadsto extensive changes in both tyrosine and serine/threonine phos-phorylation (2).The alterations in protein–protein interactions and post-

translational modifications elicited by transmembrane receptorscan in principle be characterized by mass spectrometry (MS)-based proteomics (3–6). However, under circumstances that in-volve complex cell–cell interactions, identifying physiologicallyrelevant signals using MS-based proteomics can be challenging.For example, obtaining a sufficiently robust response for MSanalysis has often required the use of cells that overexpressa receptor of interest and are subjected to a nonphysiologicalstimulus. For this reason, experiments have often focused onspecific, often stereotypic, aspects of the cellular response, rather

than taking a more global approach. Moreover, cell–cell inter-actions are often typified by the interaction of multiple trans-membrane receptor-ligand pairs, frequently involving membraneproteins that are not receptor kinases. Short-peptide motifs fromsuch receptors that serve as potential ligands for downstreamtargets can be used as affinity probes for binding partners, butthese experiments suffer from high false-positive and false-neg-ative rates (7, 8). Importantly, existing techniques are especiallyunsuitable for investigating the common situation typified bycells of the immune system, in which antigen-dependent signal-ing is induced by direct cell–cell contact, rather than by solublesecreted factors.This latter issue is exemplified by the observation that com-

munication between two distinct cell types often can be con-trolled by the interaction of two transmembrane or membrane-tethered proteins (9). Under these circumstances, investigatingsignaling events requires that cells expressing an activatingmembrane-associated ligand are cocultured with cells displaying

Significance

Intracellular signaling during complex cell–cell interactions,such as between immune cells, provides essential cues leadingto cell responses. Global characterization of these signalingevents is critical for systematically exploring and understandinghow they eventually control cell fate. However, proteome-widecharacterization of intercellular signaling under physiologicallyrelevant conditions involving multiple interacting receptorsduring cell–cell interactions remains challenging. We developedan integrated proteomic strategy for quantitatively profilingintercellular-signaling events mediated by protein phosphoryla-tion and protein–protein interaction. We applied this approachto determine the influence of a single receptor-ligand pair dur-ing T-cell stimulation by blocking the interaction of the CD28costimulatory receptor with its ligand. This approach is gen-erally applicable to other transmembrane receptors involved insignaling during complex cell interactions.

Author contributions: R.T., H.W., A.W., and T.P. designed research; R.T., H.W., G.D.G., E.P.,A.P., Y.S., M.M., and R.D.B. performed research; N.Y., T.H., and A.-C.G. contributed newreagents/analytic tools; R.T., H.W., G.D.G., E.P., A.P., Y.S., M.M., R.D.B., A.W., and T.P.analyzed data; and R.T., H.W., A.W., and T.P. wrote the paper.

The authors declare no conflict of interest.1R.T. and H.W. contributed equally to this work.2To whom correspondence may be addressed. Email: aweiss@medicine.ucsf.edu or tian.rj@sustc.edu.cn.

3Deceased August 7, 2013.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1503286112/-/DCSupplemental.

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its corresponding receptor. However, exploring the resultingsignal transduction pathways is challenging because the identityof the cell from which a particular protein is derived is lost aftercell lysis. The T.P. laboratory developed a proteomic approach[termed quantitative analysis of bidirectional signaling (qBidS)]for profiling cell-specific tyrosine phosphorylation events re-sulting from contact between cells expressing either an Eph re-ceptor tyrosine kinase or its ephrin ligand, respectively (10). Inthese experiments, the EphB2 receptor or its ephrin-B1 ligand,a transmembrane protein that itself has intrinsic signaling ac-tivity, was stably expressed in human embryonic kidney (HEK)293 cells so that the two cell types segregated in culture due torepulsive EphB2/ephrin-B1 signaling. The two distinct cell pop-ulations were then differentially labeled with stable isotopes[stable isotope labeling by amino acid in cell culture (SILAC)]and cocultured. This strategy not only can identify tyrosinephosphorylation events modulated after the interaction betweenEphB2 and ephrin-B1 but also can indicate from which cell typea specific phosphopeptide originates, and thus can infer path-ways activated in either cell type involved in EphB2/ephrin-B1bidirectional signaling. However, these experiments have limi-tations. One is that they depend on overexpression of receptorsand ligands in a model-cell system. Another is that, once twocells adhere to one another, they may interact through multipledifferent ligands and receptors, making it difficult to confidentlydefine the exclusive signaling output of a single class of receptor(9, 11).Here, we overcome these issues by using a combination of

proteomic and cell-biological approaches to systematically identifyspecific signaling events downstream of the endogenous CD28costimulatory receptor on T cells. CD28 is a type I transmembraneprotein that binds through its extracellular region to B7 proteins(CD80 and CD86), which are transmembrane proteins expressedon the surface of antigen-presenting cells (APCs) and are up-regulated by inflammatory signals. Upon the interaction of T cellsand APCs, the T-cell receptor (TCR) engages a peptide antigen-bound major histocompatibility complex molecule on the APCs,resulting in a primary signal. Simultaneously, costimulatory sig-naling is established predominantly, but not exclusively, throughthe association of CD28 on T cells with B7 ligands on the surfaceof the APCs. Both TCR and CD28 signals are required for a fullT-cell response, resulting in, for example, production of the IL-2cytokine. Although it lacks an intrinsic catalytic domain, the short41 amino acid cytoplasmic tail of CD28 has highly conservedtyrosine-based motifs that are phosphorylated by cytoplasmic ty-rosine kinases upon T-cell stimulation and that bind targets withSH2 domains in a pTyr-dependent manner, as well as proline-richsequences that potentially bind SH3 domains (12). It therefore canhelp orchestrate the response to the signal from an APC, such asa B cell.Because the complex cellular interactions of T cells and APCs

stimulate multiple receptors (e.g., the integrin receptor) in ad-dition to CD28, we have developed techniques to distinguishspecific events uniquely dependent solely on CD28 signaling toundertake a comprehensive screen of tyrosine and serine/thre-onine phosphorylation sites selectively regulated by CD28. Wehave used a complementary approach using the full cytoplasmictail of CD28 to identify the cytoplasmic targets with which itinteracts. This approach involved immunoprecipitation coupledto mass spectrometry (IP-MS) to identify a network of inter-acting proteins immediately proximal to CD28. We then in-tegrated these data to dissect CD28-based signaling networksactivated in T cells after coculture with APCs. We targeted onenewly identified CD28-regulated phosphoprotein, CapZ-inter-acting protein (CapZIP), to demonstrate its importance in theCD28-dependent functional response. Our results demonstratethe value of a combinatorial and broad-based strategy to identify

signaling pathways regulated by a specific endogenous receptoractivated by direct cell–cell contact.

ResultsGlobal Phosphoproteomic Analysis of Endogenous CD28 Signaling.We sought to develop a general proteomics approach to quan-titatively measure global signaling events mediated by an en-dogenous receptor activated by direct cell–cell contact. As a testbiological system, we systematically explored how CD28-medi-ated costimulatory signaling coordinates with TCR signaling forfull activation of T cells by antigen-presenting B cells, usinga combination of MS-based interaction-proteomics and phos-phoproteomics. To specifically explore physiological CD28-de-pendent protein phosphorylation events, we used a coculturesystem involving Jurkat leukemic T cells and Raji lymphoblastoidB cells (Fig. 1A) (13, 14). Distinct combinations of SILAC la-beling were used to differentially label T cells with either “me-dium” (M) or “heavy” (H) SILAC isotopes, and with B cellsbeing in an unlabeled “light” (L) state. After coculture of T cellsand B cells for 5 min, this differential labeling allowed us todistinguish peptides and phosphorylation sites derived from Bcells (L) or T cells (M or H), as well as those derived from T cellsthat were fully stimulated by coculture with B cells (M) or T cellsin which the TCR (and other interacting receptor/ligand pairsthat are not defined here) is engaged but CD28 is not (H) (Fig.1A). Specifically, Jurkat leukemic T cells were stimulated bycoculturing with Raji lymphoblastoid B cells presenting thesuperantigen staphylococcal enterotoxin E (SEE), which binds tothe TCR and to MHC class II molecules. Under these con-ditions, the majority of the T-cell population (up to 88.2%) wasefficiently activated, as indicated by potent ERK phosphoryla-tion, reflecting TCR signaling (Fig. 1B and SI Appendix, Fig. S1A);consistent with this result, we observed T and B cells forminga typical immunological synapse, with strong induction of tyrosinephosphorylation at the site of synaptic contact in T cells (SI Appendix,Fig. S1B).To selectively block CD28 signaling, we made use of cytotoxic

T-lymphocyte antigen-4 (CTLA4), a coreceptor expressed onactivated T cells, which binds the same family of B7 ligands on Bcells as does CD28, but with more than 20-fold higher affinity(15). CTLA4-Ig, a CTLA4-Ig recombinant fusion protein that isused in the treatment of rheumatoid arthritis, was added tospecifically block the interaction of CD28 on Jurkat leukemic Tcells with B7 ligands on the surface of Raji lymphoblastoid Bcells. Incubation with CTLA4-Ig completely inhibited CD28signaling in the coculture assay, even after 22 h, as suggested byan efficient block in the production of the cytokine IL-2, a sig-nature target of CD28-mediated costimulation (Fig. 1C). Tofocus on proximal CD28 signaling events and minimize potentialsecondary effects, a relative early time point after T-cell ac-tivation was chosen. We were guided by the kinetics of ERKphosphorylation after Raji-SEE stimulation was systematicallyinvestigated. Maximal ERK phosphorylation was observed at5 min after T-cell stimulation (SI Appendix, Fig. S1A). Therefore,5 min was chosen to activate the Jurkat T cells in the phospho-proteomic analysis. Signals that are present in M-labeled T cellscocultured with SEE-presenting B cells, but are specificallyblocked by treatment with CTLA4-Ig in H-labeled T cells, aretherefore dependent on B7-stimulated CD28.We sought to develop a sensitive MS assay to identify tyrosine

and serine/threonine phosphorylation events regulated by en-dogenous CD28 on a global scale. To this end, we optimized atwo-step phosphoproteomic approach involving tryptic digestionfollowed by either anti-pTyr IP or strong cation exchange (SCX)fractionation and subsequent titanium dioxide (TiO2) enrich-ment (Fig. 1A). By combining these enrichment steps with theuse of a high-resolution Orbitrap Elite instrument with fast se-quence speed (16), we identified 936 phosphorylation sites by

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pTyr IP, of which 778 were pTyr sites, and 15,861 phosphoryla-tion sites using the SCX/TiO2 workflow, of which 13,516 werephospho-serines (pSers), and 2,184 were phospho-threonines(pThrs) (Dataset S1). Two biological replicates gave an overlapof about 75% for sites identified by pTyr IP and 90% for theSCX/TiO2 workflow, demonstrating reasonable reproducibilityof both procedures (SI Appendix, Fig. S2A). The results obtaineddemonstrated high identification confidence and MS1 mass ac-curacy (SI Appendix, Fig. S2 B and C).To delineate T cell-specific phosphorylation sites dependent

on CD28 costimulation, we applied stringent quantification fil-ters and show the results derived from every single MS spectrum.This effort is mainly due to the fact that Jurkat leukemic T cellsand Raji lymphoblastoid B cells are completely different celltypes and that their proteome profile and expression level of thesame protein could be significantly different. Consequently, ac-curate measurement of the SILAC ratios between coelutedSILAC-labeled peptides from distinct cell types could be chal-lenging. We strictly selected phosphopeptides that were reliablyidentified (Andromeda score > 42) and quantified in at leastthree MS/MS spectra from two biological replicates, and thathad negligible peak overlap, defined by the SILAC light peakoverlapping into the medium isotope peak by less than 20%.Furthermore, we used the H/L and M/L ratios (T cells/B cells) todefine and remove B cell-specific phosphopeptides. The cutoffswere selected based on removal of phosphopeptides from PYK2,which was predominantly phosphorylated in B cells as judged bymanual inspection, and from six well-characterized B cell-spe-cific proteins, including CD19, LYN, BTK, CD22, BLNK, andPIK3AP1, which had significantly low H/L and M/L ratios. Intotal, 8,208 T cell-specific phosphorylation sites were quantified,with a probability of being assigned to the correct residue of ≥0.33and where 6,267 sites have site probability of ≥0.75 (Dataset S2).The majority of phosphorylation sites exhibited a tight distributionaround zero, indicating no significant change when CD28 signalingwas blocked, and demonstrating the high accuracy of the SILACquantification (Fig. 1D). A total of 598 sites showed significantchanges upon CTLA4-Ig treatment, with a P value (Wilcoxon test)less than 0.01 and log2 ratio H/M cutoff at ±0.95 (98.5% and 1.5%quartiles, respectively).

Previous studies indicate that multiple signaling pathways aredownstream of CD28. Our pathway analysis revealed that manyproteins involved in known CD28-related signaling pathwayswere identified by at least one phosphorylation site, and morethan 20 of those phosphorylation sites were significantly reducedupon CD28 inhibition (Fig. 2A) (12). For example, we observeda remarkable reduction of phosphorylation of Y191 in the CD28cytoplasmic tail, which is known to recruit phosphatidylinositol3-kinase (PI3K) through its SH2 domain, leading to PI3K acti-vation. Consistent with this observation, phosphorylation ofseveral proteins involved in the PI3K pathway, including PI3K,cAMP-binding protein 1 (CREB1), and mammalian target ofrapamycin (mTOR), changed after inhibiting CD28 signaling.We also observed increased phosphorylation of filamin A(FLNA), which interacts with CD28 and is involved in regulatingcytoskeleton remodeling (17). Protein kinase C theta (PKCθ)was recently reported to associate with CD28 and control NFκBactivation (18). Although we didn’t confidently detect anyphosphorylation changes of PKCθ, the phosphorylation of itsdownstream signaling targets, CARMA1, as well as the tran-scription factor NFkB, was reduced after CD28 blockade. Weobserved dynamic CD28-dependent regulation of phosphoryla-tion-signaling events in fully activated T cells. For example,phosphorylation of CD28-proximal signaling complexes [e.g.,VAV1, phospholipase Cγ1 (PLCγ1), and SHC1] tended to beconsistently decreased whereas nuclear proteins such as NFATtranscription factors mostly exhibited increased serine/threoninephosphorylation. Aside from known CD28-related signalingpathways, the global phosphorylation analysis quantitativelyidentified phosphoproteins belonging to a broad spectrum ofcanonical signaling pathways with up to 66.7% coverage of allpathway components (SI Appendix, Fig. S2D and Dataset S3).Interestingly, most of the enriched signaling pathways within the598 CD28-regulated phosphorylation sites were down-regulated(Fig. 2B and Dataset S3). TCR signaling and a number of otherimmune signaling pathways were down-regulated by CD28blockade. It is noteworthy, however, that events associated withthe TCR signaling pathway did not dominate the down-regulatedevents, suggesting that CD28 may influence events indepen-dently of the TCR. These data provide a broad map of signaling

Fig. 1. Exploring global phosphorylation events mediated by endogenous CD28 in direct cell–cell contact. (A) Schematic of the phosphoproteomic approach.(B) FACS analysis of ERK phosphorylation in resting Jurkat leukemic T cells (gray histogram) or Jurkat leukemic T cells stimulated by 30 ng/mL SEE presented byRaji lymphoblastoid B cells (solid line) after 5 min. (C) The ELISA analysis of cytokine IL-2 production by Jurkat leukemic T cells after their stimulation with Rajilymphoblastoid B cells and SEE at different concentrations in the presence of the indicated concentration of CTLA4-Ig. (D) Volcano plot of the globalquantification of T cell-specific phosphosites. Each circle represents one site scaled by frequency of peptide detection. Three CD28 pTyr sites are indicated byan “X”. (E) The identified CD28 phosphorylation sites are shown with site position and localization probability. The relative positions of each measurementagainst noise distribution are indicated. The sites were labeled according to a full-length human CD28 sequence.

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events specifically regulated by endogenous CD28 activated bycontact between Jurkat T and Raji B cells.

Identification of a Protein-Interaction Network Involving CD28. TheCD28-dependent pathways identified in the preceding phos-phoproteomic analysis are most likely controlled by proteins thatare recruited to the evolutionarily conserved pTyr-containing orproline-rich motifs on CD28 (SI Appendix, Fig. S3A). Indeed, inthe preceding analysis, we confidently identified three pTyr siteson the CD28 cytoplasmic tail that were significantly down-regulated upon CTLA4-Ig–mediated inhibition (Fig. 1E). In-terestingly, two of these pTyr sites, Y191 (located in the motifYMNM) and Y209 (located in the motif PYAPP) were relativelyabundant and strongly inhibited by CTLA4-Ig treatment whereasphosphorylation on Y218 was less abundant and more weaklyinhibited. By IP and Western blot, we observed reduced re-cruitment of the p85α subunit of phosphatidylinositol 3′-kinase(PI3K) to CD28 after CTLA4-Ig treatment, which corroborates theobservation that phosphorylation of Y191 is down-regulated uponCD28 inhibition because this residue is in a site that favors bindingto the PI3K p85α SH2 domains (SI Appendix, Fig. S3B). We alsoidentified two phosphoserine and phosphothreonine sites on theCD28 cytoplasmic tail but with low site localization probabilityscores that don’t meet the data cutoff. Other function-relatedposttranslational modifications including ubiquitination would bepossibly identified with a more dedicated workflow.To explore the targets immediately proximal to CD28, and

thus to investigate how CD28 is connected to the spectrum ofproteins whose phosphorylation is regulated by its signaling, wesought to systematically profile proteins that bind the major pTyrand Pro-rich sites on CD28 identified in the phosphoproteomicsscreen. The comprehensive identification of proteins associatedwith transmembrane receptors is technically problematic due tothe harsh protein extraction conditions needed to solubilize thereceptors for affinity purification, to which the relevant com-plexes are relatively labile (19, 20). Compared with alternativestrategies of using short peptide motifs corresponding to receptor-docking sites, we expected that the “full-length” cytoplasmic do-main with physiologically relevant modifications could recruitmore critical downstream proteins associated with an activatedreceptor (7, 8).To attempt to capture a relevant view of the receptor’s im-

mediate targets and the role of the regulated pTyr sites in me-diating interactions, we devised a proteomic approach to definethe CD28 interactome in which a chemically synthesized CD28

cytoplasmic polypeptide was used to affinity purify CD28-bind-ing proteins from T-cell lysates (Fig. 3A). The CD28 cytoplasmictail contains only 41 amino acids, of which we synthesized a 33amino acid peptide that contains all of the highly conservedmotifs, including two of the identified sites of tyrosine phos-phorylation, as well as proline-rich sequences (SI Appendix, Fig.S3A) (CD28cyto). We anticipated that, using longer syntheticpeptides corresponding to almost the entire CD28 cytoplasmicdomain, together with appropriate modifications, would providea physiologically relevant and comprehensive view of the receptor’simmediate targets. To ensure that we identified phosphorylation-dependent interactions, we synthesized several forms of CD28cyto

that were variously unmodified (YY), phosphorylated on eitherY191 or Y209 (pYY or YpY), doubly phosphorylated on bothY191/Y209 (pYpY), or had alanine substitutions for Pro208 andPro211 to destroy a potential PXXP SH3-binding motif (pYAA).To examine phosphorylation-dependent protein interactions,

the various forms of biotin-tagged CD28cyto were immobilized onstreptavidin agarose beads and incubated with SILAC-labeledT-cell lysates, and associated proteins were identified by MS. Forquantitative purposes, we applied a three-channel SILAC ap-proach that allowed us to perform two biological replicates in thesame experiment with reversed SILAC labeling (21). For thepurpose of identifying pTyr-dependent interactions on bothY191 and Y209, we did pulldowns using pYpY peptide with bothSILAC light- and heavy-labeled T-cell lysates, while using YYpeptide with SILAC medium-labeled T-cell lysate as a commoncontrol (Fig. 3A). We thus obtained two replicate ratios ofpYpY:YY peptide pulldowns as represented by H/M and L/Mratios (Fig. 3B and Dataset S4). This approach generated highlyreproducible data between two biological replicates and nicelydifferentiated pTyr-dependent interacting proteins from themajority of other proteins that bound nonspecifically or to thenonphosphorylated YY peptide. Twenty-eight CD28-bindingproteins were confidently identified, including 8 proteins pre-viously identified as associating with the CD28 cytoplasmic do-main (Fig. 3C). Based on the accurate quantification afforded bySILAC, use of the various phosphorylated and mutant forms ofCD28cyto identified three families of proteins that bound selec-tively to either the pYMNM site (group I), the PXXPP motif(group II), or the PpYAPP sequence (group III) of CD28 (Fig. 3B and C, and SI Appendix, Fig. S4 A–D). Western blots for 8CD28cyto-associated proteins that contained either pTyr-bindingSH2 domains or PXXP-binding SH3 domains also gave resultsthat were consistent with the MS data (SI Appendix, Fig. S4E).

Fig. 2. Pathway analysis of the phosphoproteomics data. (A) Pathway map of CD28 signaling-related proteins. The proteins with phosphorylation sitessignificantly regulated by CD28 inhibition are indicated. The pathway map was based on a previous report (12). (B) Global canonical pathway analysis ofsignificantly regulated phosphoproteins. Additive log2 ratio H/M of all phosphorylation sites associated with a pathway are presented.

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Pathway analysis revealed that more than 50% of the CD28interactors are involved in immune signaling pathways, includingCD28 signaling in T-helper cells, demonstrating the biologicalrelevance of the CD28 interactome obtained by the in vitroCD28cyto pulldown (Fig. 3D). Our data recapitulated previousobservations demonstrating that the YMNM and PYAPP motifsare the two dominant motifs on the CD28 cytoplasmic tail (12),including the binding of PI3K family proteins as well as theGRB2 and GADS adaptors to the CD28 pYMNM motif andGRB2 SH3 domain-mediated interaction with the PYAPP motif.In addition, we identified seven previously unidentified inter-actions to the PYAPP motif that are dependent on pTyr, theproline-rich sequence, or both. Because these newly identifiedproteins contain SH2 or SH3 domains as GRB2 and GADSadaptors, they likely bind to the CD28 cytoplasmic tail in anindirect manner. However, a more dedicated experimentalworkflow is needed to confirm their binding modes. The PYAPPmotif has been suggested to be indispensable for mediating T-cell functions such as proliferation and IL-2 secretion, in contrastto the YMNM motif (12). Therefore, our approach and findingsmay be the basis for further experiments to study the mecha-nisms underlying the importance of these motifs.Like CD28, most immune receptors and ligands contain rather

short cytoplasmic tails (Dataset S5). Peptides of this relativelyshort length, together with their posttranslational modifications,can be readily synthesized using current technology, indicatingthat the overall approach described above could have widespreadapplications.

A High-Resolution in Vivo CD28 Interactome Reveals Phosphorylation-Dependent Interaction Hubs. To validate and extend the signalingnetwork out from these CD28-binding partners identified by invitro CD28cyto pulldown experiments, we performed IP-MS fromJurkat leukemic T cells (Fig. 4A). For this purpose, we useda piggybac transposon system to efficiently generate Jurkat leu-kemic T cells stably expressing specific 3xFlag-tagged CD28-binding proteins, such as GRB2 (22). These cells were theneither left unstimulated or were stimulated by the anti-CD28antibody and lysed, and proteins associated with the Flag-taggedpolypeptide were identified by IP-MS. A three-channel SILACapproach was used to quantitatively distinguish proteins that

bound constitutively and selectively to the target, proteins whosebinding was induced by CD28 activation, and proteins thatbound nonspecifically. GRB2 was investigated because it isknown to interact with CD28 on both the YMNM and thePYAPP motifs and to function as an adaptor to link receptors tomultiple downstream signaling pathways (Fig. 4B). A group ofproteins that selectively, but constitutively, interacted withGRB2 in T cells was reliably identified with high confidence; incontrast, the association between GRB2 and CD28 requiredCD28 stimulation, consistent with an SH2–pTyr interaction.Among the constitutively GRB2-interacting proteins were CBL,SOS1, and THEMIS (23). Suppressor of T-cell receptor signal-ing 1 (STS1; gene name UBASH3B), a negative regulator ofT-cell activation, is a potential atypical tyrosine phosphatase thatcontains an SH3 domain that recognizes a proline-rich motif anda UBA domain that binds ubiquitin (24, 25). The CD28cyto

pulldown experiments have shown that STS1 binding requiredboth the PXXP and pTyr elements within the CD28 PYAPPmotif. Interestingly, our IP-MS data showed that STS1 alsointeracted with GRB2 and that both of them bound to the E3ubiquitin ligases CBL and CBL-B (Fig. 4C). The association ofSTS1 with CD28 was validated by immunoprecipitating endog-enous CD28 (SI Appendix, Fig. S5A). We also found that theinteraction between STS1 and GRB2 is enhanced upon the ac-tivation of both TCR and CD28 signaling, demonstrating itspotential recruitment to CD28-associated signaling complexesthrough GRB2 (SI Appendix, Fig. S5B).We further followed up experimentally an interaction hub that

focused on CD28, GRB2, and CBL/CBL-B, which contains 14proteins, including 8 identified by CD28cyto pulldown experi-ments (Fig. 4D). By using IP-Western blot (IP-WB) and IP-MSapproaches, we validated 12 pairs of protein–protein interactionswithin this interaction hub and found that 6 of these interactionscould be enhanced upon stimulation of both TCR and CD28signaling (Fig. 4D and SI Appendix, Fig. S5B). Interestingly, wefound that the phosphorylation level of 14 phosphorylation siteson nine proteins, as identified by our initial unbiased phospho-proteomic screen in cell lysates (Fig. 1 and Dataset S3), allconsistently decreased upon CD28 inhibition. Of these 14phosphorylation sites, 10 are pTyr sites, which is consistent withthe down-regulation of three pTyr sites on the CD28 cytoplasmic

A

D

CD28Synthetic CD28

cytoplasmic domain

YMNM

PYAPP

Cytoplasmic domainn= 41 aa n= 33 aa

pYMNM

AYAAP

YMNM

PYAPP

YY

pYMNM

PpYAPP

pYpY

pYMNM

PYAPP

pYY

YMNM

PpYAPP

YpY pYAA

B C

P

P

191

209

PIK3CDPIK3R1

PIK3R2PIK3C1

PIK3CA

GRB2RASA1/VAV3CSK/GADS

PIK3R3PLCG1/SHIP1

PTPN11 GRAP

SH2D1AVAV1STS1/ZAP70/CRKL

LCK/THEMIS/STAT5

Log 2 R

atio

H/M

(pY

pY/Y

Y 2

nd re

plic

ate)

Log2 Ratio L/M (pYpY/YY 1st replicate) 0 1 2 3 4 5 6-1-2-3-4-5-6-7

0

1

2

34

5

6

-1-2-3

-4-5-6-7

PIK3R3

GADSPIK3CA

PIK3R1

GRAPPIK3C1PIK3CD

PIK3R2

STS1

GRB2

CIN85

VAV3PTPN11

CSK

CD2AP

STS2

LCK

CRKLSH2D1A

VAV1

ZAP70PLCG1ARAP1

CBLSLP76

STAT3

CD28

STAT5

SHIP1

I II

III

pYpY YY pYpY

SILAC T cell lysates

CD28cyto pull down

1:1:1 combine

Mass spectrometry

SILAC“Light”

SILAC“Medium”

SIALC“Heavy”

On-bead digestion 0

4

8

12

16

20

0

5

10

15

20

25

30-Log10 (p-value)Proteins

# of overlapped proteins

-Log

10 (p

-val

ue)

Fig. 3. SILAC-based CD28cyto pulldown approach reveals phosphorylation-dependent interactome of CD28. (A) Schematic of the experimental workflow.(B) A 2D plot shows double phosphorylation-dependent interacting proteins to CD28. Positive hits are annotated. (C) Integrated map of the CD28 interactome.Proteins previously identified as interacting with CD28 are underlined. Motif-binding preference to pYMNM (I), PxxP (II), and PpYAPP motif (III) are high-lighted in shaded circle. The width of the lines is scaled by the number of spectral counts. (D) The top 10 pathways enriched in the CD28 interactome and thenumber of overlapping proteins are indicated.

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tail. Docking protein 1 (DOK1) has been previously indicated tobe tyrosine-phosphorylated upon CD28 activation by antigen-presenting cells (26). We characterized three phosphorylationsites with a decreased phosphorylation level on DOK1 (Y409,Y449, and T406) and identified a stimulation-dependent in-teraction of DOK1 with Crk-like protein (CRKL), which po-tentially links it to the CD28-mediated interaction network(SI Appendix, Fig. S5B). ADAP is a well-characterized adaptorprotein that positively regulates TCR signaling (27). We identi-fied a decreased phosphosite at Y813 of ADAP (Dataset S2)and showed that ADAP constitutively associated with CRKL(SI Appendix, Fig. S5B). ODIN is a newly characterized proteincontaining ankyrin repeats, a SAM domain, and a PTB domain(28). Odin constitutively bound both GRB2 and CD2-associatedprotein (CD2AP) (SI Appendix, Fig. S5B). The phosphorylationlevel of ODIN Y445 strongly decreased upon CD28 inhibition,with log2 ratio H/M of −2.2 (Dataset S3). These results validatetight and consistent phosphorylation-dependent regulation ofthe CD28 interactome, demonstrating the usefulness of thecombinatorial analysis of the phosphoproteome and interactomefor dissecting the CD28-mediated signaling network.To obtain a comprehensive view of the phosphorylation-

dependent CD28 interaction network, we queried our 28 CD28-interacting proteins for their interactors in the STRING andBioGRID databases (Fig. 5A). Ninety-three proteins wereobtained that have regulated phosphorylation sites as identifiedby the phosphoproteomic screen (Dataset S3). These proteinsare highly enriched for protein functions such as intracellular-signaling cascades and actin-cytoskeleton organization (Fig. 5Band SI Appendix, Fig. S6A). Pathway analysis of the extended

CD28 interaction network showed that immune signaling, in-cluding TCR signaling, is overrepresented and additively down-regulated by CD28 inhibition (SI Appendix, Fig. S6B and DatasetS3). The extended CD28 interaction network forms clear phos-phorylation-dependent interaction hubs around proteins such asGRB2, the PI3K family, the STAT family, CD2AP and CIN85,and CBL. Interestingly, two well-characterized CD28-interactingproteins are most notable; GRB2 has extensive connections to 37newly recruited phosphoproteins whereas PI3K p85α (PIK3R1)has broad associations with multiple components in the CD28interactome. These observations might explain a functional im-portance of GRB2 as a key adaptor for regulating critical CD28-associated downstream signaling.

Costimulation-Regulated Phosphorylation of CapZIP and Its Functionin Regulation of IL-2 Production. Our analysis of the phosphoryla-tion-dependent CD28 interaction network suggested that one ofthe main functions of CD28 signaling is to regulate actin dy-namics (Fig. 5B). Consistent with this observation, it was recentlyreported that Rltpr, an actin-uncapping protein, plays an es-sential role in CD28-mediated signaling (29). Rltpr containsa capping protein (CP) interaction (CPI) motif that interactswith the CapZ protein, thereby inhibiting actin filament cappingand promoting actin polymerization. In our global phosphopro-teomics study, no significant change of RLTPR phosphorylationwas observed. However, the phosphorylation pattern of CapZIP,another member of the CPI motif-containing protein family,was significantly changed upon blockage of CD28 costimu-lation (Dataset S2). CapZIP, highly expressed in immunecells and skeletal muscle, can be phosphorylated by multiple

Fig. 4. Expanding the CD28 interactome in vivo by both IP-MS and IP-WB approaches. (A) Schematic of the experimental workflow for the IP-MS approach.Triple Flag-tagged GRB2 (B) and STS1 (C) were stably transfected into Jurkat leukemic T cells and SILAC-labeled for pulling down interacting proteins.Stimulation-dependent interactions are labeled in red color. Bait proteins are labeled in bold. (D) Validated interaction map of a protein interaction hubfocused on CD28, GRB2, and CBL/CBLB.

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stress-activated protein kinases (SAPKs) when cells are exposedto cellular stresses (30). A previous study suggested that phos-phorylation of CapZIP may cause the dissociation of CapZIPfrom CapZ and thereby regulate actin-filament assembly (30).Among 18 CapZIP phosphorylation sites identified by our phos-phoproteomic screening, three phosphoserine sites (S132, S135,and S333) were significantly changed upon CD28 inhibition, withS132 and 135 showing significant down-regulation and S333showing significant up-regulation, suggesting that CapZIP mightbe involved in CD28-mediated costimulation (Dataset S2). Toexamine the role of CapZIP in the CD28-signaling pathway, wegenerated CapZIP-deficient cells from the Jurkat T-cell lines byusing CRISPR/Cas9 technology. The double-nicking strategy wasused to minimize off-target mutagenesis (Fig. 6A) (31). We suc-ceeded in generating four single-cell clones of CapZIP-deficient(CapZIP−/−) Jurkat cells (Fig. 6B). The deficiency of CapZIP didnot affect surface expression of CD3 and CD45 (Fig. 6C). Al-though two of four CapZIP−/− single-cell clones had reducedsurface CD28 expression level by up to 60%, the other CapZIP−/−

clones had CD28 expression that was indistinguishable from theparental Jurkat line. The reduction of CD28 expression was notstatistically significant (Fig. 6D). After stimulation with SEE pre-sented by Raji B cells, WT and CapZIP−/− Jurkat cells up-regu-lated comparable amounts of CD69, indicative of intact TCRsignaling in CapZIP−/− cells (Fig. 6E). CD69, a major activationmarker downstream of the Ras/MEK/ERK signaling pathway, canbe induced by TCR stimulation alone. However, the IL-2production in SEE-stimulated CapZIP−/− Jurkat cells was nearlyabolished (Fig. 6F). Notably, CapZIP−/− Jurkat cells were able toproduce similar levels of IL-2 compared with WT Jurkat cells,when cells were stimulated by PMA plus ionomycin, which bypassthe TCR/CD28 proximal signaling (SI Appendix, Fig. S7). Col-lectively, these data suggest that CapZIP is required for CD28costimulation-dependent IL-2 production.

DiscussionAlthough the importance of CD28-mediated costimulation forT-cell activation has been explored for more than 29 y, thedownstream signaling pathways that are regulated by CD28 arestill poorly understood. Current models derived from targetedexperimental studies have yielded controversial views, especiallyfor the functional role of the PI3K pathway in CD28 signaling(12). Our combinatorial proteomic analysis provides a windowinto the physiologically relevant CD28-regulated protein phos-phorylation and its interaction networks. The extent of protein

phosphorylation changes that were affected by blocking CD28engagement was more extensive than anticipated. The globalphosphorylation analysis using cell-based stimulation showed someintersection of CD28-mediated signaling with TCR signaling butalso involved a number of other signaling pathways, some of whichhave been associated with other immune receptors.Studying signal transduction events mediated by endogenous

receptors under physiologically relevant conditions is crucial forunderstanding fine-tuned signaling outputs and their cellulareffects. Toward this end, we have designed our SILAC approachwith the following considerations. We studied endogenouslyexpressed CD28 receptors during a physiologically relevant in-tercellular interaction. We used CTLA4-Ig to specifically in-terrupt CD28 receptor activation, leaving signaling by otherreceptors, including the TCR, intact. This physiologically rele-vant activation condition maintains the complexity of the naturalcell–cell communication. It has been well-characterized thata broad spectrum of different costimulatory and coinhibitoryreceptors and their ligands are engaged and activated duringTCR-dependent T-cell activation (9). The assumption of ourstudy is that, in terms of T-cell activation, CD28 has some key,unique functions that either work independently or are in-tegrated with other signals generated by TCR or other moleculesinvolved in cell–cell interaction. In our experimental setting, theonly difference between two activation conditions is CD28blockade. We acknowledge that other receptor-mediated signalsmay rely on or influence the CD28 signals. Our experimentsindeed aim to determine which of these pathways are CD28-dependent.To explore the multidimensional nature of receptor signaling,

a combination of different unbiased screening approaches ona global scale is required. Our combinatorial proteomic ap-proach allowed the identification of signaling events associatedwith CD28 costimulation through both a global phosphorylationanalysis and a protein-interactome screen. The phosphopro-teome analysis provides a global view of CD28 receptor activa-tion across a broad spectrum of different downstream signalingpathways. The synthetic cytoplasmic-domain pulldown approachcaptured CD28-associated protein complexes without proteinsolubilization issues. The combination of these phosphoproteo-mic approaches facilitated the study and recognition of phos-phorylation-dependent recruitment of protein complexes.The CD28 interactome analysis was characterized by a spec-

trum of associated signaling proteins that are shared with otherimportant immune signaling pathways. Prominent among these

Fig. 5. Phosphorylation-dependent CD28 interactome. (A) An interaction map of the extended CD28 interaction network. CD28 and its 28 interactingproteins are displayed in the outer ring whereas newly recruited interacting proteins are presented inside. The additive log2 ratio H/M of all regulatedphosphorylation sites associated with a protein is presented. (B) Enriched Gene Ontology (GO) term analysis of the newly recruited phosphorylation-dependent CD28 interactome (see SI Appendix, Fig. S6A for an extended version). Representative enriched biological processes are presented.

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pathways are those that involve remodeling the actin cytoskele-ton and the PI3K pathway. As a preliminary effort to explore thisdataset, we focused on the actin cytoskeleton because our recentstudies (32) and those of others (29) suggested an important rolefor CD28 in regulating the actin cytoskeleton. The genetic studyof Liang et al. (29), which identified an important role of Rlptr,led us to focus on CapZIP. Ablation of CapZIP expression led toimpaired IL-2 production in our Jurkat-SEE/Raji system, whichmimicked the effects of CD28 blockade but had no effect onTCR signaling, based on CD69 expression. Although the preciserole of CapZIP is not clear and further work is required to un-derstand its role and that of its CD28-regulated phosphorylation,these studies do support an important role for CD28 signaling inactin-cytoskeleton regulation and have identified a previouslyunappreciated role for CapZIP downstream of CD28. Our fur-ther systems-wide interactome analysis and experimental vali-dation revealed a prominent GRB2-proximal interaction hubthat is widely regulated by CD28-dependent phosphorylationand merits further exploration. Clearly, our CD28-regulated

proteome will provide an important starting point for many otherstudies aimed at understanding CD28 costimulatory signaling.Importantly, this SILAC-based phosphoproteomic approach is

generally applicable for the study of other endogenous receptorsactivated by direct cell–cell contact because blocking reagentsare available for most of the well-characterized receptors. Withadvances in synthetic biology, the synthetic cytoplasmic-domainpulldown approach could, in principle, be applied to study abroad spectrum of transmembrane receptors. As such, this com-binatorial approach provides a widely applicable workflow tosystematically understand signaling events mediated by mem-brane receptors with short cytoplasmic tails in more physiologi-cally relevant contexts.

Materials and MethodsCell–Cell Stimulation and Sample Preparation for Phosphoproteomics. Mediumand heavy SILAC-labeled Jurkat leukemic T cells were starved in SILAC mediawithout dialyzed FBS for 12 h whereas two sets of equal amounts of un-labeled Raji lymphoblastoid B cells were kept in normal culture. After that,1 × 108 medium and heavy SILAC-labeled Jurkat leukemic T cells were used

Fig. 6. Generation and characterization of CapZIP-deficient cells. (A) Schematic of targeting human CapZIP exon 2 using Cas9 double-nicking strategy. The targetregions of each sgRNA are labeled in blue, and PAM sequences are highlighted in red. (B) Immunoblot analysis with a CapZIP-specific or GAPDH-specific antibody intotal cell lysates. Single-cell clones (clones 1–7) were generated from cells transfected with Cas9-nickase and sgRNAs targeting CapZIP. CapZIP protein level remainedlargely unchanged in clones 1–3 and was completely deleted in clones 4–7. (C) Representative FACS analysis of surface expression of CD3, CD45, and CD28 on restingCapZIP-sufficient cells (blue, clone 2) and CapZIP-deficient cells (red, clone 5). Gray-filled histogram, unstained control. (D) Bar chart showing CD28 surface expressionlevel on resting CapZIP-sufficient cells (blue, WT Jurkat and clones 1–3) and CapZIP-deficient cells (red, clone 4–7). (E) Jurkat cells were stimulated by SEE (30 ng/mL)presented by Raji B cells. CD69 up-regulation on CapZIP-sufficient cells (blue, clone 2) and CapZIP-deficient cells (red, clone 5) was measured by FACS 22 h afterstimulation. Gray-filled histogram, untreated control. (F) The ELISA analysis of cytokine IL-2 production by Jurkat cells 22 h after stimulation with Raji B cells and SEEat indicated concentrations. **P < 0.01, ***P < 0.001, n = 4, unpaired t test. The CapZIP-sufficient samples (WT Jurkat and clone 1–3) are labeled in blue, and theCapZIP−/− samples (clone 4–7) are labeled in red.

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for the following steps. Two sets of 1 × 108 Raji lymphoblastoid B cells werewashed and resuspended into 1 mL of SILAC RPMI 1640. After adding 20ng/mL SEE or 20 ng/mL SEE plus 10 μg/mL CTLA4-Ig (final concentration), theRaji lymphoblastoid B cells were incubated at 37 °C for 30 min. Both Jurkatleukemic T cells and Raji lymphoblastoid B cells were then transferred to icefor 10 min, mixed together with equal volumes, quickly centrifuged at 500 ×g for 5 min at 4 °C to promote cell–cell contact, and stimulated at 37 °C for5 min without resuspending the cell pellet, to promote cell–cell contact. Afterthat, the cells were lysed in lysis buffer [50 mM Tris·HCl, pH 7.4, 150 mMNaCl, 1% Nonidet P-40, 0.1% sodium deoxycholate, 1 mM sodium ortho-vanadate, protease inhibitors mixture (Complete mini; Roche), phosphataseinhibitor mixture (PhosSTOP; Roche)], and the two sets of cell lysates werethen mixed together. The soluble proteins were centrifuged at 4 °C andwere precipitated with four volumes of acetone at −20 °C overnight. Theprotein precipitate was harvested by centrifugation at 12,000 × g, 4 °C, andsolubilized in 8 M urea sample buffer (8 M urea, 20 mM Hepes, pH 8, 1 mMsodium orthovanadate, 2.5 mM sodium pyrophosphate, 1 mM β-glycer-ophosphate) with mild sonication. The pellet fraction was solubilized in thesame 8 M urea sample buffer containing benzonase. Total protein concen-tration was measured by the BCA assay (Pierce). The obtained proteins werereduced with DTT, alkylated with iodoacetamide, and digested with trypsinas described (10). The obtained peptide sample was split for either immu-noprecipitation or strong-cation exchange (SCX) fractionation following bytitanium dioxide (TiO2) enrichment. For tyrosine-phosphorylated peptideenrichment, the peptide sample was dissolved in 100 mM Tris·HCl, pH 7.4,100 mM NaCl, and 0.3% Nonidet P-40 and enriched by anti-phosphotyrosineantibody 4G10-conjugated beads (Millipore). The rest of the sample wasfractionated into 24 fractions by SCX chromatography as described (33) andenriched by a TiO2 kit (GL Sciences).

Flow-Cytometry Analysis and ELISA. Flow-cytometry analysis of phosphory-lated ERK was performed as described (34). Briefly, after stimulation, cellswere fixed in 4% (wt/vol) formaldehyde (Polysciences Inc.) and permeabilizedin 95% (vol/vol) ice-cold methanol. Permeabilized cells were stained witha pERK antibody (Cell Signaling) followed by a PE-conjugated antibodyagainst rabbit IgG and APC-conjugated anti-CD3. The level of pERK in gatedCD3+ populations was measured by an LSRFortessa cell analyzer. For theELISA, the supernatants of the coculture system were collected 22 h afterstimulation, and IL-2 production was measured by using a Human IL-2 ELISAKit (BD Biosciences) according to the manufacturer’s instructions.

CD28cyto Pulldown and Immunoprecipitation Assays. The CD28 cytoplasmicdomain with biotin tag and various modifications was synthesized in-house,HPLC-purified, and conjugated on streptavidin agarose beads. Jurkat leu-kemic T cells were SILAC-labeled with light, medium, or heavy lysine andarginine. Then, 50 × 106 SILAC-labeled cells were lysed in Nonidet P-40 lysisbuffer [25 mM Tris·HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 1 mM so-dium orthovanadate, protease inhibitors mixture (Complete minbi; Roche),phosphatase inhibitor mixture (PhosSTOP; Roche)] and incubated with dif-ferent types of beads overnight at 4 °C. The beads were washed four timeswith washing buffer (25 mM Tris·HCl, pH 7.4, 150 mM NaCl, 0.1% NonidetP-40) and two times with 20 mM ammonium bicarbonate at 4 °C. The as-sociated proteins were digested on the beads by trypsin as described (35)and combined for MS analysis.

For IP-MS analysis of GRB2 and STS1, WT and triple Flag-tagged Jurkatleukemic T cells were SILAC-labeled as indicated in Fig. 2A. Then, 1 × 108 cellswere starved in PBS for 20 min at 37 °C, and the heavy SILAC-labeledcells were stimulated with an anti-CD28 antibody for 5 min at 37 °C. The cellswere lysed in Nonidet P-40 lysis buffer, precleared with mouse IgG beads(Sigma), and incubated with anti-Flag M2 beads (Sigma) overnight. Thebeads were washed and digested as described above.

MS Analysis and Data Analysis. Samples were analyzed on LTQ-Orbitrap Elite(for phosphoproteomics) or Classic (for part of CD28cyto pulldown) mass

spectrometers (Thermo). The obtained raw data were processed by Max-Quant (version 1.2.2.5) coupled with Andromeda for database searchingagainst the International Protein Index (IPI) human database (version 3.79;91464 entries). The assignment of phosphorylation sites to tryptic peptideswas performed automatically in the MaxQuant software environment withproper evaluation of the site localization probabilities (36). Evidence tablescontaining all of the quantified peptide information were used to generatethe final identification information as shown in Dataset S1. The R languagepackage was used to perform quantitative analysis of the global phos-phorylation dataset with data obtained from the Maxquant evidence tables.The minimum Andromeda score was set as 42. In addition, the minimum sitelocalization probability was 0.33. The pathway analysis was performed usinggene annotations from the Kyoto Encyclopedia of Genes and Genomes(KEGG) database (37) (P value cutoff 0.1; Fisher’s exact test) and IngenuityPathways Analysis (P value cutoff 0.05; Fisher’s exact test) (Fig. 3D). Furtherinteractome analysis related to Fig. 4D was performed based on STRING v9.1(38) (score cutoff 805) and BIOGRID (39).

For CD28 cytoplasmic domain pulldowns and IP-MS experiments, onlyproteins identified and quantified with at least two “Unique + Razor Pep-tides” were considered. Only proteins identified and quantified in at leasttwo out of three experiments as shown in Fig. 3B and SI Appendix, Fig. S4 Aand B were considered as positive hits. The log2 ratio cutoff was set as 1.pYMNM motif- and PpYAPP motif-dependent interactions were definedby SILAC quantification with three-peptide pulldown combinations (SIAppendix, Fig. S4 A and B), which were cross-validated by YpY peptidepulldown (SI Appendix, Fig. S4C). The result, as shown in SI Appendix, Fig.S4D, was used to define PxxPP motif-dependent interactions, and two se-lected proteins (i.e., CIN85 and STS1) were further validated by Western blot(SI Appendix, Fig. S4E).

Western Blots. For IP-WB analysis of GRB2, CRKL, DOK1, CD2AP, CIN85, ODIN,and ADAP, 50 × 106 cells were starved in PBS for 20 min at 37 °C, and cells werestimulated with anti-TCR or anti-TCR plus anti-CD28 antibody for 5 min at 37 °C.The cells were lysed in Nonidet P-40 lysis buffer and incubated with anti-FlagM2 beads (Sigma) or anti-mouse IgG beads (Sigma) overnight. The beads werewashed and boiled in 2× SDS/PAGE loading buffer for Western blot analysis.Western blot quantitation was performed with ImageJ software (version 1.47v).

Protein complexes pulled down by conjugated CD28 cytoplasmic domainor antibodies were boiled in 2× SDS/PAGE loading buffer, resolved on 10%SDS/PAGE gels, transferred to nitrocellulose, and analyzed.

Generation of CapZIP-Deficient Jurkat Cell Lines. Two single-guide RNAs(sgRNAs) targeting the CapZIP gene were cloned into pX335 (Addgene).Jurkat cells were cotransfected with these two sgRNA plasmids and a GFPreporter plasmid. At 24 h posttransfection, GFP+ live Jurkat cells were singlecell sorted into a 96-well plate. Three weeks later, single cell clones wereexpanded upon confluency.

MS Data Storage. The raw MS data files associated with this study can bedownloaded from the Mass Spectrometry Interactive Virtual Environment(MassIVE) (massive.ucsd.edu/ProteoSAFe) via the following ftp access: ad-dress, massive.ucsd.edu/ProteoSAFe; user name, CD28; password, CD28RAJI.

ACKNOWLEDGMENTS. We acknowledge K. Colwill, L. Taylor, B. Larsen,C. Zhang, and M. Tucholska for technical help and J. Jin, Y. Zheng, C. Chen,G. M. Findlay, and M. Kofler for helpful discussions. We thank A. Salomonand N. Krogan for constructive suggestions after their reading of thismanuscript. We thank J. Bluestone, who provided the CTLA4-Ig. This workwas supported by grants from Ontario Research Fund GL2 (to T.P. andA.-C.G.), the Canadian Institutes of Health Research (CIHR) (Grant MOP-84314) (to A.-C.G.), the Howard Hughes Medical Institute (to A.W.), the NIH/National Cancer Institute (Grant CA82683) (to T.H.), and a Shenzhen grant(ZDSYS20140509142721429) (to R.T.). R.T. is the recipient of a fellowshipfrom the CIHR. H.W. is the recipient of a postdoctoral fellowship from the USArthritis Foundation.

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