Phosphorylation Ε CD3 Phosphorylation/ZAP70

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Differential Src Family Kinase Activity Requirements for CD3 ζ Phosphorylation/ZAP70 Recruitment and CD3ε Phosphorylation This information is current as of September 27, 2021. Tara L. Lysechko and Hanne L. Ostergaard J Immunol 2005; 174:7807-7814; ; doi: 10.4049/jimmunol.174.12.7807 http://www.jimmunol.org/content/174/12/7807 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Differential Src Family Kinase Activity Requirements for CD3␨ Phosphorylation/ZAP70 Recruitment and CD3⑀ Phosphorylation1

Tara L. Lysechko and Hanne L. Ostergaard2

The current model of T cell activation is that TCR engagement stimulates Src family tyrosine kinases (SFK) to phosphorylate CD3␨. CD3␨ phosphorylation allows for the recruitment of the tyrosine kinase ZAP70, which is phosphorylated and activated by SFK, leading to the phosphorylation of downstream targets. We stimulated mouse CTLs with plate-bound anti-CD3 and, after cell lysis, recovered proteins that associated with the CD3 complex. The protein complexes were not preformed, and a number of tyrosine-phosphorylated proteins were inducibly and specifically associated with the TCR/CD3 complex. These results suggest that complex formation only occurs at the site of TCR engagement. The recruitment and tyrosine phosphorylation of most proteins were abolished when T cells were Downloaded from stimulated in the presence of the SFK inhibitor PP2. Surprisingly, CD3␨, but not CD3⑀, was inducibly tyrosine phosphorylated in the presence of PP2. Furthermore, ZAP70 was recruited, but not phosphorylated, after TCR stimulation in the presence of PP2, thus confirming the phosphorylation status of CD3␨. These data suggest that there is a differential requirement for SFK activity in phosphorylation of CD3␨ vs CD3⑀. Consistent with this possibility, ZAP70 recruitment was also detected with anti-CD3-stimulated, Lck- deficient human Jurkat T cells. We conclude that TCR/CD3-induced CD3␨ phosphorylation and ZAP70 recruitment do not absolutely ⑀ require Lck or other PP2-inhibitable SFK activity, but that SFK activity is absolutely required for CD3 and ZAP70 phosphorylation. http://www.jimmunol.org/ These data reveal the potential for regulation of signaling through the TCR complex by the differential recruitment or activation of SFK. The Journal of Immunology, 2005, 174: 7807–7814.

hosphorylation of CD3␨ is one of the earliest detectable This immobilized Ab capture approach is similar to the solid phase events that occurs after TCR stimulation of T cells (1). immunoisolation assay described by Tamura et al. (11), in which Ample evidence suggests that the Src family kinases Abs were immobilized to plastic to capture labeled proteins from P3 (SFK), Lck and, to a lesser extent, Fyn, phosphorylate the ITAMs cell lysates. This solid phase immunoisolation was shown to be of CD3␨ (2–4). ZAP70 is recruited to CD3␨, binding to dually highly speciﬁc, with low background binding (11). We have phosphorylated ITAMs, via its tandem Src homology 2 (SH2) do- adapted this technique to use anti-TCR/CD3 stimulatory Ab to by guest on September 27, 2021 mains (2, 5–7). Once recruited, ZAP70 is phosphorylated by Lck capture protein complexes formed in live T cells to study TCR- or Fyn, which renders it catalytically active (2, 3, 5, 8, 9), leading proximal signaling events. This approach can also be used as a tool to phosphorylation of downstream substrates, including the adap- to identify novel proteins that are associated with such protein tor protein, linker for activation of T cells (10). Tyrosine phos- complexes. phorylation of linker for activation of T cells, in turn, allows for Using solid phase immunoisolation, we have found that TCR- the recruitment of a number of proteins to the signaling associated CD3␨ can be phosphorylated and recruits ZAP70 in the complex (10). presence of the SFK inhibitor PP2, a potent inhibitor of Lck and We have developed a biochemical method to capture and ana- Fyn activity in T cells (12). By contrast, PP2 did block both CD3⑀ lyze proteins found complexed with the TCR after TCR stimula- and ZAP70 phosphorylation and all additional downstream ty- tion. In this approach, an immobilized stimulatory Ab is used to rosine phosphorylation events. These data suggest that there is a both stimulate T cells and, after cell lysis, capture protein com- differential SFK requirement for CD3␨ and CD3⑀/ZAP70 phos- plexes regardless of how they are associated with the complex. phorylation. Furthermore, we observed CD3␨ phosphorylation and ZAP70 recruitment in Lck-deﬁcient Jurkat cells. Our results indicate that during TCR stimulation, the PP2-inhibitable SFK, Lck Department of Medical Microbiology and Immunology, University of Alberta, Ed- and Fyn, are not uniquely capable of mediating CD3␨ phosphor- monton, Alberta, Canada ylation, resulting in ZAP70 recruitment. However, Lck and/or an- Received for publication October 13, 2004. Accepted for publication April 12, 2005. other SFK are absolutely required for CD3⑀ and ZAP70 phosphor- The costs of publication of this article were defrayed in part by the payment of page ylation and recruitment of additional proteins to the CD3 complex, charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. suggesting that the levels of active SFK needed for phosphoryla- 1 This work was supported by the National Cancer Institute of Canada, with funds tion of the various CD3 subunits differ substantially. from the Canadian Cancer Society. H.L.O. is an Alberta Heritage Foundation for Medical Research Scientist, and T.L.L. is supported by a studentship from Alberta Heritage Foundation for Medical Research. Materials and Methods 2 Address correspondence and reprint requests to Dr. Hanne L. Ostergaard, Depart- Cells ment of Medical Microbiology and Immunology, 670 Heritage Medical Research ϩ Center, University of Alberta, Edmonton, Alberta, Canada T6G 2S2. E-mail address: The murine CD8 CTL clone AB.1 has been described previously (13). [email protected] The clones were stimulated weekly with irradiated allogeneic C57BL/6 3 Abbreviations used in this paper: SFK, Src family kinase; cska-␨, cytoskeleton- splenocytes and IL-2 and were used for experiments 5–6 days after stim- associated ␨; MBP, myelin basic protein; NRS, normal rabbit serum; PTK, protein ulation. The degranulation assay has been described in detail previously tyrosine kinase; SH2, Src homology 2; PAK, p21-activated kinase. (14). Con A blasts were generated by culturing C57BL/6 splenocytes with

2 ␮g/ml Con A for 48 h. Human Jurkat T cells and the Lck-deficient variant associated proteins after cell stimulation with immobilized mAb. JCaM1.6 were obtained from American Type Culture Collection. Proteins that remain bound to the plate are recovered after cell Abs and reagents lysis and removal of cellular debris. We demonstrate that numer- ous tyrosine-phosphorylated proteins are enriched after stimulation The source and purification of Abs from hybridomas producing 145-2C11 of the CTL clone AB.1 and recovery of the proteins bound to the (anti-CD3⑀), H57–597 (anti-TCR␤), I3/2.3 (anti-CD45, pan-specific), MB23G2 (anti-CD45RB), MB4B4 (anti-CD45RB), M1/42.3.9.8 (anti- plate (Fig. 1A). A number of tyrosine-phosphorylated proteins that MHC class I), OKT3 (anti-CD3⑀), and PY72.10.5 (anti-phosphotyrosine) cannot be detected in the cell lysate prepared from stimulated cells have been described previously (15). The hybridoma producing H146- are enriched after capturing the complexes on the plate. As ex- 968A was obtained from Dr. M. C. Miceli (University of California, Los pected, the SFK Lck and Syk family kinase ZAP70 was found Angeles, CA) (16). Polyclonal anti-Lck (C437) used for Lck immunoprecipi- ⑀ tations was generated in our laboratory using a BSA-coupled peptide based on complexed with CD3 (Fig. 1A). We were unable to detect Fyn in aa 476–509 of Lck. Anti-ERK2, anti-Lck, anti-Nck, and anti-ZAP70 were the CD3⑀ complexes; however, these CTL clones express very low purchased from BD Biosciences. Anti-CD3⑀ (M-20), anti-CD3␨ (6B10.2), levels of Fyn (data not shown). The SH2/SH3 adapter Nck was ␣ anti-Nck1/2 (C-19), anti- p21-activated kinase (PAK) (N-20), and anti-goat also found in the CD3⑀ complexes (Fig. 1A). IgG-HRP were purchased from Santa Cruz Biotechnology. Anti-mouse IgG- HRP and anti-hamster IgG-HRP were purchased from Jackson ImmunoRe- We have performed a number of experiments to validate this search Laboratories. Protein A-HRP was purchased from Pierce. The SFK approach. To determine whether this method results in the recov- inhibitor, PP2, and the inactive analog, PP3, were purchased from Calbiochem. ery of preformed complexes or whether we are indeed detecting Myelin basic protein (MBP) was purchased from Sigma-Aldrich. inducible recruitment, we prepared lysates of unstimulated CTL Cell stimulation with immobilized Abs and recovery of clones, added the lysates to the plate, recovered CD3⑀, and ana- Downloaded from associated complexes lyzed associated proteins. There is no specific recruitment of tyrosine-phosphorylated proteins or of Lck, Nck, or ZAP70 from For immobilizing Abs on plastic, 60-mm, non-tissue culture-treated plates were incubated with 10 ␮g/ml 145-2C11 or other mAb overnight at 4°C. lysates of unstimulated cells (Fig. 1B), confirming that we are ob- The plates were washed twice with PBS, blocked with 2% BSA in PBS for serving TCR-induced complex formation. Also of note, the recov- 45 min at 37°C, and washed three times with PBS, then 1 ϫ 107 cells were ery of associated proteins is essentially identical after stimulation added to the plates. Where indicated, the cells were pretreated with PP2 or with either anti-CD3⑀ or anti-TCR␤ (Fig. 1B).

PP3 for 15 min on ice before addition to the plates. The cells were stim- http://www.jimmunol.org/ ulated at 37°C for the indicated time, after which the cells were lysed in the Because cells spread on the anti-CD3 and become tightly ad- plates for 20 min with lysis buffer (1% Nonidet P-40, 10 mM Tris (pH 7.5), hered to the mAb, it could be argued that many of these proteins 150 mM NaCl, and 1 mM sodium vanadate). Lysates were removed from are nonspeciﬁcally recovered, perhaps as a result of trapping large the plates, and a sample containing 5 ϫ 105 cell equivalents was saved for membrane fragments or cellular debris. To control for this possi- further analysis. The plates were vigorously washed three times with lysis bility, we plated cells on immobilized anti-CD45, which results in buffer to ensure that all cellular debris was removed, then the immobilized Abs and associated proteins were eluted from the plate by adding reducing dramatic cell spreading (17), and analyzed captured protein for the sample buffer and heating the plates to 55°C. The entire sample of eluted presence of CD45 or CD3⑀. CD3⑀ is only captured when cells are protein was loaded into a single lane of an SDS-PAGE gel. When used, plated on anti-CD3⑀, and CD45 is only captured when cells are PP2 was not added to the lysis buffer used to wash plates, because we have

plated on anti-CD45 (Fig. 2A). These results clearly demonstrate by guest on September 27, 2021 found that its inclusion in the washes does not impact the quality or quan- tity of phosphoproteins recruited to the TCR/CD3 complex. that there is specificity to the protein capture, and proteins do not nonspecifically adhere to, or become trapped on, the plates. As an Western blotting additional control, we plated cells on anti-class I MHC to compare Cell lysates and proteins eluted from the mAb-coated plates were run on 12 class I MHC-captured proteins with those captured with anti-CD3⑀ or 15% SDS-PAGE gels and transferred to polyvinylidene difluoride mem- and anti-TCR␤. Very few tyrosine-phosphorylated proteins were branes. Immunoblotting was performed using the appropriate HRP-coupled recovered with immobilized anti-class I MHC (Fig. 2B) compared Ab and was visualized by ECL (NEN). with anti-CD3⑀ and TCR␤. As well, no ZAP70 (Fig. 2B), Lck, or Lck in vitro kinase assays Nck (data not shown) complexed with class I MHC in these cells. AB.1 clones were washed three times with PBS, and 1 ϫ 107 cells were Because we wanted to use this procedure for the isolation of resuspended in RPMI 1640. Cells were pretreated with DMSO, PP2, or intracellular proteins that associate with the TCR complex, we PP3 for 15 min on ice. Cells were lysed with 1% Nonidet P-40 lysis buffer needed to demonstrate that intracellular proteins do not nonspe- with or without PP2/PP3. Lck or normal rabbit serum (NRS) immunopre- cifically associate with the complex. We examined two kinases cipitations were performed on postnuclear lysates. Immunoprecipitates were washed twice with radioimmunoprecipitation assay (RIPA) buffer that become activated upon TCR stimulation, but have not been with or without PP2/PP3 and once with HEPES kinase buffer (10 mM shown to associate with the TCR complex: ERK2 and ␣PAK. Im-

HEPES (pH 7.1) and 10 mM MnCl2) with or without PP2/PP3. Kinase munoblots with anti-ERK2 and anti-␣PAK demonstrate that these reactions contained Lck or NRS immunoprecipitates, 5 ␮g of MBP sub- proteins are not recovered with anti-CD3⑀ and TCR␤ complexes ␮ strate, 1 mM ATP, and either PP2 or PP3 in 50 l. Kinase reactions pro- after stimulation, even though both proteins are clearly present in ceeded for 30 min at 32°C, at which time the beads containing immunoprecipitates were subjected to centrifugation, and 40 ␮l of the supernatant the lysate (Fig. 2B). We also examined a number of additional containing the substrate was transferred to a new tube. Substrate-containing intracellular proteins and did not observe recruitment of these pro- supernatant was loaded onto a 15% SDS-PAGE gel. Proteins were trans- teins (data not shown). These results demonstrate that there is ferred, and the membrane was probed with anti-phosphotyrosine. A portion specificity to the recruitment of proteins. of the sample was stained with Coomassie Blue to detect MBP protein loading. Lck immunoprecipitates were run on an 8.5% gel and transferred, and the membrane was blotted for Lck as a loading control. CD3␨, but not CD3⑀, is tyrosine phosphorylated in the presence Results of the SFK inhibitor PP2 Stimulation of CTL clones with immobilized anti-CD3 results in The phosphorylation cascade induced by T cell activation is gen- specific recruitment of a number of tyrosine-phosphorylated erally accepted to be initiated by SFK (18). To confirm in our proteins to TCR/CD3 complex system that the bulk of the tyrosine-phosphorylated proteins that We have established a biochemical approach to evaluate proteins are recruited to the TCR/CD3 complex during immobilized mAb recruited to the TCR complex by capturing the TCR complex and stimulation are dependent on the activation of SFK, we examined The Journal of Immunology 7809 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 1. Several tyrosine-phosphorylated proteins are recruited to the TCR/CD3 complex upon stimulation of CTL clones with immobilized anti-CD3. A, AB.1 CTL clones were stimulated with plate-bound anti-CD3 (145-2C11) for the indicated time or were left unstimulated by plating on FIGURE 2. Speciﬁc complexes form in association with CD3 after BSA as a control. Cells were then lysed in the plates, the lysate was re- stimulation of CTL clones with plate-bound anti-CD3. A, Cells were plated moved, and a sample was saved for analysis (indicated as lysate). The on immobilized anti-CD3 or anti-CD45 for the indicated time, and com- proteins associated with the immobilized mAb were recovered as described plexes were recovered as described in Fig. 1A. Samples were probed with in Materials and Methods. Samples were run on SDS-PAGE gels, trans- anti-CD3⑀ or anti-CD45RB. B, CTL clones were stimulated on immobi- ferred to membranes, and probed with the indicated mAb. The dot desig- lized anti-class I MHC, anti-CD3⑀, or anti-TCR␤ for 20 min, and com- nates the Ig H chain. B, AB.1 clones were exposed to BSA or immobilized plexes were recovered as described in Fig. 1A. A sample of the lysate anti-CD3⑀ or anti-TCR␤ (indicated as cells) as in A, or lysates from un- containing the unbound proteins, which was removed from the plate, is also stimulated AB.1 clones were added to plates precoated with anti-CD3⑀ or shown. Immunoblots for phosphotyrosine, ERK2, ␣PAK, and ZAP70 are anti-TCR␤, and complexes from the lysate that bound to the plate were shown. recovered and analyzed (indicated as lysate).

hibited TCR-triggered degranulation, whereas the inactive analog, the effect of inhibiting SFK activity with PP2, a potent and selec- PP3, had no impact on degranulation (Fig. 3A). To ensure that tive inhibitor of SFK, particularly Lck and Fyn (12). We ﬁrst as- these concentrations of PP2 effectively inhibited SFK activity, we sessed the effect of PP2 on a CTL functional response, speciﬁcally examined the ability of Lck to phosphorylate a protein substrate degranulation. At concentrations of 5–20 ␮M, PP2 completely in- (MBP) on tyrosine in the presence of PP2 or PP3. We were unable 7810 SFK REQUIREMENTS FOR CD3 SUBUNIT PHOSPHORYLATION

FIGURE 3. Effect of the SFK inhibitor, PP2, on CTL functional responses, Lck kinase activity, and tyrosine phosphorylation after TCR stimulation. A, Serine esterase release (degranulation) was mea- sured from AB.1 in response to 10 ␮g/ml immobilized anti-CD3 in the presence of different concentrations of PP2 and its inert analog, PP3. B, Kinase activity of Lck immunoprecipitated from control and PP2-treated cells was assessed by an in vitro kinase assay using MBP as a substrate. AB.1 clones were pretreated with PP2 or PP3 and lysed. Lck immu- noprecipitations were performed, mixed with the MBP substrate and reaction mix, and incubated for 30 min. The Lck-bound beads were subjected to centrifugation, and a sample of the MBP-containing supernatant was removed. A phosphotyrosine blot (top Downloaded from panel) shows the degree of MBP phosphorylation, and a Coomassie stain (middle panel) is an indication of the amount of MBP in the assay. An Lck blot of the immunoprecipitate is shown in the bottom panel.An NRS immunoprecipitate was included as a control for background phosphorylation of MBP. C, Anti-

phosphotyrosine blot of postnuclear cell lysates from http://www.jimmunol.org/ AB.1 stimulated with immobilized anti-CD3⑀ in the absence or the presence 10 ␮M PP2 or PP3.

to examine Fyn activity because its expression was barely detect- tyrosine-phosphorylated proteins was recruited to the TCR/CD3 able in these cells (data not shown). Lck was immunoprecipitated complex (Fig. 4). In the presence of PP2, this recruitment was by guest on September 27, 2021 from unstimulated AB.1 clones in the presence of PP2 or PP3 with almost totally abolished, except, surprisingly, for CD3␨, which re- an anti-C-terminal Ab, which results in maximal Lck activity (data mained phosphorylated (Fig. 4). After stimulation, with or without not shown), probably by binding in such a way that the Lck is in the inert analog PP3, a substantial fraction of phosphorylated an open and active conformation. The Lck immunoprecipitates CD3␨ appeared as a 19-kDa form, which was previously described were then used in in vitro kinase reactions with MBP as a substrate (19), in addition to the 21- and 23-kDa forms of CD3␨ (Fig. 4). In in the presence of PP2 or PP3. After the reaction, Lck immuno- the presence of PP2 we detected primarily the 19-kDa and low precipitates were recovered by centrifugation, and the supernatant levels of the 21-kDa form, but never the 23-kDa form, of CD3␨ containing MBP was used to analyze the phosphorylation status of (Fig. 4 and data not shown). These results suggest that CD3␨ is MBP. In the presence of PP3, Lck is active and phosphorylates subject to tyrosine phosphorylation in the absence of detectable MBP on tyrosine residues (Fig. 3B). However, with concentrations Lck activity. ␮ of PP2 ranging from 10 to 20 M, MBP was not phosphorylated, The detection of CD3␨ phosphorylation in the presence of the indicating that Lck kinase activity was effectively inhibited in this SFK inhibitor PP2 was surprising and was investigated further. As assay system. An NRS control immunoprecipitate was used in an an additional indirect confirmation of CD3␨ phosphorylation, we identical kinase reaction to detect any kinase activity that may be determined whether ZAP70 was recruited to the complex in the nonspecifically associated with protein A-coated beads. A Coo- presence of PP2. Interestingly, ZAP70 recruitment could easily be massie stain of a gel containing a fraction of the substrate super- detected in the captured complexes isolated from cells treated with natants showed that equal amounts of MBP were present in each sample. The Lck proteins bound to the protein A-coated beads PP2 (Fig. 4). The reduced recruitment in the presence of PP2 could were also loaded onto gels and probed with anti-Lck to demon- stem from the inability of cells to strongly adhere to the immobi- strate that similar amounts of Lck were present in the kinase as- lized mAb in the presence of PP2, thereby not allowing for optimal says. We also examined the effect of SFK inhibition on general association of the cells with the immobilized mAb. Although tyrosine phosphorylation and recruitment of proteins to the TCR/ ZAP70 was clearly recruited to the complex, it was not phosphor- CD3 complex after stimulation with immobilized anti-CD3. It is ylated in the presence of PP2 (Fig. 4), thus confirming that SFK clear that 10 ␮M PP2 is sufficient to abolish nearly all detectable activity was indeed effectively inhibited. Furthermore, unlike anti-CD3-induced tyrosine phosphorylation in the lysate (Fig. 3C). CD3␨, CD3⑀ was not phosphorylated in the presence of PP2 (Fig. The impact of SFK inhibition on the recruitment of tyrosine- 4). Similar results were seen in Con A blasts prepared from phosphorylated proteins after CD3 stimulation was next assessed. C57BL/6 spleen cells (Fig. 5). We clearly detected CD3␨ phos- As shown in Fig. 1, a number of tyrosine-phosphorylated proteins phorylation in the captured CD3⑀ complexes in the presence of were captured with the plate-bound Ab in the absence of any in- PP2. ZAP70 recruitment was reduced in these cells, but was still hibitor (Fig. 4). With the inactive analog PP3, a similar profile of detectable (Fig. 5). Thus, there is a differential requirement for The Journal of Immunology 7811 Downloaded from http://www.jimmunol.org/

FIGURE 4. CD3␨, but not CD3⑀, is phosphorylated and recruits ZAP70 after anti-CD3 stimulation in the presence of the SFK inhibitor PP2. Im- munoblots are shown of complexes captured with plate-bound anti-CD3 FIGURE 5. CD3␨ is phosphorylated in the presence of the SFK inhib- after stimulation of AB.1 with immobilized anti-CD3 as described in Fig. itor, PP2, after stimulation of Con A blasts. Spleen cells from C57BL6/J ␮ 1A in the absence or the presence of 10 M PP2 or PP3. Tyrosine phos- mice were stimulated to proliferate with Con A. These Con A blasts were ⑀ ␨ phorylation of CD3 , CD3 , and ZAP70 was detected by immunoblotting stimulated with plate-bound anti-CD3 for the indicated time in the presence of ⑀ ␨ for phosphotyrosine. Blots were stripped and reprobed for CD3 , CD3 , 10 ␮M PP2 or PP3. Complexes captured with the plate-bound anti-CD3⑀ were and ZAP70. The arrowhead indicates the position of the 19-kDa form, and recovered and probed with the indicated Ab. A sample of the postnuclear by guest on September 27, 2021 ␨ the asterisks indicate the positions of the 21- and 23-kDa forms of CD3 . lysate is also shown. The arrowhead indicates the position of CD3␨.

either SFK family members or levels of active SFK in the phos- recovery of CD3␨ (Fig. 6C) in these cells compared with Jurkat phorylation of CD3 components, because CD3␨ can be phosphor- cells. These results indicate that CD3␨ is phosphorylated in the ylated in the presence of PP2, whereas CD3⑀ phosphorylation is absence of Lck in JCaM1.6 cells. Combined with the results ob- completely abolished by this SFK inhibitor. tained using the SFK inhibitor with the CTL clones, CD3␨ appeared to be phosphorylated in an Lck-independent, and possibly ␨ CD3 is phosphorylated after TCR/CD3 stimulation in Lck- a Fyn-independent, manner. deficient Jurkat T cells To further explore the apparent Lck-independent phosphorylation Discussion of CD3␨, we examined whether CD3␨ is phosphorylated in In this study we document a novel approach to analyze proteins JCaM1.6, Jurkat cells deficient in Lck expression (20). We used associated with the TCR complex after T cell activation. This different anti-CD3⑀ concentrations to stimulate Jurkat and method differs from that described by Harder and Kuhn (21), in JCaM1.6 cells and found, as previously documented (20), that in- which TCR-associated membrane fragments are isolated from ho- duction of tyrosine phosphorylation in the lysates of JCaM1.6 cells mogenates. We use detergent to disrupt the cells and to remove stimulated with anti-CD3 is severely compromised (Fig. 6A). We proteins that are nonspecifically bound to the complexes captured then examined recruitment of tyrosine-phosphorylated proteins to on the plates. This approach will probably preserve interactions the TCR/CD3 complex by capturing complexes with the stimula- that would presumably be disrupted during standard immunopre- tory anti-CD3 on plastic. Surprisingly, we found that there are a cipitation procedures. number of tyrosine-phosphorylated proteins that are recruited to We found that the process of stimulating CTL on immobilized the complex in JCaM1.6 cells, although the degree of protein re- Ab and then capturing complexes significantly enriches for en- cruitment was significantly lower than that in wild-type Jurkat gaged TCR signaling complexes as the cells spread on the immo- cells (Fig. 6B). Similar to what was found for the CTL clones bilized Ab. Stimulation with immobilized Ab also more closely treated with PP2, phosphorylated CD3␨ (Fig. 6B) and ZAP70 re- mimics stimulation by APC or target cells than does stimulation cruitment (Fig. 6C) could be detected in CD3⑀ complexes obtained with soluble cross-linked Ab. For example, immobilized Ab stim- from JCaM1.6 cells, confirming that CD3␨ is indeed phosphory- ulation generates more sustained signaling, provides a polarized lated in the absence of Lck (Fig. 6C). There are lower levels of stimulus, and leads to degranulation of CTL (22), as is observed ZAP70 recruited in these cells; however, we found that there were with target cells. Unlike stimulation with APC, in which engaged also substantially lower levels of expression (data not shown) and molecules can move freely within membranes, immobilized Ab 7812 SFK REQUIREMENTS FOR CD3 SUBUNIT PHOSPHORYLATION

FIGURE 6. CD3␨ phosphorylation and ZAP70 recruitment occur after CD3 stimulation of JCaM1.6 cells. A, Jurkat and JCaM1.6 cells were stimulated with the indicated concentration of plate-bound anti-CD3 (OKT3) for 10 min, after which a sample of the cell lysate was removed from the plate, subjected to SDS-PAGE, and probed with anti-phosphotyrosine. B, Com- plexes remaining on the mAb-coated plates after removal of lysate from A were recovered as described in Fig. 1A, run on a gel, and probed with anti-phosphotyrosine. C, Membrane from B was probed with Abs to CD3⑀, CD3␨, ZAP70, and Lck. Downloaded from The arrowhead indicates the position of CD3␨. http://www.jimmunol.org/

provides a fixed plane of stimulatory Ab that does not allow for though phosphorylation of CD3⑀ and ZAP70 is completely inhib- rearrangement of engaged TCR/CD3 complexes or formation of a ited under these conditions (Figs. 4 and 5). We could also detect mature immunological synapse/supramolecular activation cluster CD3␨ phosphorylation and ZAP70 recruitment in JCaM1.6, a Lck- (23, 24). However, in light of the recent findings demonstrating deficient variant of Jurkat cells (Fig. 6). JCaM1.6 cells still express that tyrosine kinase signaling occurs before a mature synapse Fyn, albeit at low levels (27); therefore, we cannot rule out a role by guest on September 27, 2021 forms (25, 26), this method of stimulation still enables us to study for Fyn in the phosphorylation of CD3␨ in these cells. However, the very early events in T cell activation, such as initial phosphor- the results from the CTL clones and Con A blasts treated with PP2 ylation events and recruitment of proteins to the TCR/CD3 suggest that neither SFK is responsible for this phosphorylation. complex. Therefore, our results suggest that another kinase(s), in addition to We have used a number of controls to demonstrate the speci- Lck or Fyn, is capable of phosphorylating CD3␨. It is possible that ficity of the proteins recruited to the TCR/CD3 complex with our the kinase responsible for CD3␨ phosphorylation is a SFK that is novel assay. We show that virtually no proteins bind to BSA- not inhibited by PP2. This is nonetheless interesting, because this coated plates and that very few proteins (other than the protein to would imply that this other SFK, or perhaps reduced levels of SFK which the Ab is specific) within an unstimulated lysate will bind to activity, could function to phosphorylate CD3␨, but is not able to immobilized anti-CD3⑀ or anti-TCR␤ (Fig. 1B). We have also per- compensate for the lack of Lck activity with respect to the phos- formed experiments in which stimulated lysates were plated onto phorylation of CD3⑀ or ZAP70. This study seems to contradict immobilized anti-CD3⑀ and found, as with an unstimulated lysate, other studies that concluded that Lck is required for CD3␨ phos- that there are very few proteins that can bind the immobilized Ab phorylation; however, this approach has not been used previously, (data not shown). These results clearly demonstrate that intact cells and we may be significantly enriching for TCR signaling com- must be stimulated to induce complex formation around the TCR/ plexes, thus improving the ability to recover and detect phosphor- CD3 complex. As well, we have shown that proteins do not non- ylated CD3␨. specifically adhere to or become trapped on the plates as the cells What is the nature of the CD3␨ phosphorylation that is detected spread on the immobilized stimulatory Ab (Fig. 2A). Control ex- using this approach? It has been shown previously that in 30–40% periments performed with immobilized anti-class I MHC demon- of the cell surface-expressed TCR/CD3 complexes CD3␨ can as- strate that a different and less complex set of proteins is recruited sociate with the cytoskeleton through the interaction of CD3␨ with to the immobilized Ab (Fig. 2B and data not shown). In addition to monomeric actin (28–30). This pool of CD3␨ that can interact with detecting proteins known to associate with the TCR/CD3 complex, the cytoskeleton is detergent insoluble and has been designated we were unable to detect proteins that would not be expected to cytoskeleton-associated ␨ (cska-␨). Cska-␨ differs from detergent- associate with the TCR/CD3 complex (Fig. 2B). We therefore con- soluble CD3␨ in that it is phosphorylated in resting and activated clude that our method of immunoisolation is highly specific and T cells, and phosphorylation does not change the mobility of physiologically relevant. cska-␨ during electrophoresis (30). With the method used in this Our most surprising finding is that CD3␨ can become phosphor- study to recover the TCR-associated complexes, all proteins bound ylated in the absence of detectable Lck activity. We found that to the immobilized Ab are collected; therefore, it is conceivable both CD3␨ phosphorylation and ZAP70 recruitment can occur in that we may be capturing some insoluble cytoskeletal components. normal T cells after treatment with the SFK inhibitor, PP2, even The predominant species of phosphorylated CD3␨ that we recover The Journal of Immunology 7813

from cells stimulated in the presence of PP2 migrates at 19 kDa. phorylated CD3␨. It has been shown previously that the SH2 do- This 19-kDa phosphorylated species has also been observed in a main of Lck can interact with phosphorylated CD3␨ (36, 37) and study examining the cytoskeletal recruitment of CD3␨ (19). We that a functional SH2 domain of Lck is required for the earliest can detect a minor, less phosphorylated form of CD3␨ that seems signaling responses (37). to migrate just below the p19 species and appears to have a low Another explanation for why CD3␨ might undergo Lck-inde- level of constitutive phosphorylation that may correspond to pendent phosphorylation is based on the differential signaling cska-␨. We cannot exclude the possibility that p19 is, in fact, model proposed by Pitcher and van Oers (38). In this model, the cska-␨; nevertheless, two pieces of evidence argue against the like- ␥-, ␦-, and ⑀-chains of the TCR/CD3 complex perform distinct lihood of this species being cska-␨. First, the amount of cska-␨ is functions from the CD3 ␨-chain. CD3␥,-␦, and -⑀ are responsible increased after T cell activation (29, 30), and we do not detect an for mediating activation events (signaling, proliferation, and effec- increase in the amount of complex-associated, 19-kDa ␨ protein tor functions), whereas CD3␨ may mediate distinct signaling path- after activation. Second, as mentioned previously, cska-␨ is basally ways and TCR expression and recycling. This model is based on phosphorylated (30), whereas the 19-kDa CD3␨ phosphorylation observations that CD3␨ can interact with distinct proteins that do that we are detecting is clearly inducible, with little or no pre- not bind to the other invariant chains through ITAM and non- existing phosphorylation in unstimulated cells. Furthermore, we ITAM sequences (38). That we found that CD3␨ can be phosphor- can detect ZAP70 with captured protein complexes, which is pre- ylated by another PTK(s) supports the hypothesis that TCR␨ could sumably associated with phosphorylated CD3␨, and it has not yet be involved in a distinct signaling pathway. been determined whether ZAP70 is capable of interacting with In this study we provide evidence that CD3␨ can be phosphor-

phosphorylated cska-␨. Interestingly, Caplan et al. (31) found that ylated and recruit ZAP70 in the presence of an SFK inhibitor and Downloaded from cska-␨ can be phosphorylated by a kinase other than Lck in vivo, in the absence of detectable Lck activity. In contrast, the SFK because treatment of cells with PP1, an SFK inhibitor, had no inhibitor completely blocked CD3⑀ and ZAP70 recruitment, im- effect on cska-␨ phosphorylation. However, the authors were un- plying that there is a differential SFK requirement for CD3⑀ and able to identify which kinase(s) was responsible. CD3␨ phosphorylation. Our results suggest that Lck and possibly There are two major phosphorylated intermediates of soluble Fyn, because it is also effectively inhibited by PP2, may not be

CD3␨ that form upon T cell activation, which migrate at 21 and 23 essential for CD3␨ phosphorylation and that perhaps another SFK http://www.jimmunol.org/ kDa. It has been shown that the 21-kDa (p21) intermediate is con- or another PTK may be able to phosphorylate CD3␨, but not CD3⑀. stitutively phosphorylated in thymocytes and peripheral T cells, Alternatively, if SFK are still required for CD3␨ phosphorylation, and it associates with nonphosphorylated ZAP70 as a result of in the levels and/or types of active SFK differ substantially from situ TCR interactions with peptide/MHC complexes (32, 33). van those required for CD3⑀ phosphorylation. Taken together, our data Oers et al. (34) have elegantly demonstrated that p21 is tyrosine suggest that current models of membrane-proximal, TCR-medi- phosphorylated on the two membrane-distal ITAMs. ZAP70 can ated signaling events may be incomplete, and that additional stud- associate with p21; however, this associated ZAP70 does not be- ies are required to understand the apparent differential phosphor- come phosphorylated (34, 35). After activation through the TCR, ylation requirements for CD3⑀ and CD3␨. p21 becomes phosphorylated at the most membrane-proximal by guest on September 27, 2021 ITAM, giving rise to p23, and the associated ZAP70 becomes Acknowledgments phosphorylated (34). We did not detect basally phosphorylated We thank Dr. Kevin Kane for critical review of the manuscript and helpful CD3␨ or ZAP70 association with the recovered complexes before discussions. TCR stimulation. The major phosphorylated form of CD3␨ that we detected after TCR stimulation in both the presence and the ab- Disclosures sence of PP2 inhibition was ϳ19 kDa (Fig. 4). We hypothesize The authors have no financial conflict of interest. that we may be capturing and detecting an earlier tyrosine-phosphorylated intermediate than p21. Indeed, it has been shown that References the sites of CD3␨ phosphorylation affect the migration of CD3␨ 1. Baniyash, M., P. Garcia-Morales, E. Luong, L. E. Samelson, and R. D. Klausner. ␨ during electrophoresis (34). In the absence of PP2, we can detect 1988. The T cell antigen receptor chain is tyrosine phosphorylated upon activation. J. Biol. Chem. 263: 18225–18230. both the p21 and p23 species, and in the presence of PP2, upon 2. Iwashima, M., B. A. Irving, N. S. van Oers, A. C. Chan, and A. Weiss. 1994. longer exposures, a band migrating at ϳ21 kDa can clearly be Sequential interactions of the TCR with two distinct cytoplasmic tyrosine kinases. 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