esis were 5'-GGCGTATTCAATGCACTGC-3' for tyrosine 90, 5'-. GGCCTTTTTCAGGGTC-3' ... plasmid kits (Studio City, CA) according to the manufacturer's rec-.
Vol. 267. No . 5. Issue of February 15, pp. 3375-3361,1992 Printed in U.S.A.
THEJOURNAL OF BIOLOGICAL CHEMISTRY
0 1992 by The American Society forBiochemistry and Molecular Biology,Inc.
Phosphorylation of Multiple CD3c Tyrosine Residues Leads to Formation of pp21 in Vitroand in Vivo STRUCTURAL CHANGES UPON T CELL RECEPTOR STIMULATION* (Received for publication, September 4, 1991)
Shigeo Koyasu$j$$, David J. McConkey$jll, Linda K. Clayton$j, Sheena Abraham$, Booma YandavaS, Takuya KatagiriII ,Philippe MoingeonSj, Tadashi YamamotoII,and Ellis L. Reinherz$** From the $Laboratory of Immunobiology,Dana-Farber Cancer Institute and Departments of $Pathlogy and **Medicine,Harvard Medical School, Boston, Massachusetts 02115 and the (Ilnstitute of Medical Science, The University of Tokyo, Shirogane, Minato-ku, Tokyo 108, Japan
T lymphocyte activationresulting from antigen rec- molecular complex formed by three groups of transmembrane ognition involves a protein tyrosine kinase pathway proteins: ( a ) the clonotypic antigen (Ag).major histocompatwhich triggersphosphorylation of several cellular sub- ibility complex (MHC) recognition unit, termed Tia-P (or strates including the CD3{ subunit of the T cell recep- Ti-y-6), a heterodimer which is immunoglobulin-like (1-5); ( b ) tor (TCR) to form pp21. Thehomologous TCR-associ- the highly homologous CD3y, CD36, and CD3t subunits (6ated protein,CD3q, is an alternatively spliced product of the same gene locus as CD3{. CD3q lacks oneof six 9); and( c ) the structurally distinct CD3{ and CD37 subunits 132)found in CD3{ (10, 11).Being alternatively spliced products of a single gene cytoplasmic tyrosine residues (Tyrlocus, the amino-terminal sequences of the CD3{ and CD37 and is itself not phosphorylated. Site-directed mutagenesis in conjunction with in vitro and in vivo phos- subunits share the same leader peptide and are identical phorylation studies herein demonstratesthat Tyr- 132throughamino acid 122 of each matureprotein (12-17). is required for the formation of pp21. Moreover, the However, the CD37 subunit lacks one of six possible tyrosine differential phosphorylation of CD3{ versm CD3q is phosphorylation sites (Tyr-132) as well as a putative nucleonot due to a selective association of the known TCR- tide binding site identified in CD3{ (12). CD3{ and CD37 ~ ” form disulfide-linked homo- or heterodimers (CD3f2, CD3{associated protein tyrosine kinase, ~ 5 9 ‘ ~ ~ ” ; 6 9 but ’ ~~ 6 2 is ~ associated “ with eachof the three 7, and CD3v2)important in targeting partiallyassembled Tianot ~ 5 6 ‘ or TCR isoforms containing CD3fz, CD3qz, or CD3f-q. PCD3766 TCR complexes to thecell surface and transducing This association occursthrough components of the TCR complex distinct from CD3{ or CD3q. In addition, we stimulatory signals after antigen recognition (18-22). Both the structural differences between CD3S and CD3q and the show that pp21 formation is not only dependent on abundance of CD3{ protein relative to CD37 protein within Tyr- 132 but results concomitant from phosphorylation of other CD3{ residues including Tyr-121. Mutation of the TCR complex suggest that these subunits mediate differTyr-90, -121, or -132 does not alter primary signal ent functional effects. transduction as shown by the abilityof individual CD3{ Cross-linking of the TCRby Ag-MHC is known to initiate upon TCR a series of biochemical reactions. These include activation of Tyr 4 Phe mutants to produce interleukin-2 stimulation. Thus, the substantial structural changes protein tyrosine kinase, phosphatidylinositol (PI) turnover, in CD3{ upon TCR stimulation as reflected by altera- and Ca2+mobilization (reviewed in Ref. 11).The activation tion in its mobility in sodium dodecyl sulfate-polyacryl- of the protein tyrosine kinase pathway has been considered amide gel electrophoresis may affect subsequent events to play a major role in signal transduction through the TCR such as receptor desensitization, receptor movement, (23-25). In particular, it is likely that phospholipase C”y1 is and/or protein associations. phosphorylated on tyrosine residues leading to PI turnover (26-28). One candidateproteintyrosine kinase associated with the TCR is p5@9 a member of the src family protein The T cell receptor (TCR)’ has been described as a multi- tyrosine kinase (29). A second protein tyrosine kinase found in T cells is ~ 5 6 which ” ~ is associated with CD4 and CD8 (30, * This work wassupported by National Institutesof Health Grants 31). The involvement ofCD3{ and CD37 inthese kinase AI31269, A121226, and A119807 and by agrant-in-aid from the Ministry of Education, Science and Culture of Japan. The costs of pathways is only now being defined. Using various transfecpublication of this article were defrayed in part by the payment of tants expressing different TCR isoforms, we and others have page charges. This article must therefore be hereby marked “aduer- previously observed that the CD3f but not theCD37 subunit tisement” in accordance with 18 U.S.C. Section 1734 solelyto indicate is in uiuo phosphorylated upon TCR cross-linking (22, 32). this fact. Moreover, the phosphorylation is readily detected in CD3S-< $I To whom all correspondence should he addressed. homodimers but not CD3{-7 heterodimers or CD37-7 homo7l Supported by a fellowship from the Cancer Research Institute. The abbreviations used are: TCR, T cell receptor; Ag, antigen; dimers. This finding implies either selective association of MHC, major histocompatibility complex; PI, phosphatidylinositol; one or more protein tyrosine kinases with particular CD3{/7 Hepes, 4-(2-hydroxyethyl)-l-piperazinethanesulfonicacid; TBS, dimers and/or that Tyr-132 of CD3f is a critical target for Tris-buffered saline; FCS, fetal calf serum; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraaceticacid; mAb, monoclonal antibody; phosphorylation. The present study was designed to examine SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electropho- the association of protein tyrosine kinase with different TCR resis; IL-2, interleukin-2; PDGF, platelet-derived growth factor; SH, isoforms and to begin to investigate the function of tyrosine src homology. phoshorylation of the CD3f subunit during T cell activation.
3375
3376
CD3l Phosphorylation Sites
kDa, myosin; 110 kDa, phosphorylase b;70 kDa, bovine serum albumin; 44 kDa, ovalbumin; 28 kDa, carbonic anhydrase; 18 kDa, 8Antibodies-A hamster mouse somatic B cell hybridoma 145.2C11 lactoglobulin; 13 kDa, lysozyme. producing the monoclonal antibody (mAb) 2Cll against mouse CD3c In Vitro Phosphorylation Analysis-After immunoprecipitation (33) was generously provided by J. A. Bluestone (University of was performed as described above, the immune complex was washed Chicago). 2Cll mAbwas purified from culture supernatantand 3 times with 1 mlof0.1% digitonin, twice with 0.5 mlof 100 mM coupled to CNBr-activated Sepharose CL-4B (3-5 mg/ml). A mAb NaCl, 5 mM MnCl,, 5 mM MgCI,,20 mM Hepes, pH 7.5, and A2B4.2 specificfor the Tiachain of the 2B4.11 (34) was the generous suspended in 50 pl of the same solution supplemented with 2 p~ gift of J. Ashwell (National Institutes of Health, Bethesda, MD). ATP. The solution was mixedwith 10 pCi of [y-32P]ATP(about 6000 Rabbit antiserum 387 against mouse CD3(/'/t,(35) was a generous gift Ci/mmol) and incubated for 15 min on ice. Then the beads were of R. D. Klausner (National Institutes of Health). Rabbit antibody washed twice with 1ml of 1%digitonin, 150 mM NaCl, 20 mM EDTA, against ~ 5 9 and ~ "mAb against ~ 6 2 were ~ ' described previously (36, 1 mM sodium orthovanadate, 50 mM Hepes, pH 7.5, once with 1 ml 37). Rabbit antibody against ~ 5 6 (38) " ~ was a kind gift of A. Veillette of TBS, and finally once with 1 ml of20mM Tris-HC1, pH 7.5. (McGill University, Montreal, Quebec). Proteins were then eluted in Laemmli's nonreducing sample buffer Cell Lines and Growth Conditions-MA5.8, a CD3r-7- variant of solution (41) and resolved on one- or two-dimensional SDS-PAGE. the cytochrome c-specific I-Ek-restricted T cell hybridoma 2B4.11 I n Vivo Phosphorylation Analysis by f2P]Orthophosphte Labeling (39) and a B cell hybridoma LK35.2 were cultured in RPMI 1640 and Anti-phosphotyrosine Western Blotting-Prior to labeling, cells supplemented with 10% heat-inactivated FCS (Sigma), 5 X lo-' M 2- were washed twice in phosphate-free RPMI 1640medium supplemercaptoethanol, 2 mM L-glutamine, and 1%penicillin-streptomycin mented with 5% dialyzed FCS, 15 mM Hepes, 5 X M 2-mercap(GIBCO). MAa5.4 and MA961.9 (21) were cultured with the above toethanol, and antibiotics. Cells were resuspended in the phosphatemedium containing 500 pg/ml G418 (Geneticin, GIBCO). MA(-q301 free medium (5 X 107/ml) and incubated for 3 h at 37 "C with 1 mCi/ (22) was cultured with the above medium containing 500 pg/ml G418, ml [32P]orthophosphate (Du Pont-NewEngland Nuclear). The cells 0.5 pg/ml mycophenolic acid, 15 pg/ml hypoxanthine, and 25 pg/mi were then washed 3 times and resuspended in phosphate-free medium xanthine. prior to TCR stimulation. Generation of CD3{ Mutants by in Vitro Mutagenesis and TransTo immunoprecipitate phosphorylated, TCR-associated proteins fection-The CD3( insert was subcloned from pBS23 (21) into the and analyze them by two-dimensional SDS-PAGE, cells (1 X lo7 EcoRI site of M13mp18. Mutagenesis of tyrosines at amino acids 90, cells/ml) were stimulated in the RPMI 1640 medium for 30 min at 121, and 132 to phenylalanine was carried out using the oligonucleo- 37 "C with 20 pg/ml A2B4.2plus 20 pg/ml goat anti-mouse Ig (Cappel, tide-directed in vitro mutagenesis system (Amersham Corp.) following Durham, NC), harvested by centrifugation for 20 s at 200 X g, and the manufacturer's instructions. Oligonucleotides used for mutagen- resuspended in 1 ml of a 4 "C lysis buffer containing 1%digitonin, 50 esis were 5'-GGCGTATTCAATGCACTGC-3'for tyrosine 90, 5'mM Tris, 150 mM NaCl, 10 mM EDTA, 10 mM EGTA, 1 mM GGCCTTTTTCAGGGTC-3' for tyrosine 121, and 5'-GA- orthovanadate, 1 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 5 CACCTTTGATGCC-3' for tyrosine 132. Construction of the double pg/ml leupeptin, 0.24 trypsin inhibitory units/ml aprotinin, pH 7.5. mutant atamino acids 121 and 132 wasaccomplished by carrying out Cells were allowed to lyse for 2 h at 4 "C, insoluble materials were mutagenesis with the mutant oligonucleotide for tyrosine 132 on an removed by centrifugation for 10 min at 13,000 X g, and supernatants M13 template prepared from the product of mutagenesis at amino were rotated for 16 h a t 4 "C with 50 gg of 2Cll mAb plus 100 pl of acid 121. Mutations were identified by sequence analysis, and the 10% protein A-Sepharose beads. The antigen-antibody-bead commutant inserts were excised using EcoRI, blunted with DNA polym- plexes were then washed 5 timeswith the lysis buffer solution, boiled erase I, and subcloned into the blunted XbaI site of the expression in Laemmli's nonreducing sample buffer (41), and subjected to twovector pPink2 (40). Plasmid DNAs were prepared using Qiagen dimensional nonreducing-reducing SDS-PAGE. Proteins were then plasmid kits (Studio City, CA) according to the manufacturer's rec- transferred to nitrocellulose and detected by either autoradiography ommendations. The mutations were again sequenced in the plasmid or immunoblotting using the anti-phosphotyrosine mAb 4G10 (42) DNA and theDNAs linearized with XmnI prior to transfection. Ten (kindly provided by Drs. B. Druker and T. Roberts, Dana-Farber million MA5.8 cells were transfected with 10 pg of DNA in a Hepes- Cancer Institute, Boston, MA) as a probe; immunoreactive proteins buffered saline solution (pH 7.5) by electroporation using a Bethesda were then visualized using alkaline phosphatase-conjugated goat antiResearch Laboratories cell porator system (Life Technologies, Gaith- mouse IgG (Bio-Rad). ersburg, MD) set at 200 V and 800 microfarads capacitance according Determination of ZL-2 Production-lo5 T cells were plated in 96t o the manufacturer's instructions. Cells resistant to 1 mg/ml gene- well round bottomed plates (final volume, 0.2 ml) and stimulated at ticin (G418) were subsequently sorted for high levels of TCR expres- 37 "C for 24 h with either medium alone, 145-2C11mAb at 1 pglwell sion byflow cytometry and maintainedin RPMI 1640 medium coated onto a plastic plate (Immulon, Dynatech, Chantilly, VA), or M 2-mercaptoethanol, antibisupplemented with 10% FCS, 5 X cytochrome c peptide KKANDLIAYLKQATK (5 p~ final concentraotics, and 0.5 mg/ml G418. tion) in the presence of I-E' expressing B lymphoma LK35.2 cells. Immunoprecipitations-Cells were solubilized at 1-2 X lo7 cells/ Culture supernatants were harvested and titrated in serial 2-fold ml in Tris-buffered saline (150 mM NaCl, 20 mM Tris-HC1, pH 7.5) dilutions (ranging from 1:2 to 1:2048) for their ability to support the containing 1%digitonin, 10 mM iodoacetamide, 5 pg/ml leupeptin, 1 growth of CTLL2O cells. The highest dilution of culture supernatant mM phenylmethylsulfonyl fluoride, and 0.24 trypsin inhibitory units/ able to maintain viability of 5 X lo3 CTLL2O cells (evaluated using a ml aprotinin (all from Sigma) by rotating at 4 "C for 2 h. Postnuclear trypan blue dye exclusion assay) was considered to contain one supernatant was incubated overnight at 4 "C with 20 pl of 2Cll- arbitrary unit of IL-2. coupled Sepharose CL-4B or various antibodies in the presence of 20 pl of protein A-Sepharose CL-4B (Pharmacia LKB Biotechnology RESULTS Inc.). The bead-antibody-antigen complexes were pelleted by centrifugation, the supernatant removed, and the beads washed once with CD3c and CD3q Transfectants 15 ml of 0.1% digitonin in TBS, 3 times with 1 ml of 0.1% digitonin To characterize tyrosinephosphorylation events in the in TBS, and once with 1ml of TBS and1 ml of 20 mM Tris-HC1, pH 7.5. Antigen-antibody complexes were solubilized in 40 pl of nonre- CD3{ and CD3q subunits, a series of murine T cell transfecducing Laemmli's sample buffer a t 100 "C for 3 min (41) and resolved tants was employed as summarized in Table I. These lines by two-dimensional nonreducing-reducing SDS-PAGE using 12.5% derived bywere transfecting MA5.8 (Tiaacrylamide in both dimensions. Western Blotting Analysis-After two-dimensional SDS-PAGE, TABLEI proteins were transferred to nitrocellulose (Bio-Rad) for 1h at 100 V Cell lines used in this study in a solution consisting of 25 mM Tris, pH 7.5, 192 mM glycine, and 20% MeOH. Following a 2-h room temperature incubation in TBS Cell line cDNA transfected TCR isoforms expressed Ref. containing 5% FCS and 10 mM NaN3, blots were incubated overnight "A5.8" a t 4 "C with antibody 387 diluted 1:200 in TBS containing 5% FCS MA05.4 CD3r CD3r2 21 and 10 mM NaN3. Finally, immunoreactive proteins were visualized CD372 CD3q MAs61.9 21 using alkaline phosphatase-coupled second step reagents obtained MAr-7301 CD3r CD37cD3(2 + CD3sz + C D 3 h 22 from Bio-Rad. Prestained molecular weight markers (Bethesda ReParental cell line used for transfection. search Laboratories) were co-electrophoresed with the samples: 230 MATERIALS AND METHODS
+
39
CD3{ Phosphorylation Sites
3377
@+CDS-y&+CD3{-q-)with either CD3{ cDNA, CD37 cDNA, or both CD3{and CD3q cDNAs (21,22). The MA5.8 cell line was originally derived from a cytochrome c-specific, I-Ekrestricted T cell hybridoma, 2B4.11 (39). As shown, MA05.4 expresses CD33; homodimers, MA761.9 expresses CD372 homodimers, and MA{-7301 expresses CD3{*,CD3{-7, and CD3a dimers.
unphosphorylated CD3C/q proteins as detected by Western blotting with rabbit anti-CD3{/q antibody. In MAC-7301 cells, we observed a pattern of phosphoproteins in the off-diagonal region even more complex than in MA05.4. By comparison of phosphoproteins with unphosphorylated CD3{/7 proteins, we detected two forms of pp18 and pp23 which had different molecular weights in the first dimension, reflecting the presence of phosphorylated CD3{-7 heterodimers in this cell type. In Vitro Tyrosine Phosphorylation of CD3{ In addition,pp21 was also observed. Phosphoamino acid and CD37 Subunits analyses revealed that pp18, pp21, and pp23 are phosphorylThe TCRcomplexes of MAQ5.4, MA761.9, and MA{-q301 ated exclusively on tyrosine residues (data not shown). We cells were immunoprecipitated from digitonin lysates with conclude from these results that CD3{ and CD37 are phosanti-CD3t mAb, 2Cll. In vitro kinase reactions were then phorylated in vitro on tyrosine residues by a protein tyrosine performed with the immunocomplexes in the presence of [y- kinase associated with the TCR regardless of the CD3{2, '"PIATP, and proteins were subsequently resolved on nonre- CD3{-7, or CD372 isoforms expressed. ducing-reducing two-dimensional diagonal gels and blotted Association of p 5 9 " with Different TCR Isoforms onto nitrocellulose filters. The phosphorylated proteins were detected by autoradiography of the blot (Fig. 1) while the It hasbeen recently reported that p 5 p is associated with positions of the unphosphorylated CD3{/7 proteins were iden- the TCR andtherefore may be responsible for tyrosine phostified by probing the blots with rabbit anti-mouse CD3{/7 phorylation of CD3{ (29). To next specifically examine which heteroantisera (Fig. 1, insets). A t least two major distinct TCR isoforms associate with p 5 p , in vitro kinase studies phosphoproteins, pp21 and pp18 (phosphoproteins of molec- were performed on a n t i - p 5 p antibody immune complexes of ular weights 21,000 and 18,000, respectively), were observed MA05.4 or MA761.9 lysates and, in parallel, with anti-CD3t in MAn5.4 cells as off-diagonal spots. In addition, several mAb 2Cll immune complexes from the same cells. As shown other off-diagonal spots are also evident. In contrast, only a in Fig.2A, a n t i - p 5 p as well as 2Cll immunoprecipitates single TCR-related phosphoprotein, pp23, was observed in NR MAq61.9. Note that thespot a t 32 kDa in thereduced dimenA sion is unrelated to the TCR in MA761.9 (data not shown). MAcl5.4 MAq61.9 The pp18, pp21, and pp23 spots were distinguishable since I they migrated to different positionswhen the in vitro kinased D 70 samples from MA05.4 andMA761.9 were co-electrophoresed 44 (Fig. 1, bottom right). No off-diagonal phosphoproteins were a-p59w 28 observed in MA5.8 cells, indicating that pp18 and pp21 are le phosphorylated CD3{ proteins and pp23 is a phosphorylated 13 CD37 protein (data notshown). All of the phosphorylated CD3{/7 proteins migrated more kc 7c slowly in both nonreduced and reduced dimensions than the
-
F
I
qr
N R 4
q
MAt15.4
44 kD-
MAq61.9
7
I'
1 kD
a-cD3~
2( 11 1:
' a kD 70 44 28 -
b
NR-
i
44 -
1813-
28 1813-
C
d
kD -
MAL-q301 MAt15.4 MA:61.9
FIG.1. In vitro tyrosine phosphorylation of CD3{ and CD31, subunits. 1 X 10' MAj15.4, 1 X loRMA761.9, and 0.5 X loRMAC11301 cells were immunoprecipitated with 2Cl1, and precipitateswere subjected to in vitro kinase reactions.5 X lo7 cell equivalents of MAj15.4 and MA761.9 were mixed (lower rightpanel). Proteins were then resolved innonreducing(NR)-reducing (R) two-dimensional diagonal gels, blottedonto nitrocellulosefilters, and probed with rabbit anti-CD3{/7 antibody 387 (inset). Radioactive proteins were detected by autoradiography. Squares on the diagonal show the rnigration positionsof molecular weight markers which had been mixed with samples. Squares in the off-diagonal region indicate themigration positions of CD3C and CD39 as revealed by Western blotting.
-
70 44.
28. 1813'
%
FIG.2. Association of ~ 5 9 ' ~ with " TCR. A, TCR of MA115.4 and MAq61.9 cells were immunoprecipitated with either 2Cll or antip 5 W and subjected to in vitro kinase assays. B, TCR of MAjl5.4 cellswere immunoprecipitated with anti-p6FS ( a ) , anti-p56"' ( b ) , a n t i - p 5 P (c), and 2 C l l ( d ) .After in vit;o kinase reactions, proteins were resolved in two-dimensionaldiagonal gels and visualized by autoradiography. R, reducing; NR, nonreducing.
CD3{ Phosphorylation Sites
3378
contain phosphorylated CD3{ and CD37 proteins from MA315.4 and MA761.9 cells, respectively. Because it is known that T cells express several other src family protein tyrosine kinases in addition to p 5 P including ~ 5 6 and " ~ ~ 6 2 ~ ' "we , examined the specificity of t h e ~ 5 9 ' ~association. " 2Cll and anti-p5YY"but not a n t i - ~ 5 6 "or~ anti-p62."" immunoprecipitates generated phosphorylated CD3{ (Fig. 2B) and CD37 (data notshown) after inuitro kinase reactions. These results are consistent with earlier studies defining a direct and specific association between p59"^ and the TCR (29). Furthermore, the present results extend those observations by indicating thatp59"" is associated withTCR isoforms containing CD3r2 aswell as CD3q2dimers. It is worth commenting on the fact that the pattern of CD3{ phosphoproteins detected inthe kinase reaction of the anti-p59"" immune complex is distinct from that found in the 2 C l l immune complex. In the anti-p59"" complex, all CD3{ is in the form of pp21 whereas in the 2Cll complex, both pp21 and pp18 were observed. The basis of this disparity is presently unknown. It is possible that CD3{ protein is more completely phosphorylated in anti-p59@" complexes relative to 2Cllcomplexes. This would be consistent with the known low stoichiometry of ~ 5 9 ' in ~ "the TCRcomplex (29).
possibility that there may also exist a between ~ 5 9 ' and ~ " CD3{ and CD371.
physical association
Analysis of Tyrosine Phosphorylation Sites of CD3{ by Site-directed Mutagenesis
I n Vitro Phosphorylation Analysis-Given that the above study excluded differential association of ~ 5 9 ' ~with " CD3{ versus CD3q, we turned our attention tospecific phosphorylation sites in the cytoplasmic domain ofCD3{ and CD3o which might account for the more complex in uitro phosphorylation pattern of CD3{ and thelack of detectable phosphorylation of CD371 in uiuo. In particular, we focused on Tyr132 of CD3{ since this residue is the tyrosine not found in CD37. In addition, we chose to evaluate two other tyrosine residues: Tyr-90 which is part of a consensus sequence for tyrosine phosphorylation (43)and Tyr-121located within the presumed nucleotide binding site of CD3{ (12). To this end, we performed site-directed mutagenesis on the CD3{ cDNA to replace each of these tyrosine residue with a phenylalanine residue. Wild-type or mutagenized CD3{ cDNA was then subcloned into theeukaryotic expression vector pPink-2 and transfected into MA5.8 cells. As schematically shown in Fig. 4, we generated variants with single mutations a t tyrosine residues 90 (MAPOYF), 121 (MAjlZlYF), or 132 ~ 5 9 ~Is' Also ' Associated with Partially Assembled (MAjl32YF) and one dou%le mutant which lacks both TyrTia-/3CDSy& TCR Complexes 121 andTyr-132 (MAD21/132YF). The TCR complex of these mutants as well as wild-type The observation that CD3{ and CD371 could be phosphorylMA315.4 cells were immunoprecipitated and subjected to in atedin uitro inimmune complexes from MA315.4 and vitro kinase reactions as described above. As shown in Fig. MA761.9, respectively, raised the possibility that p59"" might 5A, the Tyr-90 +Phe mutationdid not affect the phosphorylassociate with the TCR independently of these subunits. To ation of CD3{ (Fig. 5A, panel b and Fig. 5B, panel a); both test thispossibility, we employed the MA5.8 line which lacks pp18 and pp21 were observed. This result indicates that TyrCD3{ and CD3q but expressespartially assembled Ticu- 90 is not phosphorylated in uitro. In contrast, pp18 but not PCD3y6c complexes on its surface (18).Digitonin lysates of pp21 was observed in the Tyr-132 + Phe mutant (Fig. 5A, MA5.8, MA{-7301, or, as a control, LK35.2 (B cell line) were panel a and Fig. 5B, panel c). Likewise, the Tyr-121 + Phe immunoprecipitated with 2Cl1, and in uitro kinase reactions mutation selectively resulted in loss of pp21 (Fig. 5A, panel c were performed with the immunocomplexes. Phosphorylated and Fig. 5B, panel b ) . Co-electrophoresis of in uitro kinased proteins in 2Cll immunoprecipitates were eluted by SDS and TCR materialfrom MAfl21YF and MAjl32YF showed that reimmunoprecipitated with anti-p59"" antibody (Fig. 3). As the pp18 of both mutants was identical (Fig. 5A, panel d ) . In shown, in comparison to the complex precipitated directly addition, the double mutant MAU21/132YF yielded an in with p 5 P (lane I), p 5 P protein was also detected after uitro kinased pp18spot which was indistinguishable from that reimmunoprecipitationwith anti-p5gY"from 2 C l l precipi- of either of the single mutants (Fig. 5B, panel d ) . These in tates of MA5.8 (lane 3) and MA{-7301 (lane 4 ) but not from uitro findings indicatethat pp21 results from the phosphorylLK35.2 cells (lane 2). The absence of detectable p59"" in ation of multiple tyrosines in the cytoplasmic tail ofCD3{. LK35.2 is not due to a lack of ~ 5 9 ' in ~ "these cells since p59"" The involved residues include Tyr-121, Tyr-132, and yet to was readily immunoprecipitated withspecific anti-p59"" from be defined tyrosines exclusive of Tyr-90. I n Vivo Phosphorylation Analysis-To examine the effect LK35.2 (data not shown). These resultsindicate that the TCR of MA5.8 is able to associate with p59"" in the absence of these mutations on in vivo tyrosine phosphorylation, the of CD3{ and CD37 and suggest that other CD3 subunits or mutants aswell as wild-type MA315.4 cells were labeled with yet to be defined co-associated structures are involved in the ["P]orthophosphate and stimulated with anti-Ti mAb, ~59'~ linkage. " These findings, however, do not exclude the A2B4.2. The TCR complexes were immunoprecipitated with kD -
1 2 3 4
MAcl5.4
a.
a a.
a
a.
a.
a
(Wild Type)
230-
MAfSOYF 110-
70-
MAf121YF
11 I
a.
a .
MAcl32YF
II 1
.a
a a.
MAc1211132YF
I I I a.
a a
a
p59W"44-
FIG.3. p59"" association with the T C R is independent of CD3{ and CD3q. The TCR complexes were immunoprecipitated with 2Cll mAbfrom LK35.2 (2).MA5.8 (3), and MAC-a301 ( 4 ) . ~ 5 9 ~was " " directlyimmunoprecipitated from MAC-15.4 cells (1).After in uitro kinase assays, samples for 2-4 were mixed with SDS and boiled. The eluted proteins were diluted with 1% Triton buffer solution and subjected to reimmunoprecipitation with anti-p59"".
MAq61.9
a.
a a.
(CD31)
FIG. 4. Schematic model of CD3C cytoplasmic domain Tyr + Phe mutations. Closed circles represent cytoplasmictyrosine
residues. Transmembrane regions are highlighted.
CD3{ Phosphorylation Sites A a
b-
3379 b
NR-
NR1
i
e 44-
I
28 1813 -
4
C
..
,
.-
b
d
~
44-
28 18 13 -
B
33 24
NR-
28 -
i
1813-
a
kD 44-
16
e
~-
f
47 33
44-
24
28 1813-
b
44-
28 1813-
c
16
kD -
C
a
b
C
d
i
47-
33 24-
44-
28
-
1813-
NR-
16-
d
FIG. 5. E f f e c to fT y r Phe mutations on tyrosine phosphorylation. TCRof the mutants as well as wild-type MA05.4 cells were immunoprecipitated with 2 C l l followed by an i n vitro kinase reaction. A : ( a ) left, MAcYF132, right, MAc15.4; ( b ) MAcYFSO; ( c ) MAcYF121, and ( d ) MAcYF121 plus MAcYF132. B: leftside, MAu5.4; right side ( a ) MACYFSO, ( b ) MAu21YF, ( c ) MAu32YF, and ( d ) MAn21/132YF. R, reducing; NR, nonreducing.
47-
33
7
-
24 -
16-
anti-CD3t mAb, 2Cl1, resolved on two-dimensional diagonal gels, and phosphorylated CD3f protein was detected by autoradiography. In other experiments, gels were blotted onto nitrocellulose filters and probed with anti-p-Tyr mAb 4G10. As reported previously, pp21 was observed in the immunoprecipitates from the wild-type MA05.4 cells (Fig. 6A, a, c, e; Fig. 6B, a, c). On the other hand, pp18 but not pp21 was observed in MAfl21YF (Fig. 6B, b ) , MAfl32YF (Fig. 6A, d; Fig. 6B, d ) , and MAg21/132YF (Fig. 6A, f ) as observed in the in vitro kinase reactions shown in Fig. 5. MAPOYF also showed pp18 but not pp21 upon TCR stimulation (Fig. 6A, b ) ,in contrast to thein vitro kinase reaction. It is thus likely from these results that the CD3f subunit is phosphorylated on multiple tyrosine residues to form pp21 upon stimulation. The observed disparity between the i n vitro and in vivo phosphorylation results withthe MAPOYF cell line indicates that the mutation at Tyr-90 affected the i n vivo phosphorylation of CD3f protein in the absence of in vitro phosphorylation of this residue.
FIG.6. Analysis of in vivo phosphorylated CD3t following TCR triggering in cells expressing wild-type or tyrosine mutant CD31 molecules. A , detection of phosphorylation using [‘”PI orthophosphate. Cells labeled with [R2P]orthophosphatewere stimulated with the anti-Ti mAb A2B4.2 for 30 min, and anti-TCR immunoprecipitates were prepared and analyzed by two-dimensional nonreducing (NR)-reducing( R )SDS-PAGE asdescribed under “Materials and Methods.” Phosphorylated proteinswere then detectedby autoradiography. Panels a, c, e : MAn5.4 cells; panel b, MAPOYF; panel d, MAD32YF; panel f, MAQ21/132YF. Exposure time, 6 days. B, detection of tyrosine phosphorylation by Western blotting. AntiTCR immunoprecipitates were prepared from cells stimulated with the anti-TimAb A2B4.2 for 30min andsubjected to two-dimensional nonreducing-reducing SDS-PAGE. Proteins were transferred to nitrocellulose membranes and probed with the anti-phosphotyrosine mAb 4G10 as described under “Materials and Methods.” Panels a and c, MAu5.4 cells; panel b, MAn21YF; panel d, MAjl32YF. Positions of tyrosine-phosphorylated CD3e proteins areindicated by closed (wild-type)or open (mutant) arrows, and squares indicate positions of prestained molecular weight markers that were mixed with samples priorto electrophoresis.
Signal Transduction Capacityof CD3f Tyr Mutants Revealed by IL-2 Production
the IL-2-dependent CTLLcell line (Table 11). As a control, mutants were stimulated inparallel with2Cll. TableI1 shows that MAPOYF, MAJlZlYF, MA032YF, andMAU21/132YF each produced significant amounts of IL-2 upon Ag.MHC stimulation. In contrast and as expected, no IL-2 was induced from Ag.MHC stimulation of MA5.8. These results indicate
To examine whether tyrosine phosphorylation of CD3f is involved in signal transduction after Ag. MHC stimulation, the individual mutants were stimulated with Ag-MHC, and IL-2 production from these cells was tested by bioassay using
CD3fPhosphorylation Sites
3380
TABLEI1 CD3{ tyrosine mutations do not affect ZL-2 production Cells were incubated for 24 h with various stimuli. Supernatants wereharvestedand tested induplicate in serial dilution for their capacity to support the growth of the IL-2-dependent murine Tcell line CTLL2O. Results areexpressed in arbitrary units/ml of IL-2 secreted (with arbitrary 1 unit equivalentto -1.2 units of recombinant IL-2 from Biogen, Cambridge,MA)andare representative of four independent experiments. IL-2production Cells
Media
2Cll (1 pglwell)
MA5.8
