Leo, O., Foo, M., Sachs, D. H., Samelson, L. E., and Bluestone, J. A. (1987). Cheng,,S. H., Harvey, .R., Espino, P. C., Semba, K., Yamamoto, T., To-. Kypta, R. M.
Vol. 268, No. 22, Issue of August 5. pp. 16537-16543,1993 Printed in U.S. A.
THEJOURNALOF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Bimhemistry and Molecular Biology, Inc.
A 72-Kilodalton fyn-related Polypeptide (~72~""-") Binds to the Antigen-Receptor/CD3 (TcR/CD3) Complex* (Received for publication, December 21, 1992, and in revised form, March 17, 1993)
Antonio J. da Silvas and ChristopherE. Rudds From the Division of Tumor Immunology, Dana-Farber Cancer Institute and the Departmentof Pathology, Haruard Medical School, Boston, Massachusetts 02115
p56ICk, p59fyn, and p6W8could initiate this cascade. A role for Protein-tyrosine kinases playcrucial roles inthe activation andtransformationof T lymphocytes. In these src family members in signalling was first shown by the this study, we have identified a variant of the f y n demonstration that the p56lCkcan associate with CD4/CD8 kinase at 70-72 kDa (termed ~ 7 2 ' ~ ' " that ~ ) can pref- receptors on T lymphocytes (4-6) (for review,see Ref. 7). erentially associate with the TcR/CD3 complex incer- ~ 5 6 ' binds '~ via an N-terminal region to a specific sequence tain T cells. Phosphoamine acidanalysis revealed that within the CD4/CD8a cytoplasmic tail (8, 9). Antibody-inthe CD3-associatedp72fyn-R is labeled on bothtyrosine duced dimerization has also been reported to activatethe andserinelthreonineresidues.TcR/CD3-associated kinase (10). The formation of the CD4/p56lCk complex appears p7ZrYmaR could specifically be reprecipitatedusing anti- essential inthe response to antigen in certain T-cell lines (11, " . 12). This requirement may be mediated by the kinase and/or fyn antisera to both theN and C terminus of ~ 5 9 ' ~In addition, two-dimensional phosphotryptic peptide map the associated p32 (13) or p l l 0 proteins (14). CD4 and TcR(/ patterns of TcR/CD3-associated ~ 7 2 " "and anti-fyn- CD3 also physically associate, introducing the possibility that precipitable p72 were identical. By contrast, a comparison of p72wn-R and p62'Y" showed similarities and p56Ickmay directly respond to CD3 stimulation (15). Despite the importance of p56Ick,the protein-tyrosine kidifferences. p7ZfyneR possesses a peptide corresponding nase ~ 5 9 ' ~also " appears to play a key role in T-cell signalling. to the autophosphorylation site that migrates in the An altered form of ~ 5 9 is~ expressed " in T cells (~59""'~'), same positionas found for p59/62"". However, p7ZfYnsR possessed at least four novel phosphorylated sites la- generated by the alternate splicing of exon 7 (16). ~ 5 9 ~ " ' ~ ' beled onserine and threonine residues that are absenthas been reported to associate with TcR/CD3 (17).TcR/CD3 in the ~ 6 2 pattern. ~ " Phosphatase digestion experi- cross-linking stimulates fyn activity resulting in the phosments indicated that p72fyr"-R is more resistant to de- phorylation of a fyn-associated p120/130 protein (18, 19). In phosphorylation than ~59162"". Two-dimensional addition, studies with transgenic mice have shown a correlaphosphotryptic analysis indicated that the novel tion between fyn expression and response to CD3 stimulation serine/threonine phosphorylationsites were responsi- in thymocytes (20-22). TcR/CD3 and CD4 engagement have blefortheresistancetophosphatasedigestion. A1- also been reported to induce the phosphorylation of distinct though the exact nature of the relationship between substrates (23, 24). p72fyn-R and ~ S 9 / 6 2 ' ~remains " undetermined, these In this study, we report the identification of a novel TcR/ data indicate that TcR/CD3 may utilize novel variants CD3- associated polypeptide at 72 kDa (termed for of src-related kinases inthegenerationof signals fyn-related) which exhibits some structural similarity to p59/ which regulateT-cell growth. 62&"in T cells. p72'y"'R was identified in both anti-CD3 and anti-fyn immunoprecipitates labeled in an in uitro kinase assay. The inter-relationship between the two molecules was Engagement of the oligomeric antigen-receptor (TcR/ revealed by reprecipitation of p72'y"-R by anti-fyn antisera to CD3)' complex of T lymphocytes with antigen or anti-CD3 the N and C terminusof ~59""and phosphopeptide mapping. possesses at least four antibodies triggers the rapid (5-10 s) tyrosine phosphorylation Peptide mapping showed that p72fy"-R novel phosphorylation sites, not found in ~ 6 2 ~ " . is a of a myriad of substrates.Theseevents precede andare fyn-related kinase with the potential to modulate transducing required for the activation of phospholipase C y 1 (1-3), which hydrolyzes phosphatidylinositol 4,5-b1phosphate into thesec- signals from the TcR/CD3 complex. ond messengers diacylglycerol and inositol triphosphate. MATERIALS AND METHODS Three candidate protein-tyrosine kinases expressed in T cells
* This work was supported in partby National Institutes of Health Grant R01 12069 (to C. E. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Supported by a postdoctoral fellowship from the National Cancer Center. § Recipient of the Cancer Research Institute/Benjamin Jacobson Family Investigator Award, New York and the Claudia Adams Barr Award, Boston. The abbreviations used are: TcR/CD3, T-cell antigen receptor complex; PAGE, polyacrylamide gel electrophoresis; NEPHGE, nonequilibrium pH gradient electrophoresis; CIP, calf intestinal phosphatase.
Celk and Antibodies-The CD8+ DC27.10 (F6) murine T-cell hybridoma was grown in RPMI supplemented with 10% fetalcalf serum, 1%penicillin and streptomycin, and 1%L-glutamine, in some cases supplemented with geneticin 418 (1 mg/ml) (25). Antibodies used include the hamster anti-murine CD3c chain monoclonal antibody 145-2C1l (gift of J. Bluestone, Univ. of Chicago) (26), the CFN-2 (27) antisera that were raised against a synthetic peptide containing residues 16-30 from the unique N-terminal region of p59W" (gift of S. Cheng, Genzyme Corp., Framingham, MA), the anti-fyn 1 and antifyn 2 antisera that were raised against synthetic peptides containing residues 22-35 and 35-51, respectively, of the N-terminal region of p59"", and the CST-1 antisera that were raised against a synthetic peptide containing residues 527-533 of p60"" and were cross-reactive against fyn and yes ((28), generous gift of Dr. S. Courtneidge, EMBL, Heidelberg, FRG).
16537
TcRICD3-associated p 7 F n R
16538
Immunoprecipitation and Kinuse Assays-The reactions were carried out aspreviously described (18). Briefly, 50 X lo6cells per sample were washed I Xwith ice-cold phosphate-buffered saline and lysed in 1 ml of digitonin lysis buffer (1%w/v) in 20 mM Tris, pH 8.2, containing 150 mM NaCl and 1 mM phenylmethylsulfonyl fluoride. Following incubation of the cell lysates with antibodies for 1 h a t 4 "C, immunoprecipitates were incubated for 10 min in kinase buffer (30 mlof25 mM Hepes, pH 7.2, containing 10 mM Mg(%, 10 mM MnC12, and 1-10 mCi of [y3'P]ATP (ICN Chemicals Ltd.)) and subjected to SDS-PAGE (29). Two-dimensional nonequilibrium gel analysis (NEPHGE/SDS-PAGE) was conducted using ampholines of a pH range between 2 and 11as described (4,5,30). For reprecipitation analysis, bands containing the specific phospholabeled proteins were excised from unfixed gels, the proteins were eluted for 8-12 h in 50 mM ammonium acetate, 0.1% SDS, concentrated with Centricon filters (Amicon), and diluted to 1 ml of lysis buffer prior to immunoprecipitation with the antibodies. The immunoprecipitates were then analyzed by SDS-PAGE and autoradiography. Phosphatase Digestions and Phosphoamino Acid Analysis-Phosphatase digestions were conducted by incubating labeled anti-fyn precipitates a t 37 "C in 50 p1 of alkaline phosphatase buffer (30 mM triethanolamine, pH 9.3; 100 mM ZnC12; 1 mM MgC12; 3 mM NaCl) containing 1unit of calf intestinal alkaline phosphatase (CIP, Boehringer Mannheim). Phosphoamine acid analysis was conducted by eluting phospholabeled bands from polyacrylamide gels and precipitating with trichloroacetic acid as described previously (31, 32). The precipitate was washed in acetone and hydrolyzed in 6 N hydrochloric acid at 110 "C for 2 h. The individual phosphoamine acids were separated by electrophoresis (1000 V, 30 min) at pH 3.5 in pyridine:acetic acidwater (1:10189, v/v). Nonradioactive standards were detected by ninhydrin, while radiolabeled phosphoamine acids were observed by autoradiography or scanned using a PhosphorImager (Molecular Dynamics). Cleveland Phosphopeptide Map-Partial Cleveland phosphopeptide map analysis was performed essentially as described previously (33). Phospholabeled proteins were purified by two-dimensional gel electrophoresis and eluted from gel slices with 300 pl of a solution containing 20% sucrose, 10 mM sodium bicarbonate, 0.1% SDS, 0.1% 2-mercaptoethanol, by incubation at 37 "C. Equal 50-p1 fractions of the eluate were then digested with addition of varying amounts of the protease, for 1 h a t 37 "C, and the resulting products were separated by 12% SDS-PAGE. Two-dimensional Phosphotryptic Map Analysis-To perform phosphotryptic peptide maps, labeled material was eluted from gel slices and then digested with 1 mg of trypsin, as previously described (34). The peptides were separated onthin layer cellulose plates by electrophoresis in 50 mM ammonium bicarbonate, pH 8.9, in the first
dimension and ascending chromatography in the second dimension (H20:butanol:acetic acidpyridine). Phosphopeptides were visualized by autoradiography. RESULTS
Samelson et al. (17) have previously reported the association of p5gfY",a member of the src family of protein-tyrosine kinases, with the TcR/CD3 complex. In an attempt identify to other TcR/CD3-associated proteins, anti-CD3 precipitates were prepared in a digitonin-based cell lysate derived from the mouse T-cell hybridoma DC27.10 (F6) and subjected to kinase labeling with [y-32P]ATP.As seen in Fig. L 4 , antiCD3 precipitations revealed the presence of phospholabeled CD3 subunits from 16-26 kDa, two proteins at 55 and 62 kDa ( ~ 6 2 ~a )heavily , labeled band at 72 kDa, and, occasionally, a faint proteindoublet at 120/130 kDa (Fig. LA, lane 2 ) . Each of the 55-, 62- ( ~ 6 2 ' ~72-, ) , and 120/130-kDa bands were also observed in the anti-fyn immunoprecipitates (lanes 3), albeit with a different relative intensity of labeling. In particular, anti-CD3 immunocomplexes consistently precipitated more of the CD3 subunits and a 72-kDa band than observed in anti-fyn precipitates. CD3 chains could be faintly detected in the anti-fyn phospholabeled complexes upon overexposure (data notshown (18)).The p120/130 proteins have previously been shown to associate with the TcR/CD3 complex indirectly via an interaction with ~ 5 9 (18). ' ~ In order to establish the identity of the prominent TcR/ CD3-associated 72-kDa polypeptide, phospholabeled antiCD3 complexes were denatured by boiling in the presence of 2% SDS, diluted to less than 0.1% SDS with lysis buffer, and subjected to reprecipitation analysis with several anti-fyn antisera. AsFig. 1B shows, both CST-1 (which was raised against the C-terminal end of src and is cross-reactive with fyn and yes) (lane 9)and theN-terminal, fyn-specific antisera anti-fyn 1,anti-fyn 2, and CFN 2 (lanes 10-12, respectively), recognized three species at 59, 62, and 72 kDa. Three control antibodies which include rabbit anti-mouse (lane 6 ) , antiCD4 (lane 7),and anti-transferrin receptor (lane 8) failed to recognize these polypeptides. As a control for the efficiency of the denaturation procedure, an anti-CD3e antibody was found to precipitate a single protein at 26 kDa (lane 5). To further characterize the nature of the protein, p72 was found
C.
B.
A.
d
97-
c
p1201130
8 0
P-ser P-thr cp-tyr
+
32 c CD3e 8
21
13 14 4
5 6 7 8
9101112
b pH3.5
1 2 3
FIG. 1. Detection of fyn- and CD3-associated phosphoproteins. A, cell lysates of murine T-cell hybridoma D27.10 (F6) were prepared using 1%digitonin lysis buffer, immunoprecipitated with the indicated antibodies, labeled in a phosphotransferase assay, and separated by 10% SDS/PAGE. Lane I , rabbit anti-mouse control; lane 2, 145-2C11 (anti-CD3); lane 3, anti-fyn 1. B , reprecipitation analysis of labeled anti-CD3 complexes with anti-fyn antisera. Anti-CD3 complexes were phospholabeled as in A, and theindividual proteins were denatured by boiling with 2% SDS andsubjected to reprecipitation with the indicated antibodies. Lane I represents the initial pattern of bands from the anti-CD3 complex. C, solubilized anti-CD3-labeled complexes were immunoprecipitated with anti-phosphotyrosine monoclonal antibody (4GI0, lane 1 4 ) , and purified p72- was subjected to a phosphoamino acid analysis (right panel).
TcR/CD3-associatedp 7 P R C.
B.
A.
16539
1
Mr ( 1 0
)
9766-
45-
32-
32-
32-
21-
21-
21-
Control
Anti-lyn
Anli-CD3
FIG.2. Two-dimensional electrophoretic analysis of CD3- and fyn-associated proteins. Profiles of 3ZP-labeledrabbit anti-mouse (panel A ) , anti-fyn (panel B ) , and anti-CD3 (panel C ) immunoprecipitates revealed by two-dimensional NEPHGE/SDS-PAGE analysis.
ually eluted from two-dimensional gel slices and subjected to reprecipitation with anti-fyn 2 (lanes 3 and 7) and CST-1 (lanes 4 and 8).As shown in Fig. 3, both anti-fyn 2 and CST1 specifically recognized the purified ~ 7 2 ' ~protein. " Interestingly, during the reprecipitation procedure, a significant portion of ~ 7 2 ' ~converted " back to the p62 isoform of fyn. This p72fyn-5 62-kDa material was absent from an aliquot of the purified (lane 5 ) . This introduced the possibility that p72 might be derived from p62via some post-translational ~ 6 2 ' ~ ~ changes sensitive to extraction/reprecipitation conditions. To explore further the relationship between p72 and the 45 fyn kinase, a comparison of phospholabeled peptide patterns from ~62"" andp72 of anti-fyn precipitates as well as p72 from anti-CD3 and anti-fyn immunoprecipitates were conducted (Fig. 4). Individual spots were eluted from two-dimensional NEPHGE/SDS-PAGE gels and subjected to digestion 1 2 3 4 5 6 7 8 with increasing amounts of two distinct proteases. Fig. 4 FIG.3. Purified p72" is specifically recognized by anti-fyn shows a comparison of patterns ofp72'y".R from anti-CD3 antisera. p62* ( l o n e 1 ) and ~ 7 2 ~( l"o n' e ~5 ) peptides were eluted relative to ~62'"" from anti-fyn digested with V8 protease (left from a SDS/PAGE gel and subjected to reprecipitation with control panel) or elastase (middlepanel). The p72h.R and p62 patantibody (rabbit anti-mouse, lones 2 and 6),anti-fyn 2 (lanes 3 and terns showed both similarities and differences. Bands at 29, 7), and CST-1(hnes 4 and 8). 22,20, and 14 kDa derived with V8 protease, and bands at 31, 26,20, 15, and 12 kDa with elastase were shared between the to be reprecipitable with an anti-phosphotyrosine antibody two samples. However, significant differences in the relative (Fig. IC, lane 1 4 ) . Phosphoamine acid analysis further re- intensities of these shared peptides were noted. In addition, vealed that p72was labeled on bothtyrosine and serine several major different labeled peptides were also observed. residues (Fig. IC, right panel). For example, a prominent band at 35 kDa was found in the The reprecipitation analysis suggested that the TcR/CD3 p72 pattern using 0.1 pg of enzyme that was not observed in complex was associated with three fyn-related molecules a t the p62 pattern. In contrast, the patterns of p72 derived from 59,62, and 72 kDa. To furtherverify the similarity inpatterns anti-CD3 or anti-fyn precipitates were virtually identical of labeled proteins in the CD3 and the fyn complexes, we (rightpanel), indicating a similarity between these polypepsubjected the immunoprecipitates to two-dimensional gel electrophoresis (NEPHGE-SDS/PAGE) analysis (Fig. 2). Under tides. To further establish the relationship between the 62- and these conditions, the CD3-associated ~ 6 2 ' ~spot " co-migrated with that precipitated by anti-fyn antiserum (panel C uersus 72-kDa versions of the fyn kinase, anti-fyn precipitates were B ) . In addition, the prominent 72-kDa spot associated with labeled in an in uitro kinaseassay and subjected to twothe TcR/CD3 complex co-migrated with a p72 protein co- dimensional phosphotryptic map analysis. As shown in Fig. 5 " a single spot precipitated by anti-fyn antiserum. p72 focussed in a slightly (panel A ) , trypsin digestion of ~ 6 2 ' ~yielded corresponding to the fyn autophosphoryiation site Tyr-420 more acidic position relative to ~ 6 2 An ~ even . more acidic spot at 55 kDa shared in both anti-CD3 and anti-fynprecip- (33). A parallel digestion of p72 showed the same autophositates is a unique molecule that is not recognized by the anti- phorylation spot (panel B ) . Significantly, despite the similarity, additional phospholabeled spots were found in the ~ 7 2 ' ~ " fyn antisera.* ~ . the number T o further demonstrate the relatedness of the 72-kDa pro- pattern that were absent in the ~ 6 2However, tein to p62@", proteins were phospholabeled from anti-fyn of spots and their relative intensity varied to some degree ' ~ and anti-CD3 complexes by an in uitro kinase assay, individ- between experiments. These data indicate that ~ 7 2 undergoes phosphorylation a t sites not phosphorylated in ~ 6 2 ' ~ " . * A. J. da Silva and C. E. Rudd, manuscript in preparation. The above observation argued that ~ 7 2 might ' ~ be derived
66-• m ,
TcR/CD3-associated p72h.nR
16540
-
& - 0 0
Mr(10 'I
o o o
~n
0 0 - 0
-
0 - 0 0
0 0
0 0 0 - v )
-
~n
6 6 .. 45
-
32
-
0
a
L
0 - , 0 0 0 0 - . 0
0 0 - 0 " 0 0 0
0 0 0 " v ) - 0 0 0
66 45 -
66-
32
32 -
45-
21
21
-
-
L
21
-
14
14-
14 -
(anl1.CD3I p72
p62
fvn
p72 (anll-CD3)
p62
fyn
p72 (anti-CD3)
p72 (ant&-lvnl
FIG. 4. Comparative phosphopeptide map analysis of p72* with ~ 6 2~ ~ 6.generated 2 ~ from anti-fjm and ~ 7 peptides 2 ~purified from either anti-fyn or anti-CD3 immunoprecipitates were eluted from SDS-PAGE gels and subjected to a partial Cleveland analysis with increasing amounts of the elastase and V8 proteases as indicated.
A.
n
f
P62
B.
fyn
P72
FIG. 5. Two-dimensional phosphotrmtic map analysis of p62"" and p72fy"-R. Phospholabeled p62"" and p72'v" proteins gencrated from anti-fyn immunoprecipitates were eluted from SDS/ PAGE gels, digested with trypsin, separated by electrophoresis and chromatography in two dimensions, and analyzed by autoradiography. Panel A, ~62"";p a n e l B, ~ 7 2 ' ~ " .
from p62@"due to phosphorylation. Anti-fyn precipitates were therefore digested with alkaline phosphatase in order to assess whether might convert t o~ 6 2 ' ~ "A . titration of phosphatase concentrations were initially conducted (data not shown). Representative resultsare presented in Fig. 6A. Anti( l a n e 1). Digesfyn precipitated ~ 6 2 ' ~ " , ~ 7 2and ' ~ "p120/130 .~, tion of the labeled immune complexes with calf intestine phosphatase (CIP) for 10 and20 min resulted in a 50 to 60% loss in the intensity of ~ 6 2 ' ~with . only a slight effect on ~ 7 2 @ "(lanes . ~ 3 and 4 ) , relative to the addition of buffer
without enzyme (lane 2). Occasionally, complete CIP digestion of ~ 6 2 ' ~and " p120/130 could be accomplished with only partial loss of detectable p72'y"-R-labeledmaterial (lanes 5-8). A plot of values of relative intensity further emphasized the resistance of p72'yn-Rto digestion relative to ~ 6 2 ' ~(left " panel). To determine whether these observations could be extended to denatured proteins, we purified the ~ 6 2 ' ~and " proteins from SDS-PAGE and subjected them to digestion with CIP. As shown in Fig. 6B, under conditionswhere ~ 6 2 and p120/130were completely dephosphorylated (after 10 min), although the denatured form of ~ 7 2 ' ~ was ' " ~ more sensitive to theactivity of CIP, itstill exhibitedpartial resistance when compared to ~ 6 2 ' These ~. data suggest that ~ 7 2 ' ~ 'is' ~ comparatively resistant to theaction of phosphatases. Previously we showed that is phoshorylated at sites that are not normally found to be labeled in vitroin ~ 6 2 ' ~ " . To address the question of whether these additional phosphorylated sites were more resilient to alkaline phosphatase digestion, we then subjected ~ 7 2 ~ samples ' " ~ equivalent to those shown in Fig. 6A to phosphotryptic mapanalysis. Tryptic cleavage of ~ 6 2 ' ~(Fig. " 7A, left panel) resulted in the labeling at the single autophosphorylation site (Tyr-420). Digestion of ~ 7 2 ' ~ 'resulted "~ in the loss of detection of the autophosphorylation site. In contrast, four phosphorylated peptides showed a relative degree of resistance to the action of the phosphatase (middle panel). Two of the novel phosphorylation sites (spots 1 and 2) exhibited the greatest resistance to digestion relative to the other phosphorylated sites (spots 3 and 4 ) . Given that ~ 7 2 was ' ~found ~ to be labeled predominantly on serine (Fig. IC, right panel), we then purified representative spots from a TLC plate equivalent to the one shown in the right panel of Fig. 7 and subjected them to phosphoamine acid Due to the very weak degree Of labeling at this stage, the PhosPholabeled residues Were detected by scanning the plateswith a PhosphorImager scanner (Fig. 7B). ~ 6 2 ' ~and " samples purified from a phospholabeled anti-fynprecipitate were also included in this analyis for control. Hence, whereas ~ 6 2is~almost " exclusively labeled on tyrosine,p72'yn.Ris predominantly labeled on serine and threonine with a limited degree of labeling on tyrosine. Analysis of the Tyr-420-containing site from ~ 6 2 revealed ' ~ labeling exclusively on tyrosine. In contrast, the CIP-resistant -~ sites 1 and 2 from thetryptic digestion of ~ 7 2 @ "were exclusively labeled on serine. Sites 3 and 4 accounted for the phosphorylationonthreonine. Thus,the phosphorylated serine/threonine residues appear to account for most of the relative resistance of p72'rn-Rto digestion to CIP.
~
-
TcRICD3-associated p 7 P n R
A.
FIG. 6. Alkalinephosphatase di6 and2~ 7 2~ ‘ ~ A, ” -anti~. gestion of ~ fyn precipitates were subjected to an in vitro kinase assay, and thekinase buffer was aspirated to remove unincorporated [y-32P]ATP,digested with 1unit of CIP in 50 pl of CIP buffer, separated by SDS/ PAGE, and analyzed by autoradiography. Lanes 1 and 5, control samples; lunes 2 and 6,samples incubated for 20 min a t 37 “C in the presence of CIP buffer alone; lunes 3 and 7 and lunes 4 and 8 were incubated with CIP at37 “C for 10 and 20 min, respectively. Lanes 14 and 5-8 are from two separate experiments. R@t panel is a plot of the relative intensity of the p62” and ~ 7 2 ” ‘ ~ bands from CIP-digested samples compared tothe undigested sample, presented in the left paneel. B, phospholabeled p62- and p72” proteins generated from anti-fyn precipitates were eluted from SDS/PAGE gels, digested with CIP, and analyzed as in Fig. 7A.
16541
-
Anti-lyn
Anti-tyn
TIME COURSE
5 61 2 3 4
B.
7 8
Anti-lyn
I
I
120%,
._”,
97
-
-
66-
~
c-
-
-
I
p1201130
-‘
p 7 P p6Zfyn
45
-
pp Sp7Z12f2Y ‘0 yn1 n1 3 0
PAGEoccasionally converted to a 62-kDaform that comigrated with ~ 6 2 Interestingly, ~ . attempts to convert Engagement of the TcR/CD3 complex induces the rapid p72’y”-R to p62@” demonstrated that ~ 7 2 “ ”is- ~ relatively retyrosine phosphorylation of numerous substrates in the T cell (1-3). A major question has been to identify the natureof the sistant to phosphatase digestion. Although there was some receptor-associatedkinases responsible forinitiating this cas- variability in the efficiency of digestion from experiment to - ~ significantly more cade.Besides p56Ick and p60Yes, T lymphocytes express a experiment, the native form of ~ 7 2 ” ”was unique form of ~ 5 9 ~which ‘ ~ differs ) from~ 5 9 ” ” ‘by ~ ’ virtue resistant to the action of the phosphatase than ~62””.The - ~ readily dephosphoof exclusive alternate splicing within exon 7 (16). The iden- autophosphorylation site of ~ 7 2 ” ”was rylated, while the phosphoserine/threonine residues unique tification of an additional fyn-related protein at 72 kDa as‘ ~ resistant to the action of the phosphatase. sociated with the TcR/CD3 complex increases the diversity to ~ 7 2 “ ” were of receptor-associated kinases with a potential capacity to CIP is well established in its ability to dephosphorylatemulgenerate signals in T cells. The association with TcR/CD3 tiple phospholabeled amino acids, and, furthermore, readily resistance was varied from cell to cell, being most prevalent in the T-cell dephosphorylatedp120/130 (Fig. 6). Moreover, hybridoma DC27.10(Fig.2). ~ 7 2 ” was ~ reprecipitated as not the result of enzyme concentration since a titration of the eluted material from two-dimensionalgel electrophoresis(Fig. enzymeyielded similar results (data not shown). It is an 3) or from denatured, labeled anti-CD3 immunoprecipitates attractive hypothesis that this resistance could be related to alterations within ~ 7 2 @ ”however, ~; this re(Fig. 1B).Antisera to N- and C-terminal regions of ~ 5 9 ~conformational ” were effective in recognizing p72fy”-R. Peptide map analysis mains to be determined. The native kinase was generally confirmed its similarity to p59/62@” (Figs.4 and 5). Intrigu- found to be more resistant to dephosphorylation than SDS ~ ~ . residual resistance of SDS-PAGEingly, anti-CD3-associated~ 7 2 ”was ~ moreintensely labeled denatured ~ 7 2 ” ” The than thatprecipitated by the anti-fyn antisera. This discrep- purified p72’y”-R couldbe due to refolding of some of the ancy in the relative labeling may relate to differences in the protein in solution. Lesslikely is the possibility that the ” - ~ be inherently activity of ~ 7 2 @when ” - ~ associated with the TcR/CD3 com- phosphoserine residues within ~ 7 2 @ could resistant to thephosphatase. Another less likely possibility is plex. Despite its relation to p59/62@”,the exact identity of that another protein interacts with fYn rendering it resistant p72fy”-R remains to be elucidated. Two-dimensionalphospho- to phosphatases and dissociation on SDS-PAGE. Isoformshould resolve this issue. Other tryptic mapping showed a peptide pattern that included the specific antibodies to p72fy”-R autophosphorylation site (Y-420), found in p62* (Fig. 5). kinases such as p56Ick have been shown to undergo serine However, the p72@”pattern also showed several additional phosphorylation (35); however, the difference in molecular serine/threonine-labeled phosphopeptides that were not ob- size (62 kDato 72 kDa) and theunusual properties related to served in the ~ 6 2 “ ”pattern. This observation, together with the action of phosphatases conveys a potentially unique feathe fact that ~ 7 2 ” is~ slightly more acidic than ~ 6 2 is~ ,ture to thismember of the src family. Espino et al. (36) have recently described the presence of a consistent with an inter-relationship between these molecules due to phosphorylation.~ 7 2 ”that ~ was extracted from SDS- p75 polypeptide derived from the in vitro translation of a fyn DISCUSSION
16542
TcRICD3-associated p 7 P n R A.
fYn p62
fY n p72
6.
- CIP
fY n P72 + CIP
C.
-
3
2
1
1
3
i
4
FIG.7. Two-dimensional phosphotryptic map analysis of alkaline phosphatase-digested ~72"". Phos-
..
,
?
1
Y42( A
A
pholabeled ~62""and p72" proteins generated from anti-fyn precipitates were eluted from SDS/PAGE gels and subjected to phosphotryptic analysis. Panel A, ~62""; panel B, undigested ~ 7 2 ~ " ; p a nC,e l~72""digested with 1unit of CIP at37 "Cfor 10 min. Lower panel, phosphoamine acid analysis of various spots as indicated. Y420 is from the ~ 6 2 " "trypsin-digested sample; the two left lanes are from intact phospholabeled p62* and ~72"" polypeptides.
a
cDNA clone in rabbit reticulocytes. The nature of this invitro TcR/CD3-associated proteins in the 70-72-kDa range which It will be important to modification is dependent on the unique N-terminal sequence may contain zap, CD5, or " - ~ from ~62""in of the kinase. The addition of N-terminal fyn sequences to determine the manner in which ~ 7 2 @ differs pp60"" allowed for the generation of p75, while the substitu- the generation of signals emanating from the TcR/CD3 comtion of the N-terminal region of src to ~ 5 9 %prevented the plex. Lastly, the presence of serine phosphorylation intro"~~ associates appearance of this isoform. It is an interesting possibility that duces the possibility that ~ 7 2 ' ~preferentially alterations related to the N-terminal region of ~ 5 9 may ' ~ with an unidentified serine kinase. We are presently investiregulate its interaction with the TcR/CD3 complex. In fact, gating this possibility. ~ 5 appears 9 ~to interact with the CD3 and { chains of the thank Drs. Pearl Espino and Seng Cheng antigen receptor via the N-terminal region (37). Attempts to forAcknowledgments-We stimulating and helpful discussions. establish the identity of our ~ 7 2 " ' "with ~ the in vitro-transREFERENCES lated ~ 7 were 5 hindered ~ by the fact that in vitro translated 1 . June, C. H., Fletcher, M. C., Ledbetter, J. A., Schieven, G. L., Siegel, J. N., p59" and p75 failed to label in an in vitro kinase assay. Phillips, A. F., and Samelson, L. E. (1990)Proc. Natl.Acad. Sci. U. S.A. Conversely, p72 was very difficult to label and detect from T 87,7722-7726 2. June, C. H., Fletcher, M. C., Ledbetter, J. A., and Samelson, L. E. (1990) cells labeled in vivo with [35S]Met.Therefore, we could only J. I ~ ~ u w 144,1591-1599 J~. compare the peptide maps of [32P]ATP-labeledp72 with [35S] 3. Weiss, A,, Koretzky, G., Schatzman, R. C., and Kadlecek, T. (1991)Proc. Natl. Acad. S C ~U. . S.A. 88,5484-5488 Met-labeled p75. Despite the difference in the labeling pro4. Rudd, C. E., Trevillyan, J. M., Dasgupta, J. D., Wong, L. L., and Schlosscedure, they shared some bands, suggesting similarity (data man, S. F. (1988)Proc. Natl. Acad. SCL.U. S. A. 85,5190-5194 5. Barber, E. K., Dasgupta, J. D., Schlossman, S. F., Trevillyan, J. M.,and not shown). Rudd, C.E. (1989)Proc. Natl. Acad. Sci. U. S.A. 86,3277-3281 6. Veillette, A,, Bookman, M. A,, Horak, E. M., and Bolen, J. B. (1988)Cell Recent studies have described several proteins in the 7065,301-308 72 kDa molecular size range that associate with the TcR{/ 7. Rudd, C.E. (1990)Immunol. Today 11,400-404 8. Shaw, A. S., Amrein, K. E., Hammond, C., Stern, D. F., Sefton, B. M., and CD3 complex.These include the TcRl-associated zap protein Rose, J. K. (1989)Cell 59,627-636 tyrosine kinase (38), as well as the CD5 antigen (39, 40). 9. Turner, J. M., Brodsky, M. H., Irving, B. A,, Levin, S. D., Perlmutter, R. M., and Littman, D. R. (1990)Cell 60,755-765 Although we cannot exclude a relationship between ~ 7 2 " " - ~ 10. Veillette, A., Bookman, M. A., Horak, E. M., Samelson, L. E., and Bolen, and zap, various data argue against this possibility. Anti-fyn J. B. (1989)Nature 338,257-259 N., Miceli, M. C., Parnes, J. R., and Veillette, A. (1991)Nature antiserum does not react with the zap protein, nor does zap 11. Abraham, liRn.fi9" _"," _co-migrate with polypeptides precipitated by anti-fyn anti- 12. Glaichenhaus, N.,Shastri,,~N.,,~Littman, D. R., and Turner, J. M. (1991) . . Cell 64,511-520 serum. Furthermore, zap appears to require receptor ligation 13. Telfer, J. C., and Rudd, C. E. (1991)Science 264,439-441 before it can associate with the receptor (38). ~ 7 2 " appears ~ 14. Prasad, K. V. S., and Rudd, C. E. (1992)Mol. Cell. Bid. 12,5260-5267 K. E.. E.,Odysseos, Odysseos, A. D., Zalvan, C., Druker, B. J., Anderson, P., to be constitutively associated with its receptor, as observed 15. Burgess, Schlossman, S. F., and Rudd,, C. E. (1991)Eur. J. Immunol. 21, 16631668 for ~59"".Caution, therefore, should be exercisedin analyzing ~
~~~~
~~
TcR/CD3-associatedp7PR 16. Cooke, M. P., and Perlmutter, R. M. (1989) The New Biologist 1,66-74 17. Samelson, L. E., Phillips, A. F., Luong, E. T., and Klausner, R. D. (1990) Proc. Natl. Acad. Sci. U. S. A. 8 7 , 4358-4362 18. Da Silva, A. J., Yamamoto, M., Zalvan, C. H., and Rudd, C. E.(1992) Mol. IMmUIWl. 2 9 , 1417-1425 19. Tsygankov, A. Y., Broker, B. M., Fargnoli, J., Ledbetter, J. A., and Bolen, J. B. (1992) J. Biol. Chem. 267,18259-18262 20. Cooke, M. P., Abraham, K.M., Forbush, K. A., and Perlmutter, R. M. (1991) Cell 66,281-291 21. Stein, P. L., Lee, H.-M., Rich, S., and Soriano, P. (1992) Cell 70, 741-750 22. Appleby, M. W., Gross, J. A., Cooke, M. P., Levin, S. D., Qlan, X., and Perlmutter, R. M. (1992) Cell 70, 751-763 23. Luo, K., and Sefton, B. M. (1990) Mol. Cell. Biol. 10,5305-5313 24. Veillette, A,, Bolen, J. B., and Bookman, M. B. (1989) Mol. Cell. Biol. 9 , 4441-4446 25. Zamoyska, R., Derham, P., Gorman, S. D., von Hoegen, P., Bolen, J. B., Veillette, A., and Parnes,J. R. (1989) Nature 342,278-281 26. Leo, O., Foo, M., Sachs, D. H., Samelson, L. E., and Bluestone, J. A. (1987) Proc. Natl. Acad. Sci. U. S. A . 84,1374-1378 27. Cheng,,S. H., Harvey, .R., Espino, P. C., Semba, K., Yamamoto, T., Toyoshrma, K.,and Smlth, A. E. (1988) EMBO J. 7,3845-3855 28. Kypta, R. M., Hemming, A,, and Courtneidge, S. A. (1988) EMBO J. 7 , 3837-3844
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01 I-OLL
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