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The Syk protein tyrosine kinase can function independently of CD45 or Lck in T cell antigen receptor signaling.

The Syk protein tyrosine kinase can function independently of CD45 or Lck in T cell antigen... The EMBO Journal vol.15 no.22 pp.6251-6261, 1996 The Syk protein tyrosine kinase can function independently of CD45 or Lck in T cell antigen receptor signaling and ZAP-70. The recruitment and activation of these David H.Chu, Hergen Spits1, kinases is important for phosphorylation of downstream R.Bruce Rowley3, Peyron2, Jean-Fran9ois substrates. Joseph B.Bolen4 and Arthur Weiss5 Genetic evidence from both mice and human T cells Departments of Microbiology and Immunology and of Medicine, and underscores the importance of Src PTK family members the Howard Hughes Medical Institute, University of California, in TCR signaling. T cell lines and clones lacking Lck are San Francisco, CA 94143, USA, 1Department of Immunology, unable to signal through the TCR (Karnitz et al., 1992; Netherlands Cancer Institute, Plesmanlaan 121, Amsterdam 1060 CX, Straus and Weiss, 1992). Lck-deficient and Fyn-deficient The Netherlands, 2INSERM Unit 364 Molecular and Cellular mice have T cells defective in TCR-mediated activation Immunology, Faculte de Medicine Pasteur, 06107 Nice, France, 3Department of Oncology, Bristol Myers Squibb, Princeton, NJ 08543 (Appleby et al., 1992; Molina et al., 1992; Stein et al., and 4Department of Cellular Signaling, DNAX Research Institute of 1992; van Oers et al., 1996). Similarly, the importance of Molecular and Cellular Biology, Palo Alto, CA 94304, USA ZAP-70 has been demonstrated by genetic studies. Human author 5Corresponding with a ZAP-70 deficiency have a defect in T cell patients development, and the resulting CD4+ T cells that do The protein tyrosine phosphatase CD45 is a critical develop are unable to signal through the TCR (Arpaia component of the T cell antigen receptor (TCR) sig- et al., 1994; Chan et al., 1994a; Elder et al., 1994). ZAP- naling pathway, acting as a positive regulator of Src deficient mice have a more profound block in T cell family protein tyrosine kinases (PTKs) such as Lck. (Negishi et al., 1995). development Most CD45-deficient human and murine T cell lines The role of Syk in T cell signaling is not as clearly are unable to signal through their TCRs. However, although in B cells Syk interacts with the phos- defined, there is a CD45-deficient cell line that can signal ITAMs present in the Ig-cx and Ig-f3 chains to phorylated through its TCR. We have studied this cell line to in a manner analogous to ZAP-70 in T cells function a TCR signaling pathway that is independent identify et al., 1993; Saouaf et al., 1994). A potential role (Law of CD45 regulation. In the course of these experiments, in TCR signaling was suggested by studies in for Syk we found that the Syk PTK, but not the ZAP-70 PTK, was found to associate with a CD8/; chimera which Syk is able to mediate TCR signaling independently of cells (Chan et al., 1994b). In addition, Syk in COS-18 CD45 and of Lck. For this function, Syk requires with the phosphorylated 4 chain in murine can associate functional kinase and SH2 domains, as well as intact and in the Jurkat T cell line (Chan et al., thymocytes phosphorylation sites in the regulatory loop of its Thome et al., 1995; van Oers et al., 1995). 1994b; kinase domain. Thus, differential expression of Syk is does not appear to be required for T cell However, Syk likely to explain the paradoxical phenotypes of different mice deficient in Syk expression are deficient development: CD45-deficient T cells. Finally, these results suggest B cells but do not show any gross a,B T cell in mature differences in activation requirements between two defect, although y6 T cell development is developmental closely related PTK family members, Syk and ZAP- et al., 1995; Turner et al., 1995; Mallick- (Cheng impaired 70. The differential activities of these two kinases Wood et al., 1996). suggest that they may play distinct, rather than com- have a characteristic tyrosine at their kinases Src family redundant, roles in lymphocyte signaling. pletely ends; phosphorylation of this residue carboxy-terminal CD45/Syk/TCRityrosine kinase/ZAP-70 Keywords: a negative regulatory role via an to play is thought or intermolecular association with the SH2 intramolecular of the kinase (Cooper and Howell, domains and SH3 of the Src family kinases One regulator 1993). proposed Introduction is the transmembrane protein receptor signaling in antigen CD45. This phosphatase is believed tyrosine phosphatase, of the T cell antigen receptor (TCR) results Stimulation of Src family kinases by as a positive regulator to act in the activation of a series of protein tyrosine kinases the regulatory tyrosine, thus negative dephosphorylating and culminates in a variety of distal events which (PTKs) kinase to become activated (Weiss and the permitting include transcriptional activation of the interleukin-2 CD45 can specifically dephosphorylate 1994). Littman, gene (reviewed in Weiss and Littman, 1994). (IL-2) Lck in vitro at the negative regulatory and both Fyn According to a sequential model of tyrosine kinase activ- (Mustelin et al., 1989, 1992). tyrosine carboxy-terminal ation, the Src family kinase members Lck or Fyn phos- the tyrosines of in negative regulatory vivo, Furthermore, phorylate the immunoreceptor tyrosine-based activation in cell lines that are and hyperphosphorylated Lck Fyn contained within the CD3 and 4 subunits motifs (ITAMs) et 1989; Hurley et al., 1993; al., CD45 (Ostergaard lack TCR complex (Burkhardt et al., 1994; Iwashima of the Sieh et al., 1993). In the CD45- et McFarland al., 1993; et al., 1994). Phosphorylation of these tyrosines creates the SH2 domain of the hyper- cell line J45.01, deficient docking sites for the Syk family member kinases, Syk Oxford University Press et D.H.Chu al. J.E6-1 J.D _2 , 0a, .--- ..- --- .0.0 It OCR PP9 '14p Imet -.I -1 0) 0) 'oI I' -1it1 II t0 0) -Il II -0 a) a) 'I P4 P4 A 191 192 19 104 g4 i5i> la, 1m 1091 i2 CD45 CD45 JS-7 J45.01 I*94 , I 12, 1222 14pg rlpg IDPR 1.4 1. -- 0 - ~4- 0)- 0) - UI' "4 X 11, 0) 01) tI LI. I \. ..... W- 4 1-- 11 I.....".I. . - - J1 --I 1T[ 0,- ies, in, 12C 19 1le4 114 0° 191 19B2 1a2 CD45 CD45 of CD45 on and Jurkat cells. Cells were stained with an mAb Fig. 1. Cell surface expression wild-type CD45-deficient 1O5 cells) CD45 (5x against or with an control mAb and (solid lines), isotype-matched flow The cell lines stained, either (dotted lines) analyzed by cytometry. wild-type (J.E6-1 or their variants lower are indicated above each and J.D; upper panels) CD45-deficient (J45.01 and JS-7, respectively; panels), histogram. to a differential of is to phosphorylated Lck is inaccessible expression Syk likely explain the phosphopeptide paradoxical of different that the signaling phenotypes CD45-defi- encompasses carboxy-terminal tyrosine, presum- is the of the cient cells. ably because it occupied by phosphotyrosine These results that Lck in negative regulatory site. suggest CD45-deficient cells is in an inactive conformation those Results et (Sieh al., 1993). As a of the Src CD45 The CD45-deficient JS-7 cell line can signal positive regulator family kinases, is a critical of T cell In most through its TCR component early signaling. T cell lines deficient in CD45 stimulation of The two CD45-deficient Jurkat cell J45.01 and expression, lines, the antigen receptor fails to result in transduction JS-7, have been described et signal (Koretzky al., 1991;' Peyron (Pingel and Thomas, 1989; Koretzky et et al., 1991). Both cell lines have almost undetectable al., 1990, 1991; et Volarevic et McFarland Shiroo al., 1992; al., levels of CD45 compared with their respective 1992; wild-type et in mice that have a J.E6-1 as al., 1993). Furthermore, targeted parental lines, and J.D, assessed by cell surface of a selected exon of the CD45 most and disruption gene, staining immunoblotting (Figure 1, and Figure 6, are arrested in their and those lanes 2 and the thymocytes development, 5). Importantly, JS-7 cell line is able to T cells that do are unable to be stimulated in to TCR stimulation develop through signal response despite the absence their TCR (Kishihara et al., of CD45 (Peyron et The 1993). al., 1991). J45.01 cell line, on Intriguingly, a T cell line exists whose TCR can the other is not et signal hand, (Koretzky al., 1991). the lack of despite CD45 (Peyron et al., 1991). This To address the reason why the CD45-deficient JS-7 variant of the Jurkat cell CD45-negative line has been cells are able to respond to TCR stimulation, we first shown to induce tyrosine phosphoproteins, hydrolyze examined the status of Lck in these cells. In the JS-7 cells, inositol phosphates and mobilize calcium in response to Lck is hyperphosphorylated on the negative regulatory TCR stimulation. The mechanism by which this cell line, tyrosine and its SH2 domain is inaccessible to a phospho- JS-7, is able to signal is unknown. In these studies, we peptide representing the negative regulatory carboxy- examined the CD45-independent TCR signaling pathway terminus of Lck (data not shown). These results suggest in the JS-7 cell line. It was found that the signaling- that Lck is in an inactive conformation. Furthermore, competent JS-7 cells express Syk, in contrast to the DNA sequencing of the Lck SH2 domain revealed no signaling-incompetent CD45-deficient cell line J45.01 mutation (data not shown). We next reasoned that the loss (Koretzky et al., 1991). Most interestingly, we discovered of a positive regulator of Lck such as CD45 may have been that the Syk PTK is able to mediate TCR signaling in a compensated for by the absence or decreased expression of CD45-independent and Lck-independent manner and that the negative regulator of Src family kinases, the Csk PTK 6252 Differences in Syk and ZAP-70 activation c:.'ll I'l h-I I1-1;.01 (i I. i. Ccli Linie I;.7 JYr) ICR . 9-l-; > t 3 1: i 3 Stimulation (miI -I 5 1 { - 1 -' 1l TCR Stimnu l .1WIon ;(miTl) - 11. - 1 1 } '~~~~~~~~~~~~~~~ 'I 1 1.1 1DI1 Mh _ - is * 3 4 nt) Fig. 2. The CD45-deficient Jurkat line JS-7 can signal through its TCR. (A) Jurkat cells (2X 106 cells) were left unstimulated (-) or were stimulated for the indicated times with an anti-TCR mAb. Whole cell lysates of J.E6-1 (lanes 1-4), J45.01 (lanes 5-8) and JCaMl.6 (lanes 9-12) were resolved by 12.5% SDS-PAGE and immunoblotted with an anti-phosphotyrosine mAb. (B) J.D (lanes 1-4) and JS-7 (lanes 5-8) Jurkat cells were stimulated and analyzed as described above. (Chow et al., 1993). However, immunoblotting of whole cell lysates indicated that Csk was expressed at levels \',A- t,MI l - r'-- V "! 'i,o J :^ .1- !t1\1 .. D. comparable with wild-type Jurkat cells in the JS-7 cell ii- at :1- line not shown). (data n- 7, the TCR-mediated signals in JS-7 To examine further -1 cells, we analyzed one of the earliest steps in the TCR signaling pathway, the induction of tyrosine phosphoryl- ation of cellular proteins. A time course of tyrosine phosphoprotein induction in whole cell lysates from stimu- lated cells is presented in Figure 2. The anti-phospho- revealed that the pattern and kinetics tyrosine immunoblot induction in the JS-7 cell line of tyrosine phosphoprotein to that seen in its parental wild-type are highly similar In marked contrast, the CD45- cell line, J.D (Figure 2B). does not show any significant deficient cell line J45.01 phosphoproteins (Figure 2A, lanes induction of tyrosine 23)4 R - U be noted that the wild-type parental cell 5-8). It should of TCR-1 following TCR stimulation. Fig. 3. Phosphorylation shows a pattern and time course of line of J45.01, J.E6-1, were left unstimulated (lanes 3. 5. 7 Jurkat cells (5X 107 cells) 1, (A) similar to that seen for J.D and phosphoprotein induction stimulated for 2 min with an anti-TCR mAb (lanes 2, and 9) or were lanes The defective signaling JS-7 cells 2A, 1-4). Whole cell of J.E6-1 (lanes 1 and 2). J45.01 (Figure 4, 6, 8 and 10). lysates 5 and 6). J.D (lanes 7 and 8) and JS-7 cell line is similar to that of the (lanes 3 and 4), JCaMl.6 (lanes phenotype of the J45.01 were then immunoprecipitated with an anti-TCR-; (lanes 9 and 10) cell JCaMl.6, consistent with Lck-deficient Jurkat line, The were resolved by 12.5% SDS-PAGE mAb. immunoprecipitates of Lck in J45.01 cells (Figure 2A, lanes the inactive state mAb. The blot in immunoblotted with an anti-phosphotyrosine (B) and Sieh et 1993). 9-12; al., with an anti-TCR-; was stripped and reprobed (A) subsequently mAb. is phosphorylated upon TCR The TCR-4 chain stimulation in JS-7 cells 4 chain associates with the phosphorylated the status of the earliest substrates ZAP-70 We next assessed in JS-7 cells TCR stimulation TCR engagement, the ITAMs upon phosphorylated following model of kinase of the TCR-; to the sequential tyrosine subunit. Immunoprecipitations According in the TCR-; of and stimulated cells revealed that in the TCR signaling cascade, phosphorylation from unstimulated activity chain leads to the sub- in the TCR-CD3 J.D the ITAMs complex in three of the signaling-competent lines, J.E6-1, all member. of a family was in to recruitment Syk/ZAP-70 and the chain phosphorylated response sequent JS-7, 4 of ZAP-70 were performed using Although the level of 4 Immunoprecipitations TCR stimulation (Figure 3). from unstimulated and stimulated in JS-7 to be lower in this cell lysates prepared appears phosphorylation of ZAP-70 immunoblotting this result was not observed consistently cells. Anti-phosphotyrosine experiment, that ZAP-70 was revealed inducibly the induction of TCR-1 immunoprecipitates not shown). Therefore, (data TCR stimulation in the sig- following in the CD45-deficient JS-7 cell line phosphorylated phosphorylation JS-7 cell line 4, CD45-deficient (Figure to be naling-competent appears preserved. 6253 D.H.Chu et aL CtIlllmin.eNfE. .0-1 451 7 4.; D) Cal ill,> 4-.01 .6 1.1i6-O 1..6ID-- Cell R i a t 1( tilr lait - t - ICR + - + - + - F - Sti;rilllain + lOb~~~~~~~~~~~~~~~~~~: 80e _ _ -OSyk .4 /) - I-I.k :1 1,!H 4q.3 3_. 5 ~Z-PO4- * ( 1-. IIs I 3 4 h ~ -.. o~E '4 61| som- :- lI) -4-- Syk 9 O ~~7 A- Fig. 4. of ZAP-70 TCR Phosphorylation following stimulation. (A) Jurkat cells were left unstimulated 7 and (lanes 1, 3, or 5, 9) Fig. Phosphorylation of Syk following TCR stimulation. (A) Jurkat were stimulated (lanes 8 and as in 2, 4, 6, 10) Figure 3. cells were left unstimulated 7 and or were (lanes 1, 3, 5, 9) stimulated Immunoprecipitations were anti-ZAP-70 antisera performed using and 8 and (lanes 2, 4, 6, 10) as in Figure 3. Immunoprecipitations were blotted with an mAb. The blot was anti-phosphotyrosine (B) stripped performed antisera and blotted with an anti- using anti-Syk and reprobed with anti-ZAP-70 antisera. Lanes J.E6-1 represent: (lanes mAb. phosphotyrosine (B) The blot was stripped and reprobed with and 2), J45.01 (lanes 3 and JCaM1.6 5 and J.D 4), (lanes 6), (lanes antisera. Lanes anti-Syk represent: J.E6-1 and (lanes 1 2), J45.01 7 and 8) and JS-7 (lanes 9 and 10). 3 and JCaM1.6 (lanes 4), (lanes 5 and 6), J.D (lanes 7 and and 8) JS-7 9 and (lanes 10). lane 10), like the response observed with the two wild- Jurkat cell type lines, J.E6-1 and J.D Further- (Figure 4). more, the phosphorylated 4 chain was associated with immunoprecipitates from any of the cell lines derived phosphorylated ZAP-70 (Figure 4). the Therefore, early from the E6-1 Jurkat line, J.E6-1, J45.01 or JCaM1.6 TCR signal transduction events appear to be normal in (Figure 5B, lanes 1-6). Although no Syk was detected in the CD45-deficient JS-7 cell line. the anti-Syk immunoblot of wild-type J.E6-1 Jurkat cells, very weak tyrosine phosphorylation of a 72 kDa band can Syk is and phosphorylated associates with the be detected in occasionally anti-Syk immunoprecipitation phosphorylated chain TCR upon stimulation only of stimulated of lysates these cells, indicating that there in J.D and JS-7 cells be low may very levels of Syk expressed (Figure The 5B, Syk PTK, like has also been ZAP-70, implicated in lanes 1-2 and data not shown). These results are consistent some TCR-mediated signaling processes (Chan et al., with the recent findings that E6-1-derived Jurkat cells 1994b; Couture et al., 1994a; van Oers et al., 1995). To express mutant Syk transcripts containing a nucleotide determine whether Syk was involved in the signaling insertion resulting in a frameshift and premature events following TCR stop stimulation in the JS-7 cell line, codon (Fargnoli et al., we 1995). initially examined the phosphorylation status of Syk Importantly, Syk expression and TCR phosphorylation were following stimulation. Immunoprecipitations using detected in the an signaling-competent CD45-deficient anti-Syk antiserum were performed cell on unstimulated or line, JS-7, and not in the stimulated signaling-incompetent lysates prepared from the CD45- five different cell deficient cell lines. Syk was found line, J45.01. Thus, there is a to be tyrosine correlation phosphorylated between Syk expression and inducibly only in J.D and the TCR signaling JS-7 cells (Figure SA, lanes capability 7- 10). In of CD45-deficient cells. addition, phosphorylated Consistent with this Syk was also found to interpretation, associate with the another CD45-deficient phosphorylated 4 T cell line that is unable to chain in J.D and JS-7 signal, cells (Figure 5A). H45.052, a derivative of The phosphorylated bands of the HPB.ALL line, also 40 kDa expresses in the J45.01 low to undetectable immunoprecipitates are non-specific and amounts of Syk protein not (Koretzky seen reproducibly (Figure 5A, et al., 1990; Law et lanes 3 and 4). al., 1994; and data not shown). To determine why Syk was not A phosphorylated in all smaller, 55 kDa phosphorylated band was found to of the signaling-competent Jurkat be associated lines, we blotted the with Syk after stimulation in both the J.D anti-Syk immunoprecipitates for Syk and expression. Notably, JS-7 cell lines. At least a portion of this band Syk protein was only detected in immunoprecipitates of represents the Lck PTK (data not shown), consistent with the cells derived from the J.D Jurkat line, J.D and previous JS-7 reports of Syk and ZAP-70 association with Lck (Figure SB). In contrast, Syk was not detected in anti-Syk (Duplay et al., 1994; Thome et al., 1995). 6254 Differences in Syk and ZAP-70 activation N i' does not. We hypothesized, therefore, that the N introduction of Syk into the Syk-deficient, CD45-deficient line J45.01 might be able to restore TCR signaling. To assess this possibility, human Syk was transfected CD45. into J45.01 cells. Syk was immunoprecipitated from the transfected cells and its phosphorylation status assessed by anti-phosphotyrosine immunoblotting. Syk was expressed TCR-K q_w and inducibly phosphorylated upon TCR stimulation only _wqw in J45.01 cells transfected with Syk (Figure 7A and B). Furthermore, phosphorylated Syk was found to be associated with phosphorylated TCR-; in these transfect- Lck - __ _ gum ants, as was observed for the Syk-expressing Jurkat lines J.D and JS-7 (Figures 7A and 5A). More distal tyrosine phosphorylation events appeared to be restored in the Syk- transfected cells as well. ZAP-70' J45.01 The tyrosine phosphoryl- ation pattern in anti-phosphotyrosine immunoprecipitates from stimulated J45.01 cells transfected with Syk appears to be enhanced as compared with cells J45.01 transfected Svk with an empty vector (Figure 7C). Comparison of the anti- phosphotyrosine immunoprecipitates from Syk-transfected J45.01 cells with those from the signaling-competent JS-7 1 2 3 4 cells reveals a pattern of tyrosine phosphoproteins that is virtually identical. The higher level of phosphoprotein Fig. 6. Expression of proteins involved in early TCR signaling events induction in JS-7 cells reflects the result of the low in various Jurkat cell lines. Cells (2X 106 cells) were lysed as efficiency of transient transfection into J45.01 cells, described. One hundred ,tg of total cell lysate of J.E6-1 (lane 1). J45.01 (lane JCaMl.6 (lane 3), J.D (lane 4) and JS-7 (lane 5) were 2), resulting in a smaller number of J45.01 cells that express resolved by 12.5% SDS-PAGE and immunoblotted with the antibodies Syk compared with the JS-7 cells. indicated: anti-CD45; anti-Lck; anti-ZAP-70; anti-Syk. anti-4; In order to study, in more detail, the restoration of TCR signaling in these Syk-transfected J45.01 cells, we Levels of various proteins involved in proximal examined a more distal event, activation of an NF-AT TCR signaling This reporter construct. reporter construct consists of three The unexpected difference in levels of Syk expression in tandem of the NF-AT repeats binding site derived from the various Jurkat lines we were studying prompted us to the IL-2 gene controlling the expression of the luciferase compare the relative expression in these cells of a number gene (NFAT-Luc). NF-AT-driven transcription is respons- of the proteins involved in the earliest TCR signaling ive to TCR stimulation (Durand et Rat al., 1988). Syk, events, CD45, TCR-4, Lck, ZAP-70 and Syk. As men- with a of the epitope-tagged portion hemagglutinin (HA) tioned above, neither of the described CD45- protein, was transfected into the J45.01 cell line previously together deficient cell lines, J45.01 and JS-7, expressed CD45 by with this reporter construct. Figure 8 shows that NF- immunoblotting and only small amounts were expressed AT-regulated luciferase activity was restored in a dose- by cell surface and lanes 2 dependent, stimulation-dependent manner when staining (Figure 1, Figure 6, Syk and 5). The wild-type parental cell lines, J.E6- 1 and cDNA was transfected into J45.0 1. In transfection contrast, J.D, expressed equivalent amounts of CD45 both by of comparable amounts of human ZAP-70 cDNA did not In this immunoblotting and by cell surface staining (Figure 1, reconstitute the response (Figure 8). experiment, and Figure 6, lanes 1 and 4), as did the Lck-deficient maximal NF-AT induction in Syk-transfected J45.01 cells was 15-fold over derivative of JCaMl.6 lane and data in response to TCR stimulation J.E6-1, (Figure 6, 3; basal, induction in the not shown). Lck was found to be at similar to the 11-fold parental expressed equivalent wild-type which J45.01 was transfected levels in all cell lines except the Lck null mutant, JCaM 1.6, J.E6-1 cells, from selected, alone not The decrease which expressed no Lck by immunoblotting (Figure 6). with vector (data shown). slight chain and ZAP-70 showed that in NF-AT fold induction at the level of Immunoblots of the 4 highest expression at levels gg of DNA was not due to an these proteins were (40 transfected) inhibitory expressed roughly equivalent as in the effect on but rather reflects a rise in in all five cell lines tested signaling activity, (Figure 6). However, was in the basal NF-AT when is at these activity Syk expressed Syk immunoprecipitations, Syk only expressed as and JS-7 levels. Rat was as effective J.D-derived cell J.D Syk non-epitope-tagged lines, (Figure 6). Wild-type these human cell T cell cells and mutants derived from this Jurkat line human in reconstituting lines J.E6-1 Syk not and JCaM low to undetectable levels (data shown). (J45.01 1.6) expressed of the PTK Syk protein (Figure 6). of can restore TCR in the Expression Syk signaling in cell line JCaM1.6 of can restore TCR the Lck-deficient Expression Syk signaling in TCR the block in CD45- cell line J45.01 Because presumed CD45-deficient signaling one clear difference between deficient cells is at the level of Lck activation, we next Based on the above data, if of in an Lck-deficient line that can and the determined the CD45-deficient cell JS-7, expression Syk signal, restore TCR-mediated As is shown is that the cell could one that signaling. cannot, J45.01, signaling-competent a similar stimulation- whereas the line in Figure 9A, line dose-dependent, expresses Syk signaling-incompetent 6255 D.H.Chu et aL .)\ .\ 0 1t1' ' li 'lt. i -EwiW - 4- L;0 r-I!I l.lll.lt 1 .- Plasmid Expression (,ug) can reconstitute TCR in J45.01 Fig. Syk signaling cells in a dose- manner. J45.01 dependent, stimulation-dependent cells were co- transfected with NFAT-Luc with either an together vector or empty various amounts of (0) or epitope-tagged Syk ZAP-70 epitope-tagged Transfected cells were then either left unstimulated or (LO). were stimulated with an anti-TCR mAb and then B for luciferase assayed Results are as the fold induction activity. expressed of luciferase after stimulation with the activity compared unstimulated state for each condition. The results ' K shown are of four representative S\ independent experiments. restoration of NF-AT dependent was seen with activity of in overexpression cells, similar to Syk JCaM1.6 that of C J45.01 cells. In the case of JCaM1.6 cells transfected with maximal TCR-mediated Syk, NF-AT induction was with 36-fold, 15-fold compared induction for the vector- transfected J.E6-1 cells not parental In (data shown). this both experiment, and HA- using HA-epitope tagged Syk levels - epitope tagged ZAP-70, of equivalent li, am expressed ZAP-70 were unable to restore protein signaling (Figure This effect was a 9B). as specific one, overexpression of in another Jurkat Syk signaling mutant, JCaM2.5, of a distinct representative complementation group and (Goldsmith Weiss, was not able to _- _* _ 1987), reconstitute l.. TCR as measured signaling, NF-AT by activation (data not shown). the SH2 Syk requires kinase domains, domain and the domain regulatory phosphorylation sites to function in TCR signaling To address which functional of are regions Syk required for the reconstitution of in signaling these various E6-1- Fig. 7. Induction of tyrosine in Syk-transfected J45.01 derived Jurkat Ohosphoproteins and to mutants, determine whether cells. this (A) J45.01 cells (2X 10 were cells) transfected with an empty mechanism the vector (lanes 1 and or parallels well-characterized ZAP-70 2) with a Syk expression plasmid (lanes 3 and path- in T 4). A total of 107 cells and the live cells were way recovered per Syk pathway in B we transfectant and left cells, unstimulated (lanes 1 and 3) or stimulated with an anti-TCR antibody transiently various mutants of expressed in the Lck- Syk (lanes 2 and 4). Lysates from these cells were immunoprecipitated deficient cell line JCaML.6. with anti-Syk antisera and resolved by 12.5% SDS-PAGE. The Three mutants of were Syk used. The immunoprecipitates first, a kinase- were then transferred to membranes and probed inactive with an contains a anti-phosphotyrosine mAb. mutant, mutation of a critical (B) The blot in lysine to (A) was stripped and reprobed with anti-Syk antisera. (C) J45.01 cells arginine in the were transfected (K395R) putative ATP binding site of rat with empty vector (lanes 3 and 4) or with a Syk expression plasmid The second mutant contained Syk. in the changes (lanes 5 and YYKAQ 6). Cells were left unstimulated (lanes 1, 3 and 5) or were of sequence in the Syk, present putative stimulated with regulatory an anti-TCR antibody (lanes 2, 4 loop and 6). Lysates were of the kinase domain immunoprecipitated et with an (Hubbard al., 1994). anti-phosphotyrosine The two mAb covalently coupled to protein in this A-Sepharose. Immunoprecipitates were tyrosines sequence, then required for maximal Syk resolved by 12.5% SDS-PAGE and blotted with an anti- and phosphorylation kinase activity (Couture et al., 1994b), phosphotyrosine mAb. JS-7 1 cells (lanes and 2) were transfected with were mutated to phenylalanine (Y519F and Y520F, vector YYFF empty as a positive control. In the panel on the right, a shorter The final mutant). mutant is one in exposure of the which critical immunoprecipitates from the JS-7 arginines cells is shown for within the comparison of the pattern of phosphotyrosine phosphotyrosine bands binding pockets of seen in the anti- the two phosphotyrosine immunoprecipitations SH2 from transfected domains were J45.01 cells. mutated to alanine (R41A and R194A). The results shown are representative of three independent experiments. These mutants of Syk were transiently co-transfected 6256 Differences in Syk and ZAP-70 activation _ v \ i -_- 7- 7- DNA: ~~~ ~;1 ~ ~ ~ ~ ~ ~ ~ ~ ~~1 -1 .. ; - -X )I' L' S i0Il t)IPL 911 id Lt" , I Cell Line: vvt Lck-deficient IDNA Transfected.: vector HA --ZAP 71 FIA-\-Svk .g DN I10 20 1A0 40 20 40 -, -. X S,% 1; DNA Transfected: -- M-__mm o __qmt Fig. 9. Syk can reconstitute TCR signaling in JCaM1.6 cells in a dose- dependent, stimulation-dependent manner. (A) JCaMl.6 cells were Fig. 10. Syk requires functional SH2 and kinase domains, as well as transfected and analyzed as described for J45.01 transfectants in intact regulatory phosphorylation sites, to restore TCR signaling in Figure 8. The results shown are representative of four independent JCaMl.6. (A) JCaM1.6 cells were co-transfected with NFAT-Luc and (B) Aliquots of the cells (5x 105 cells/lane) transfected in experiments. vector alone, or with 10 ,ug of epitope-tagged wild-type Syk were lysed and immunoblotted with an mAb against the HA empty (A) (WT Syk), kinase-inactive (Kinase Inact.), regulatory phosphorylation Lane 1 represents JCaMI.6 cells transfected with an empty epitope. site mutant (YYFF Mut.) or SH2 mutant (SH2 Mut.) plasmid DNA. vector. Lanes 2-5 represent JCaM1.6 cells transfected with 5, 10, 20 respectively. The transfectants were then treated and analyzed as and 40 ,ug of epitope-tagged ZAP-70 DNA. respectively. Lanes 6-9 described in Figure 8. NF-AT fold induction of wild-type J.E6-1 cells JCaMl.6 cells transfected with 5. 10, 20 and 40 tg of represent transfected with empty vector alone is shown as a reference (shaded Syk DNA, respectively. epitope-tagged bar). The results shown are representative of four independent Aliquots of cells (5X 105 cells/lane) from the experiments. (B) with the NFAT-Luc reporter construct into the Lck- in were lysed and immunoblotted with an anti-Syk transfectants (A) Lanes represent cells transfected with empty vector antiserum. deficient JCaMl.6 cells. Functional TCR signaling was wild-type HA-Syk (lane 2); the kinase-inactive mutant (lane 1); measured by assaying NF-AT-driven transcriptional the regulatory phosphorylation site mutant (YYFF Mut.; (lane 3): activity following TCR stimulation. Although wild-type and the SH2 mutant (lane 5). lane 4); was able to restore signaling, the kinase-inactive Syk mutant of Syk could not, nor could the autophosphorylation expression was found to correlate with the Syk protein mutant of Syk (Figure IOA). Moreover, functional SH2 of CD45-deficient Jurkat cells to signal through ability domains of Syk were required for restoration of TCR their TCR. Overexpression of Syk was able to restore since the Syk SH2 mutant, unable to bind to signaling, TCR-mediated signaling in a dose-dependent manner in tyrosine residues and therefore unable to phosphorylated two Jurkat E6-1-derived mutants, J45.01 and JCaMl.6. bind to a phosphorylated ITAM, was incapable of restoring This effect was specific since overexpression of equivalent NF-AT responsiveness (Figure 1OA). As can be seen in ZAP-70 was unable to restore TCR signaling amounts of lOB, the mutants of Syk were expressed in this Figure our findings demonstrate that the two function. Thus, transient assay at levels equal to or greater than that of related PTK family members Syk and ZAP-70 closely the wild-type kinase. Thus, the SH2 domains, kinase in their requirements for activation in differ significantly domain and regulatory phosphorylation sites within the T cells. of Syk are required for the restoration of kinase domain component of the TCR and B is an important CD45 in JCaM 1.6. The same requirements were TCR signaling signaling pathways (Pingel and cell antigen receptor for reconstitution of TCR signaling in the J45.01 found Koretzky et al., 1990, 1991; Justement Thomas, 1989; line not shown). cell (data Shiroo et al., 1992; Volarevic et al., 1992). et 1991; al., to be a positive regulator of Lck and Fyn It is proposed Discussion on the carboxy-terminal negative acting in T cells by in these Src family kinases. In present regulatory tyrosine The results presented here provide evidence that the Syk stimulation of the TCR is presumed CD45, cells expressing PTK is able to function in the TCR signaling pathway or to tyrosines contained induce Lck Fyn phosphorylate to independently of CD45 and Lck in the Jurkat T cell line. 6257 D.H.Chu et aL ITAM et within TCR ITAMs. This in (Bu al., 1995; Neumeister et al., 1995). Instead, phosphorylation, turn, allows it that an increase in subsequent recruitment of ZAP-70 or to the appears ZAP-70 kinase is Syk stimulated activity observed after receptor complex. in the absence of phosphorylation of tyrosines in the However, CD45, regu- of its kinase most investigators have found that Lck and latory loop domain Lck et al., Fyn are by (Chan at their Whereas substantial data hyperphosphorylated sites 1995). that maximal negative regulatory and suggest Syk that functional is influenced the initial events associated with TCR stimulation activity by interactions do involving Src it is not occur (Ostergaard et al., and family kinases, that can overcome 1989; Pingel Thomas, possible Syk 1989; et Shiroo et the of Lck-or the Koretzky al., 1990, 1991; deficiency loss of Lck function due to al., 1992; Volarevic et al., et the absence of CD45-since its 1992; Hurley al., 1993; McFarland catalytic function can be et Sieh et the activated ITAM al., 1993; al., 1993). Therefore, of directly by binding. ability the CD45-deficient JS-7 line to to TCR Our studies that and respond suggest Syk ZAP-70 are not signals represented a paradox. Our studies that the redundant. Whereas can suggest higher functionally Syk reconstitute Lck- or level of Syk expression in the CD45-deficient JS-7 CD45-deficient a Jurkat cells, comparable level of ZAP-70 line can overcome the requirement for both CD45 expression cannot. These results are and consistent with the Lck. contain a mutation Although Syk transcripts which observations of Kolanus et al. who (1993), examined the the relative of function explains deficiency in Jurkat of chimeric Syk clone receptors containing Syk or ZAP-70. E6-1-derived mutants et most found (Fargnoli al., 1995), mature They that cross-linking a CDl16-Syk chimera alone T cells and lines also have low levels was sufficient generally of to induce signal transduction events, whereas Syk (Chan et for the a al., 1994b), accounting similar CD16-ZAP-70 chimera required cross-linking with requirements for CD45 and Lck in TCR transduction a chimera signal containing a Src member to family induce that have been observed several other by laboratories comparable signaling events. However, Kong et al. (1995) (Pingel and Thomas, 1989; et that and Koretzky al., 1990, 1991; suggested Syk ZAP-70 are functionally equivalent. Karnitz et al., 1992; Shiroo et al., Straus and It is that the 1992; Weiss, noteworthy latter studies involved the recon- 1992; McFarland et al., stitution of an avian 1993). B cell line, not a T cell. The functional Previous reports have suggested that both and difference between Syk Syk and ZAP-70 revealed in our T ZAP-70 are recruited to tyrosine-phosphorylated ITAMs cell mutants have been may masked in the context of the in in antigen-receptor complexes (reviewed Weiss and B cell system, perhaps because the presence of active B Littman, 1994). The requirement for the SH2 domains of cell Src kinases such as family Lyn is sufficient to activate Syk in the reconstitution of both CD45- and Lck-deficient ZAP-70. E6-1-derived lines that suggests the phosphorylation of Interestingly, ZAP-70 is phosphorylated at wild-type an ITAM is still for to levels necessary Syk function in these in the CD45-deficient JS-7 cells that contain Syk. cells, for its presumably allowing recruitment. The kinase This is an unexpected finding since in these cells, Lck responsible for the of phosphorylation ITAMs in the (the kinase thought to phosphorylate ZAP-70) is in an absence of Lck or CD45 is not known. While it is possible inactive conformation. It may be that Syk is able to that itself is Syk responsible for the ITAM phosphorylate phosphorylation, ZAP-70 in trans. In support of this model, this seems since unlikely Syk has not been we have reported observed that Syk can phosphorylate a kinase- to interact with an unphosphorylated ITAM, and the inactive version of ZAP-70 when both proteins are over- requirement for its SH2 domains would suggest that it in expressed COS-7 cells (data not shown). However, the only interacts with an ITAM which has already been specific sites of phosphorylation on ZAP-70, and what phosphorylated (Rowley et al., 1995). Moreover, 4 ITAMs effect this phosphorylation may have on the kinase activity are poor substrates for in vitro Syk (A.Weiss, unpublished of ZAP-70, have not been determined. Alternatively, Lck data). A more likely is that possibility Fyn, which appears bound to Syk may be able to phosphorylate ZAP-70. to be less dependent on the presence of CD45, may Indeed, in JS-7 cells, phosphorylated Syk and Lck are partially compensate for the loss of Lck or CD45 (Sieh found to be associated. The Lck SH2 domain has been et al., 1993). Finally, it remains possible that another, as implicated in binding Syk and ZAP-70 (Duplay et al., yet unidentified, kinase may be responsible for ITAM 1994; Aoki et al., 1995; Thome et al., 1995). Therefore, phosphorylation. This is an intriguing possibility since the the phosphorylated negative regulatory tyrosine of the Lck stable association of either Lck or Fyn with the TCR that is bound to Syk may be displaced from its SH2 complex has been observed only at low stoichiometry domain, allowing Lck to phosphorylate other substrates, (Samelson et al., 1990; Burgess et al., 1991; Duplay despite its hyperphosphorylation on the carboxy-terminal et al., 1994). tyrosine. Again, this mechanism of Lck activation would Once Syk and ZAP-70 are recruited to the phosphoryl- have to be specific for Syk and not for ZAP-70. ated ITAM, their functions appear to be regulated in Couture et al. (1994a,b) have proposed that Syk operates distinct ways. Phosphorylated but not unphosphorylated upstream of Src family members in TCR signaling by ITAM peptides from either FcERI or Ig-odf are able to phosphorylating and activating Lck. However, in those increase the catalytic activity of Syk in vitro, independent studies, signaling was only assessed as the phosphorylation of an interaction involving a Src family kinase (Rowley of a 70 kDa band (Couture et al., 1994a). Furthermore, et al., 1995; Shiue et al., 1995). This activation may occur some of those studies were performed using JCaM1.6 by a mechanism of transphosphorylation involving two cells, which have a mutation in Syk transcripts and Syk molecules bound to neighboring ITAMs. Although therefore are deficient in endogenous Syk expression Syk and ZAP-70 were reported to have similar binding (Couture et al., 1994a; Fargnoli et al., 1995). Additionally, affinities for association with the CD3£ ITAM, the catalytic Syk is not a critical upstream activator of Lck or ZAP-70 activity of ZAP-70 is not increased when it binds to an in TCR signaling because most mature T cells and lines, 6258 Differences in Syk and ZAP-70 activation which are able to signal normally through their TCRs, described (Straus and Weiss, 1992). After normalizing for protein content using the Bio-Rad protein assay, 3.6 mg of total cell lysate were express low levels of Syk. Furthermore, T cells from mice immunoprecipitated per sample. Immunoprecipitations were carried out deficient in Syk are able to signal normally following as previously described (Qian et al., 1993). For immunoprecipitations TCR stimulation (Turner et al., 1995). On the other hand, 1 lt using ascites, of ascitic fluid was used For using per sample. those Syk may be sufficient to mediate TCR signaling in the rabbit heterosera, 4 of rabbit antisera were used For per sample. pl immunoblots of whole cell lysates, 100 of were loaded absence of ZAP-70. This is suggested by observations ,ug protein per lane. Immunoprecipitates or lysates were resolved SDS-PAGE by that a cell line made from peripheral T cells of human and transferred to polyvinylidene difluoride membranes (Immobilon) ZAP-70-deficient patients is able to signal in response to (Millipore Ltd, Bedford, MA). The membranes were blocked with 5% TCR stimulation, and these cells have an increased level dry milk and in powder 3% bovine serum albumin 10 mM Tris (BSA) (pH 7.6), 500 mM and or in NaCl 0.05% with 5% BSA of Syk expression as compared with a peripheral T cell Tween-20, phosphate-buffered saline. Blots were then incubated with primary line derived from normal patients (Gelfand et al., 1995). and washed with 10 mM Tris 500 mM NaCl and antibody (pH 7.6), Thus, the findings described here demonstrate the ability 0.05% Tween-20. After incubation of blots with secondary antibody of Syk to function independently of CD45 or Lck. Further- coupled to horseradish or alkaline results were peroxidase phosphatase, more, differential expression of Syk in various cell lines visualized by either enhanced chemiluminescence (ECL) (Amersham, Arlington Heights, IL) or alkaline detection by phosphatase is likely to explain the variety of signaling phenotypes (Zymed, South San Blots were to manufac- Francisco, CA). stripped according seen in CD45-deficient cells. Finally the fact that Syk, but turer's instructions and as described above. (Amersham) reprobed not ZAP-70, is able to function in the absence of CD45 or Lck is a clear difference between two closely related Luciferase assays family members and indicates that these two kinases have A total of 107 cells were co-transfected with 20 of NFAT-Luc reporter plasmid and amounts of or ZAP-70 significantly different requirements for activation. The varying empty vector, Syk plasmids at 250 V and 960 tF using a Bio-Rad Gene by electroporation differential activities of these two kinases suggest that Pulser Transfected cells were (Bio-Rad Laboratories, Hercules, CA). they may play distinct, rather than completely redundant, fetal bovine cultured for 24 h in RPMI-1640 with 10% supplemented roles in lymphocyte signaling. were then counted serum, penicillin, and Cells streptomycin L-glutamine. bottom and 105 live cells were well in 96-well round plated per plates or stimulated with in 100 Cells were left unstimulated (Coming) ,ul. Materials and methods of ascitic or C305 ascites (1:1000 dilution fluid), phorbol myristate for 6 h. were then acetate (50 ng/ml) and ionomycin (1.0 ltM) Samples Cells and antibodies of 100 mM 5.0 mM lysed in a 100 volume KPO4 (pH 7.8), pl The human leukemic Jurkat T cell lines J.E6-1 (Weiss et al., 1984), 1% Triton and this was mixed with dithiothreitol and X-100, lysate J45.01 (Koretzky et al., 1991), JCaMl.6 (Straus and Weiss, 1992), J.D id mM 10 mM 20 mM 100 of assay buffer [200 ATP, KPO4 (pH 7.8), (Peyron et al., 1991) and JS-7 (Peyron et al., 1991) were maintained in of 1.0 mM luciferin. Luciferase MgCl2] followed 100 by ,ul activity, RPMI-1640 medium supplemented with 10% fetal calf serum, penicillin, was determined in for each expressed in units, arbitrary duplicate streptomycin and glutamine (Irvine Scientific, Irvine, CA). The J.D cell Fold induction was calculated as the ratio of experimental condition. line is the wild-type parental Jurkat line described by Peyron et al. stimulation divided the in the luciferase activity following by activity (1991). JS-7 refers to 'Jurkat sublcone #7,' a subclone of the CD45- unstimulated state for each condition. deficient Jurkat clone #25 described in the same paper. Note that J.D is a wild-type Jurkat line that is distinct from the wild-type J.E6-1 Jurkat line (Weiss et al., 1984) that has been described previously. Murine Acknowledgements monoclonal antibodies (mAbs) and their specificities include: C305, Jurkat Ti n-chain (Weiss and Stobo, 1984); 6B10.2, 4 (van Oers et al., NFAT-Luc D.Cantrell We thank G.Crabtree for the reporter plasmid; 1995); 2F3.2, ZAP-70 (Iwashima et al., 1994); 4GI0, phosphotyrosine and Drs A.DeFranco and N.van for for the Oers pEF-BOS plasmid; (Upstate Biotechnology, Incorporated, Lake Placid, NY); 1F6, Lck critical of the This work discussions and helpful reading manuscript. (Burkhardt et al., 1994); 9.4, CD45 (HB 10508, American Type Culture the Medical Scientist was in (D.H.C.) supported part by Training Program Collection, Rockville, MD); and 12CA5, hemagglutinin epitope Institutes of Health Grant GM39553 and National (to A.W.). by (Boehringer-Mannheim, Indianapolis, IN). Rabbit antisera and their specificities are 1373, Syk (van Oers et al., 1995) and 1222, ZAP-70 (Chan et al., 1992). The 4G10 mAb was covalently coupled to protein References A-Sepharose CL-4B (Pharmacia LKB, Piscataway, NJ) using dimethyl- The and Pillai,S. pimelimidate (Harlow and Lane, 1988). Kim,T.J. (1995) SH2 Aoki,Y., Kim,Y.-T., Stillwell,R., with Biol. kinases associate Clhem., domains of Src J. family Syk. 15658-15663. Plasmids 270, and Levin,S.D., cDNAs encoding wild-type human ZAP-70, wild-type rat Syk or mutants Gross,J.A., Cooke,M.P., Qian,X. Appleby,M.W., T cell in mice Defective of Syk were subcloned into the mammalian expression vector pEF-BOS Perlmutter,R.M. (1992) receptor signaling of 751-763. the isoform Cell, 70, (Mizushima and Nagata, 1990) for transient transfections. Mutants of lacking thymic p59f"". and Roifman,C.M. (1994) Cohen,A. HA epitope-tagged rat Syk (Rowley et al., 1995) were constructed by Arpaia,E., Shahar,M., Dadi,H., and CD8+ selection in T cell site-directed mutagenesis using the p-Alter system (Promega, Madison, Defective thymic receptor signaling kinase. 947-958. WI). The kinase-inactive mutant was generated by mutating Lys395 to humans ZAP-70 76, lacking Cell, of the interaction and Arg. The double SH2 mutant has been described (Rowley et al., 1995). Bu,J.-Y., Shaw,A.S. Chan,A.C. (1995) Analysis kinases with the T-cell For the YYFF mutant, point mutations were introduced to both of ZAP-70 and antigen change syk protein-tyrosine Proc. Natl Acad. Sci. USA, 92, resonance. of the Tyr residues at positions 518 and 519 to Phe. The XbaI fragment receptor by plasmon 5106-5110. of the C-terminal epitope-tagged ZAP-70 cDNA in pSV7d was subcloned Drucker,B., Zalvan,C., into pEF-BOS. For the N-terminal epitope-tagged Syk constructs, the Anderson,P., Burgess,K.E., Odysseos,A.D.. Biochemical identification of and the HA-Syk sequence in the Rudd,C.E. (1991) SalI-EcoRI fragment containing pMEX Schlossman,S.F. and between the Ti(TCR)/ interaction vector was subcloned into pEF-BOS. The NF-AT reporter construct a direct CD4:pS61ck physical 1663-1668. CD3 Eur Immunol., 21, (NFAT-Luc) was a generous gift from G.Crabtree. J. complexes. Prendergast,M., Burkhardt,A.L., Mahajan,S., Stealey,B., Rowley,R.B., of non- and (1994) regulation Fargnoli,J. Bolen,J.B. Temporal Cell stimulations, immunoprecipitations, electrophoresis kinase T following transmembrane activity and immunoblots enzyme protein tyrosine Biol. Chern., 269, 23642-23647. dilution cell J. cells were stimulated with anti-TCR antibodies (1:1000 antigen receptor Jurkat engagement. and (1992) ZAP-70: a Iwashima,M., Turck,C.W. Weiss,A. of C30537IC ascitic fluid) at for 2 mi. 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Turner,M., Mee,P.J., Costello,P.S., Williams,O., Price,A.A., Duddy,L.P., Furlong,M.T., Geahlen,R.L. and Tybulewicz,V.L.J. (1995) Perinatal lethality and blocked B-cell development in mice lacking the tyrosine kinase Syk. Nature, 378, 298-302. van Oers,N.S.C., von Boehmer,H. and Weiss,A. (1995) The pre-T cell receptor (TCR) complex is functionally coupled to the TCR-1 subunit. J. Erp. Med., 182, 1585-1590. van Oers,N.S.C., Killeen,N. and Weiss,A. (1996) Lck regulates the tyrosine phosphorylation of the TCR subunits and ZAP-70 in murine thymocytes. J. Exp. Med., 183, 1053-1062. Niklinska,B.B., Burns,C.M., Yamada,H., June,C.H., Volarevic,S., Dumont,F.J. and Ashwell,J.D. (1992) The CD45 tyrosine phosphatase homeostasis and its loss reveals a novel regulates phosphotyrosine of late T cell Ca2+ oscillations. J. pattern receptor-induced Exp. Med., 176, 835-844. Weiss,A. and Littman,D.R. (1994) Signal transduction by lymphocyte antigen receptors. Cell, 76, 263-274. Weiss,A. and Stobo,J.D. (1984) Requirement for the coexpression of T3 and the T cell on a human T cell line. antigen receptor malignant J. Exp. Med., 160, 1284-1299. The role of T3 surface Weiss,A., Wiskocil,R. and Stobo,J.D. (1984) T cells: a two stimulus molecules in the activation of human events at a requirement for IL-2 production reflects occurring pre- 123-128. translational level. J. Immunol., 133, revised on 1996 Received on January July 5, 2, 1996; http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The EMBO Journal Springer Journals

The Syk protein tyrosine kinase can function independently of CD45 or Lck in T cell antigen receptor signaling.

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Copyright © European Molecular Biology Organization 1996
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10.1002/j.1460-2075.1996.tb01015.x
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The EMBO Journal vol.15 no.22 pp.6251-6261, 1996 The Syk protein tyrosine kinase can function independently of CD45 or Lck in T cell antigen receptor signaling and ZAP-70. The recruitment and activation of these David H.Chu, Hergen Spits1, kinases is important for phosphorylation of downstream R.Bruce Rowley3, Peyron2, Jean-Fran9ois substrates. Joseph B.Bolen4 and Arthur Weiss5 Genetic evidence from both mice and human T cells Departments of Microbiology and Immunology and of Medicine, and underscores the importance of Src PTK family members the Howard Hughes Medical Institute, University of California, in TCR signaling. T cell lines and clones lacking Lck are San Francisco, CA 94143, USA, 1Department of Immunology, unable to signal through the TCR (Karnitz et al., 1992; Netherlands Cancer Institute, Plesmanlaan 121, Amsterdam 1060 CX, Straus and Weiss, 1992). Lck-deficient and Fyn-deficient The Netherlands, 2INSERM Unit 364 Molecular and Cellular mice have T cells defective in TCR-mediated activation Immunology, Faculte de Medicine Pasteur, 06107 Nice, France, 3Department of Oncology, Bristol Myers Squibb, Princeton, NJ 08543 (Appleby et al., 1992; Molina et al., 1992; Stein et al., and 4Department of Cellular Signaling, DNAX Research Institute of 1992; van Oers et al., 1996). Similarly, the importance of Molecular and Cellular Biology, Palo Alto, CA 94304, USA ZAP-70 has been demonstrated by genetic studies. Human author 5Corresponding with a ZAP-70 deficiency have a defect in T cell patients development, and the resulting CD4+ T cells that do The protein tyrosine phosphatase CD45 is a critical develop are unable to signal through the TCR (Arpaia component of the T cell antigen receptor (TCR) sig- et al., 1994; Chan et al., 1994a; Elder et al., 1994). ZAP- naling pathway, acting as a positive regulator of Src deficient mice have a more profound block in T cell family protein tyrosine kinases (PTKs) such as Lck. (Negishi et al., 1995). development Most CD45-deficient human and murine T cell lines The role of Syk in T cell signaling is not as clearly are unable to signal through their TCRs. However, although in B cells Syk interacts with the phos- defined, there is a CD45-deficient cell line that can signal ITAMs present in the Ig-cx and Ig-f3 chains to phorylated through its TCR. We have studied this cell line to in a manner analogous to ZAP-70 in T cells function a TCR signaling pathway that is independent identify et al., 1993; Saouaf et al., 1994). A potential role (Law of CD45 regulation. In the course of these experiments, in TCR signaling was suggested by studies in for Syk we found that the Syk PTK, but not the ZAP-70 PTK, was found to associate with a CD8/; chimera which Syk is able to mediate TCR signaling independently of cells (Chan et al., 1994b). In addition, Syk in COS-18 CD45 and of Lck. For this function, Syk requires with the phosphorylated 4 chain in murine can associate functional kinase and SH2 domains, as well as intact and in the Jurkat T cell line (Chan et al., thymocytes phosphorylation sites in the regulatory loop of its Thome et al., 1995; van Oers et al., 1995). 1994b; kinase domain. Thus, differential expression of Syk is does not appear to be required for T cell However, Syk likely to explain the paradoxical phenotypes of different mice deficient in Syk expression are deficient development: CD45-deficient T cells. Finally, these results suggest B cells but do not show any gross a,B T cell in mature differences in activation requirements between two defect, although y6 T cell development is developmental closely related PTK family members, Syk and ZAP- et al., 1995; Turner et al., 1995; Mallick- (Cheng impaired 70. The differential activities of these two kinases Wood et al., 1996). suggest that they may play distinct, rather than com- have a characteristic tyrosine at their kinases Src family redundant, roles in lymphocyte signaling. pletely ends; phosphorylation of this residue carboxy-terminal CD45/Syk/TCRityrosine kinase/ZAP-70 Keywords: a negative regulatory role via an to play is thought or intermolecular association with the SH2 intramolecular of the kinase (Cooper and Howell, domains and SH3 of the Src family kinases One regulator 1993). proposed Introduction is the transmembrane protein receptor signaling in antigen CD45. This phosphatase is believed tyrosine phosphatase, of the T cell antigen receptor (TCR) results Stimulation of Src family kinases by as a positive regulator to act in the activation of a series of protein tyrosine kinases the regulatory tyrosine, thus negative dephosphorylating and culminates in a variety of distal events which (PTKs) kinase to become activated (Weiss and the permitting include transcriptional activation of the interleukin-2 CD45 can specifically dephosphorylate 1994). Littman, gene (reviewed in Weiss and Littman, 1994). (IL-2) Lck in vitro at the negative regulatory and both Fyn According to a sequential model of tyrosine kinase activ- (Mustelin et al., 1989, 1992). tyrosine carboxy-terminal ation, the Src family kinase members Lck or Fyn phos- the tyrosines of in negative regulatory vivo, Furthermore, phorylate the immunoreceptor tyrosine-based activation in cell lines that are and hyperphosphorylated Lck Fyn contained within the CD3 and 4 subunits motifs (ITAMs) et 1989; Hurley et al., 1993; al., CD45 (Ostergaard lack TCR complex (Burkhardt et al., 1994; Iwashima of the Sieh et al., 1993). In the CD45- et McFarland al., 1993; et al., 1994). Phosphorylation of these tyrosines creates the SH2 domain of the hyper- cell line J45.01, deficient docking sites for the Syk family member kinases, Syk Oxford University Press et D.H.Chu al. J.E6-1 J.D _2 , 0a, .--- ..- --- .0.0 It OCR PP9 '14p Imet -.I -1 0) 0) 'oI I' -1it1 II t0 0) -Il II -0 a) a) 'I P4 P4 A 191 192 19 104 g4 i5i> la, 1m 1091 i2 CD45 CD45 JS-7 J45.01 I*94 , I 12, 1222 14pg rlpg IDPR 1.4 1. -- 0 - ~4- 0)- 0) - UI' "4 X 11, 0) 01) tI LI. I \. ..... W- 4 1-- 11 I.....".I. . - - J1 --I 1T[ 0,- ies, in, 12C 19 1le4 114 0° 191 19B2 1a2 CD45 CD45 of CD45 on and Jurkat cells. Cells were stained with an mAb Fig. 1. Cell surface expression wild-type CD45-deficient 1O5 cells) CD45 (5x against or with an control mAb and (solid lines), isotype-matched flow The cell lines stained, either (dotted lines) analyzed by cytometry. wild-type (J.E6-1 or their variants lower are indicated above each and J.D; upper panels) CD45-deficient (J45.01 and JS-7, respectively; panels), histogram. to a differential of is to phosphorylated Lck is inaccessible expression Syk likely explain the phosphopeptide paradoxical of different that the signaling phenotypes CD45-defi- encompasses carboxy-terminal tyrosine, presum- is the of the cient cells. ably because it occupied by phosphotyrosine These results that Lck in negative regulatory site. suggest CD45-deficient cells is in an inactive conformation those Results et (Sieh al., 1993). As a of the Src CD45 The CD45-deficient JS-7 cell line can signal positive regulator family kinases, is a critical of T cell In most through its TCR component early signaling. T cell lines deficient in CD45 stimulation of The two CD45-deficient Jurkat cell J45.01 and expression, lines, the antigen receptor fails to result in transduction JS-7, have been described et signal (Koretzky al., 1991;' Peyron (Pingel and Thomas, 1989; Koretzky et et al., 1991). Both cell lines have almost undetectable al., 1990, 1991; et Volarevic et McFarland Shiroo al., 1992; al., levels of CD45 compared with their respective 1992; wild-type et in mice that have a J.E6-1 as al., 1993). Furthermore, targeted parental lines, and J.D, assessed by cell surface of a selected exon of the CD45 most and disruption gene, staining immunoblotting (Figure 1, and Figure 6, are arrested in their and those lanes 2 and the thymocytes development, 5). Importantly, JS-7 cell line is able to T cells that do are unable to be stimulated in to TCR stimulation develop through signal response despite the absence their TCR (Kishihara et al., of CD45 (Peyron et The 1993). al., 1991). J45.01 cell line, on Intriguingly, a T cell line exists whose TCR can the other is not et signal hand, (Koretzky al., 1991). the lack of despite CD45 (Peyron et al., 1991). This To address the reason why the CD45-deficient JS-7 variant of the Jurkat cell CD45-negative line has been cells are able to respond to TCR stimulation, we first shown to induce tyrosine phosphoproteins, hydrolyze examined the status of Lck in these cells. In the JS-7 cells, inositol phosphates and mobilize calcium in response to Lck is hyperphosphorylated on the negative regulatory TCR stimulation. The mechanism by which this cell line, tyrosine and its SH2 domain is inaccessible to a phospho- JS-7, is able to signal is unknown. In these studies, we peptide representing the negative regulatory carboxy- examined the CD45-independent TCR signaling pathway terminus of Lck (data not shown). These results suggest in the JS-7 cell line. It was found that the signaling- that Lck is in an inactive conformation. Furthermore, competent JS-7 cells express Syk, in contrast to the DNA sequencing of the Lck SH2 domain revealed no signaling-incompetent CD45-deficient cell line J45.01 mutation (data not shown). We next reasoned that the loss (Koretzky et al., 1991). Most interestingly, we discovered of a positive regulator of Lck such as CD45 may have been that the Syk PTK is able to mediate TCR signaling in a compensated for by the absence or decreased expression of CD45-independent and Lck-independent manner and that the negative regulator of Src family kinases, the Csk PTK 6252 Differences in Syk and ZAP-70 activation c:.'ll I'l h-I I1-1;.01 (i I. i. Ccli Linie I;.7 JYr) ICR . 9-l-; > t 3 1: i 3 Stimulation (miI -I 5 1 { - 1 -' 1l TCR Stimnu l .1WIon ;(miTl) - 11. - 1 1 } '~~~~~~~~~~~~~~~ 'I 1 1.1 1DI1 Mh _ - is * 3 4 nt) Fig. 2. The CD45-deficient Jurkat line JS-7 can signal through its TCR. (A) Jurkat cells (2X 106 cells) were left unstimulated (-) or were stimulated for the indicated times with an anti-TCR mAb. Whole cell lysates of J.E6-1 (lanes 1-4), J45.01 (lanes 5-8) and JCaMl.6 (lanes 9-12) were resolved by 12.5% SDS-PAGE and immunoblotted with an anti-phosphotyrosine mAb. (B) J.D (lanes 1-4) and JS-7 (lanes 5-8) Jurkat cells were stimulated and analyzed as described above. (Chow et al., 1993). However, immunoblotting of whole cell lysates indicated that Csk was expressed at levels \',A- t,MI l - r'-- V "! 'i,o J :^ .1- !t1\1 .. D. comparable with wild-type Jurkat cells in the JS-7 cell ii- at :1- line not shown). (data n- 7, the TCR-mediated signals in JS-7 To examine further -1 cells, we analyzed one of the earliest steps in the TCR signaling pathway, the induction of tyrosine phosphoryl- ation of cellular proteins. A time course of tyrosine phosphoprotein induction in whole cell lysates from stimu- lated cells is presented in Figure 2. The anti-phospho- revealed that the pattern and kinetics tyrosine immunoblot induction in the JS-7 cell line of tyrosine phosphoprotein to that seen in its parental wild-type are highly similar In marked contrast, the CD45- cell line, J.D (Figure 2B). does not show any significant deficient cell line J45.01 phosphoproteins (Figure 2A, lanes induction of tyrosine 23)4 R - U be noted that the wild-type parental cell 5-8). It should of TCR-1 following TCR stimulation. Fig. 3. Phosphorylation shows a pattern and time course of line of J45.01, J.E6-1, were left unstimulated (lanes 3. 5. 7 Jurkat cells (5X 107 cells) 1, (A) similar to that seen for J.D and phosphoprotein induction stimulated for 2 min with an anti-TCR mAb (lanes 2, and 9) or were lanes The defective signaling JS-7 cells 2A, 1-4). Whole cell of J.E6-1 (lanes 1 and 2). J45.01 (Figure 4, 6, 8 and 10). lysates 5 and 6). J.D (lanes 7 and 8) and JS-7 cell line is similar to that of the (lanes 3 and 4), JCaMl.6 (lanes phenotype of the J45.01 were then immunoprecipitated with an anti-TCR-; (lanes 9 and 10) cell JCaMl.6, consistent with Lck-deficient Jurkat line, The were resolved by 12.5% SDS-PAGE mAb. immunoprecipitates of Lck in J45.01 cells (Figure 2A, lanes the inactive state mAb. The blot in immunoblotted with an anti-phosphotyrosine (B) and Sieh et 1993). 9-12; al., with an anti-TCR-; was stripped and reprobed (A) subsequently mAb. is phosphorylated upon TCR The TCR-4 chain stimulation in JS-7 cells 4 chain associates with the phosphorylated the status of the earliest substrates ZAP-70 We next assessed in JS-7 cells TCR stimulation TCR engagement, the ITAMs upon phosphorylated following model of kinase of the TCR-; to the sequential tyrosine subunit. Immunoprecipitations According in the TCR-; of and stimulated cells revealed that in the TCR signaling cascade, phosphorylation from unstimulated activity chain leads to the sub- in the TCR-CD3 J.D the ITAMs complex in three of the signaling-competent lines, J.E6-1, all member. of a family was in to recruitment Syk/ZAP-70 and the chain phosphorylated response sequent JS-7, 4 of ZAP-70 were performed using Although the level of 4 Immunoprecipitations TCR stimulation (Figure 3). from unstimulated and stimulated in JS-7 to be lower in this cell lysates prepared appears phosphorylation of ZAP-70 immunoblotting this result was not observed consistently cells. Anti-phosphotyrosine experiment, that ZAP-70 was revealed inducibly the induction of TCR-1 immunoprecipitates not shown). Therefore, (data TCR stimulation in the sig- following in the CD45-deficient JS-7 cell line phosphorylated phosphorylation JS-7 cell line 4, CD45-deficient (Figure to be naling-competent appears preserved. 6253 D.H.Chu et aL CtIlllmin.eNfE. .0-1 451 7 4.; D) Cal ill,> 4-.01 .6 1.1i6-O 1..6ID-- Cell R i a t 1( tilr lait - t - ICR + - + - + - F - Sti;rilllain + lOb~~~~~~~~~~~~~~~~~~: 80e _ _ -OSyk .4 /) - I-I.k :1 1,!H 4q.3 3_. 5 ~Z-PO4- * ( 1-. IIs I 3 4 h ~ -.. o~E '4 61| som- :- lI) -4-- Syk 9 O ~~7 A- Fig. 4. of ZAP-70 TCR Phosphorylation following stimulation. (A) Jurkat cells were left unstimulated 7 and (lanes 1, 3, or 5, 9) Fig. Phosphorylation of Syk following TCR stimulation. (A) Jurkat were stimulated (lanes 8 and as in 2, 4, 6, 10) Figure 3. cells were left unstimulated 7 and or were (lanes 1, 3, 5, 9) stimulated Immunoprecipitations were anti-ZAP-70 antisera performed using and 8 and (lanes 2, 4, 6, 10) as in Figure 3. Immunoprecipitations were blotted with an mAb. The blot was anti-phosphotyrosine (B) stripped performed antisera and blotted with an anti- using anti-Syk and reprobed with anti-ZAP-70 antisera. Lanes J.E6-1 represent: (lanes mAb. phosphotyrosine (B) The blot was stripped and reprobed with and 2), J45.01 (lanes 3 and JCaM1.6 5 and J.D 4), (lanes 6), (lanes antisera. Lanes anti-Syk represent: J.E6-1 and (lanes 1 2), J45.01 7 and 8) and JS-7 (lanes 9 and 10). 3 and JCaM1.6 (lanes 4), (lanes 5 and 6), J.D (lanes 7 and and 8) JS-7 9 and (lanes 10). lane 10), like the response observed with the two wild- Jurkat cell type lines, J.E6-1 and J.D Further- (Figure 4). more, the phosphorylated 4 chain was associated with immunoprecipitates from any of the cell lines derived phosphorylated ZAP-70 (Figure 4). the Therefore, early from the E6-1 Jurkat line, J.E6-1, J45.01 or JCaM1.6 TCR signal transduction events appear to be normal in (Figure 5B, lanes 1-6). Although no Syk was detected in the CD45-deficient JS-7 cell line. the anti-Syk immunoblot of wild-type J.E6-1 Jurkat cells, very weak tyrosine phosphorylation of a 72 kDa band can Syk is and phosphorylated associates with the be detected in occasionally anti-Syk immunoprecipitation phosphorylated chain TCR upon stimulation only of stimulated of lysates these cells, indicating that there in J.D and JS-7 cells be low may very levels of Syk expressed (Figure The 5B, Syk PTK, like has also been ZAP-70, implicated in lanes 1-2 and data not shown). These results are consistent some TCR-mediated signaling processes (Chan et al., with the recent findings that E6-1-derived Jurkat cells 1994b; Couture et al., 1994a; van Oers et al., 1995). To express mutant Syk transcripts containing a nucleotide determine whether Syk was involved in the signaling insertion resulting in a frameshift and premature events following TCR stop stimulation in the JS-7 cell line, codon (Fargnoli et al., we 1995). initially examined the phosphorylation status of Syk Importantly, Syk expression and TCR phosphorylation were following stimulation. Immunoprecipitations using detected in the an signaling-competent CD45-deficient anti-Syk antiserum were performed cell on unstimulated or line, JS-7, and not in the stimulated signaling-incompetent lysates prepared from the CD45- five different cell deficient cell lines. Syk was found line, J45.01. Thus, there is a to be tyrosine correlation phosphorylated between Syk expression and inducibly only in J.D and the TCR signaling JS-7 cells (Figure SA, lanes capability 7- 10). In of CD45-deficient cells. addition, phosphorylated Consistent with this Syk was also found to interpretation, associate with the another CD45-deficient phosphorylated 4 T cell line that is unable to chain in J.D and JS-7 signal, cells (Figure 5A). H45.052, a derivative of The phosphorylated bands of the HPB.ALL line, also 40 kDa expresses in the J45.01 low to undetectable immunoprecipitates are non-specific and amounts of Syk protein not (Koretzky seen reproducibly (Figure 5A, et al., 1990; Law et lanes 3 and 4). al., 1994; and data not shown). To determine why Syk was not A phosphorylated in all smaller, 55 kDa phosphorylated band was found to of the signaling-competent Jurkat be associated lines, we blotted the with Syk after stimulation in both the J.D anti-Syk immunoprecipitates for Syk and expression. Notably, JS-7 cell lines. At least a portion of this band Syk protein was only detected in immunoprecipitates of represents the Lck PTK (data not shown), consistent with the cells derived from the J.D Jurkat line, J.D and previous JS-7 reports of Syk and ZAP-70 association with Lck (Figure SB). In contrast, Syk was not detected in anti-Syk (Duplay et al., 1994; Thome et al., 1995). 6254 Differences in Syk and ZAP-70 activation N i' does not. We hypothesized, therefore, that the N introduction of Syk into the Syk-deficient, CD45-deficient line J45.01 might be able to restore TCR signaling. To assess this possibility, human Syk was transfected CD45. into J45.01 cells. Syk was immunoprecipitated from the transfected cells and its phosphorylation status assessed by anti-phosphotyrosine immunoblotting. Syk was expressed TCR-K q_w and inducibly phosphorylated upon TCR stimulation only _wqw in J45.01 cells transfected with Syk (Figure 7A and B). Furthermore, phosphorylated Syk was found to be associated with phosphorylated TCR-; in these transfect- Lck - __ _ gum ants, as was observed for the Syk-expressing Jurkat lines J.D and JS-7 (Figures 7A and 5A). More distal tyrosine phosphorylation events appeared to be restored in the Syk- transfected cells as well. ZAP-70' J45.01 The tyrosine phosphoryl- ation pattern in anti-phosphotyrosine immunoprecipitates from stimulated J45.01 cells transfected with Syk appears to be enhanced as compared with cells J45.01 transfected Svk with an empty vector (Figure 7C). Comparison of the anti- phosphotyrosine immunoprecipitates from Syk-transfected J45.01 cells with those from the signaling-competent JS-7 1 2 3 4 cells reveals a pattern of tyrosine phosphoproteins that is virtually identical. The higher level of phosphoprotein Fig. 6. Expression of proteins involved in early TCR signaling events induction in JS-7 cells reflects the result of the low in various Jurkat cell lines. Cells (2X 106 cells) were lysed as efficiency of transient transfection into J45.01 cells, described. One hundred ,tg of total cell lysate of J.E6-1 (lane 1). J45.01 (lane JCaMl.6 (lane 3), J.D (lane 4) and JS-7 (lane 5) were 2), resulting in a smaller number of J45.01 cells that express resolved by 12.5% SDS-PAGE and immunoblotted with the antibodies Syk compared with the JS-7 cells. indicated: anti-CD45; anti-Lck; anti-ZAP-70; anti-Syk. anti-4; In order to study, in more detail, the restoration of TCR signaling in these Syk-transfected J45.01 cells, we Levels of various proteins involved in proximal examined a more distal event, activation of an NF-AT TCR signaling This reporter construct. reporter construct consists of three The unexpected difference in levels of Syk expression in tandem of the NF-AT repeats binding site derived from the various Jurkat lines we were studying prompted us to the IL-2 gene controlling the expression of the luciferase compare the relative expression in these cells of a number gene (NFAT-Luc). NF-AT-driven transcription is respons- of the proteins involved in the earliest TCR signaling ive to TCR stimulation (Durand et Rat al., 1988). Syk, events, CD45, TCR-4, Lck, ZAP-70 and Syk. As men- with a of the epitope-tagged portion hemagglutinin (HA) tioned above, neither of the described CD45- protein, was transfected into the J45.01 cell line previously together deficient cell lines, J45.01 and JS-7, expressed CD45 by with this reporter construct. Figure 8 shows that NF- immunoblotting and only small amounts were expressed AT-regulated luciferase activity was restored in a dose- by cell surface and lanes 2 dependent, stimulation-dependent manner when staining (Figure 1, Figure 6, Syk and 5). The wild-type parental cell lines, J.E6- 1 and cDNA was transfected into J45.0 1. In transfection contrast, J.D, expressed equivalent amounts of CD45 both by of comparable amounts of human ZAP-70 cDNA did not In this immunoblotting and by cell surface staining (Figure 1, reconstitute the response (Figure 8). experiment, and Figure 6, lanes 1 and 4), as did the Lck-deficient maximal NF-AT induction in Syk-transfected J45.01 cells was 15-fold over derivative of JCaMl.6 lane and data in response to TCR stimulation J.E6-1, (Figure 6, 3; basal, induction in the not shown). Lck was found to be at similar to the 11-fold parental expressed equivalent wild-type which J45.01 was transfected levels in all cell lines except the Lck null mutant, JCaM 1.6, J.E6-1 cells, from selected, alone not The decrease which expressed no Lck by immunoblotting (Figure 6). with vector (data shown). slight chain and ZAP-70 showed that in NF-AT fold induction at the level of Immunoblots of the 4 highest expression at levels gg of DNA was not due to an these proteins were (40 transfected) inhibitory expressed roughly equivalent as in the effect on but rather reflects a rise in in all five cell lines tested signaling activity, (Figure 6). However, was in the basal NF-AT when is at these activity Syk expressed Syk immunoprecipitations, Syk only expressed as and JS-7 levels. Rat was as effective J.D-derived cell J.D Syk non-epitope-tagged lines, (Figure 6). Wild-type these human cell T cell cells and mutants derived from this Jurkat line human in reconstituting lines J.E6-1 Syk not and JCaM low to undetectable levels (data shown). (J45.01 1.6) expressed of the PTK Syk protein (Figure 6). of can restore TCR in the Expression Syk signaling in cell line JCaM1.6 of can restore TCR the Lck-deficient Expression Syk signaling in TCR the block in CD45- cell line J45.01 Because presumed CD45-deficient signaling one clear difference between deficient cells is at the level of Lck activation, we next Based on the above data, if of in an Lck-deficient line that can and the determined the CD45-deficient cell JS-7, expression Syk signal, restore TCR-mediated As is shown is that the cell could one that signaling. cannot, J45.01, signaling-competent a similar stimulation- whereas the line in Figure 9A, line dose-dependent, expresses Syk signaling-incompetent 6255 D.H.Chu et aL .)\ .\ 0 1t1' ' li 'lt. i -EwiW - 4- L;0 r-I!I l.lll.lt 1 .- Plasmid Expression (,ug) can reconstitute TCR in J45.01 Fig. Syk signaling cells in a dose- manner. J45.01 dependent, stimulation-dependent cells were co- transfected with NFAT-Luc with either an together vector or empty various amounts of (0) or epitope-tagged Syk ZAP-70 epitope-tagged Transfected cells were then either left unstimulated or (LO). were stimulated with an anti-TCR mAb and then B for luciferase assayed Results are as the fold induction activity. expressed of luciferase after stimulation with the activity compared unstimulated state for each condition. The results ' K shown are of four representative S\ independent experiments. restoration of NF-AT dependent was seen with activity of in overexpression cells, similar to Syk JCaM1.6 that of C J45.01 cells. In the case of JCaM1.6 cells transfected with maximal TCR-mediated Syk, NF-AT induction was with 36-fold, 15-fold compared induction for the vector- transfected J.E6-1 cells not parental In (data shown). this both experiment, and HA- using HA-epitope tagged Syk levels - epitope tagged ZAP-70, of equivalent li, am expressed ZAP-70 were unable to restore protein signaling (Figure This effect was a 9B). as specific one, overexpression of in another Jurkat Syk signaling mutant, JCaM2.5, of a distinct representative complementation group and (Goldsmith Weiss, was not able to _- _* _ 1987), reconstitute l.. TCR as measured signaling, NF-AT by activation (data not shown). the SH2 Syk requires kinase domains, domain and the domain regulatory phosphorylation sites to function in TCR signaling To address which functional of are regions Syk required for the reconstitution of in signaling these various E6-1- Fig. 7. Induction of tyrosine in Syk-transfected J45.01 derived Jurkat Ohosphoproteins and to mutants, determine whether cells. this (A) J45.01 cells (2X 10 were cells) transfected with an empty mechanism the vector (lanes 1 and or parallels well-characterized ZAP-70 2) with a Syk expression plasmid (lanes 3 and path- in T 4). A total of 107 cells and the live cells were way recovered per Syk pathway in B we transfectant and left cells, unstimulated (lanes 1 and 3) or stimulated with an anti-TCR antibody transiently various mutants of expressed in the Lck- Syk (lanes 2 and 4). Lysates from these cells were immunoprecipitated deficient cell line JCaML.6. with anti-Syk antisera and resolved by 12.5% SDS-PAGE. The Three mutants of were Syk used. The immunoprecipitates first, a kinase- were then transferred to membranes and probed inactive with an contains a anti-phosphotyrosine mAb. mutant, mutation of a critical (B) The blot in lysine to (A) was stripped and reprobed with anti-Syk antisera. (C) J45.01 cells arginine in the were transfected (K395R) putative ATP binding site of rat with empty vector (lanes 3 and 4) or with a Syk expression plasmid The second mutant contained Syk. in the changes (lanes 5 and YYKAQ 6). Cells were left unstimulated (lanes 1, 3 and 5) or were of sequence in the Syk, present putative stimulated with regulatory an anti-TCR antibody (lanes 2, 4 loop and 6). Lysates were of the kinase domain immunoprecipitated et with an (Hubbard al., 1994). anti-phosphotyrosine The two mAb covalently coupled to protein in this A-Sepharose. Immunoprecipitates were tyrosines sequence, then required for maximal Syk resolved by 12.5% SDS-PAGE and blotted with an anti- and phosphorylation kinase activity (Couture et al., 1994b), phosphotyrosine mAb. JS-7 1 cells (lanes and 2) were transfected with were mutated to phenylalanine (Y519F and Y520F, vector YYFF empty as a positive control. In the panel on the right, a shorter The final mutant). mutant is one in exposure of the which critical immunoprecipitates from the JS-7 arginines cells is shown for within the comparison of the pattern of phosphotyrosine phosphotyrosine bands binding pockets of seen in the anti- the two phosphotyrosine immunoprecipitations SH2 from transfected domains were J45.01 cells. mutated to alanine (R41A and R194A). The results shown are representative of three independent experiments. These mutants of Syk were transiently co-transfected 6256 Differences in Syk and ZAP-70 activation _ v \ i -_- 7- 7- DNA: ~~~ ~;1 ~ ~ ~ ~ ~ ~ ~ ~ ~~1 -1 .. ; - -X )I' L' S i0Il t)IPL 911 id Lt" , I Cell Line: vvt Lck-deficient IDNA Transfected.: vector HA --ZAP 71 FIA-\-Svk .g DN I10 20 1A0 40 20 40 -, -. X S,% 1; DNA Transfected: -- M-__mm o __qmt Fig. 9. Syk can reconstitute TCR signaling in JCaM1.6 cells in a dose- dependent, stimulation-dependent manner. (A) JCaMl.6 cells were Fig. 10. Syk requires functional SH2 and kinase domains, as well as transfected and analyzed as described for J45.01 transfectants in intact regulatory phosphorylation sites, to restore TCR signaling in Figure 8. The results shown are representative of four independent JCaMl.6. (A) JCaM1.6 cells were co-transfected with NFAT-Luc and (B) Aliquots of the cells (5x 105 cells/lane) transfected in experiments. vector alone, or with 10 ,ug of epitope-tagged wild-type Syk were lysed and immunoblotted with an mAb against the HA empty (A) (WT Syk), kinase-inactive (Kinase Inact.), regulatory phosphorylation Lane 1 represents JCaMI.6 cells transfected with an empty epitope. site mutant (YYFF Mut.) or SH2 mutant (SH2 Mut.) plasmid DNA. vector. Lanes 2-5 represent JCaM1.6 cells transfected with 5, 10, 20 respectively. The transfectants were then treated and analyzed as and 40 ,ug of epitope-tagged ZAP-70 DNA. respectively. Lanes 6-9 described in Figure 8. NF-AT fold induction of wild-type J.E6-1 cells JCaMl.6 cells transfected with 5. 10, 20 and 40 tg of represent transfected with empty vector alone is shown as a reference (shaded Syk DNA, respectively. epitope-tagged bar). The results shown are representative of four independent Aliquots of cells (5X 105 cells/lane) from the experiments. (B) with the NFAT-Luc reporter construct into the Lck- in were lysed and immunoblotted with an anti-Syk transfectants (A) Lanes represent cells transfected with empty vector antiserum. deficient JCaMl.6 cells. Functional TCR signaling was wild-type HA-Syk (lane 2); the kinase-inactive mutant (lane 1); measured by assaying NF-AT-driven transcriptional the regulatory phosphorylation site mutant (YYFF Mut.; (lane 3): activity following TCR stimulation. Although wild-type and the SH2 mutant (lane 5). lane 4); was able to restore signaling, the kinase-inactive Syk mutant of Syk could not, nor could the autophosphorylation expression was found to correlate with the Syk protein mutant of Syk (Figure IOA). Moreover, functional SH2 of CD45-deficient Jurkat cells to signal through ability domains of Syk were required for restoration of TCR their TCR. Overexpression of Syk was able to restore since the Syk SH2 mutant, unable to bind to signaling, TCR-mediated signaling in a dose-dependent manner in tyrosine residues and therefore unable to phosphorylated two Jurkat E6-1-derived mutants, J45.01 and JCaMl.6. bind to a phosphorylated ITAM, was incapable of restoring This effect was specific since overexpression of equivalent NF-AT responsiveness (Figure 1OA). As can be seen in ZAP-70 was unable to restore TCR signaling amounts of lOB, the mutants of Syk were expressed in this Figure our findings demonstrate that the two function. Thus, transient assay at levels equal to or greater than that of related PTK family members Syk and ZAP-70 closely the wild-type kinase. Thus, the SH2 domains, kinase in their requirements for activation in differ significantly domain and regulatory phosphorylation sites within the T cells. of Syk are required for the restoration of kinase domain component of the TCR and B is an important CD45 in JCaM 1.6. The same requirements were TCR signaling signaling pathways (Pingel and cell antigen receptor for reconstitution of TCR signaling in the J45.01 found Koretzky et al., 1990, 1991; Justement Thomas, 1989; line not shown). cell (data Shiroo et al., 1992; Volarevic et al., 1992). et 1991; al., to be a positive regulator of Lck and Fyn It is proposed Discussion on the carboxy-terminal negative acting in T cells by in these Src family kinases. In present regulatory tyrosine The results presented here provide evidence that the Syk stimulation of the TCR is presumed CD45, cells expressing PTK is able to function in the TCR signaling pathway or to tyrosines contained induce Lck Fyn phosphorylate to independently of CD45 and Lck in the Jurkat T cell line. 6257 D.H.Chu et aL ITAM et within TCR ITAMs. This in (Bu al., 1995; Neumeister et al., 1995). Instead, phosphorylation, turn, allows it that an increase in subsequent recruitment of ZAP-70 or to the appears ZAP-70 kinase is Syk stimulated activity observed after receptor complex. in the absence of phosphorylation of tyrosines in the However, CD45, regu- of its kinase most investigators have found that Lck and latory loop domain Lck et al., Fyn are by (Chan at their Whereas substantial data hyperphosphorylated sites 1995). that maximal negative regulatory and suggest Syk that functional is influenced the initial events associated with TCR stimulation activity by interactions do involving Src it is not occur (Ostergaard et al., and family kinases, that can overcome 1989; Pingel Thomas, possible Syk 1989; et Shiroo et the of Lck-or the Koretzky al., 1990, 1991; deficiency loss of Lck function due to al., 1992; Volarevic et al., et the absence of CD45-since its 1992; Hurley al., 1993; McFarland catalytic function can be et Sieh et the activated ITAM al., 1993; al., 1993). Therefore, of directly by binding. ability the CD45-deficient JS-7 line to to TCR Our studies that and respond suggest Syk ZAP-70 are not signals represented a paradox. Our studies that the redundant. Whereas can suggest higher functionally Syk reconstitute Lck- or level of Syk expression in the CD45-deficient JS-7 CD45-deficient a Jurkat cells, comparable level of ZAP-70 line can overcome the requirement for both CD45 expression cannot. These results are and consistent with the Lck. contain a mutation Although Syk transcripts which observations of Kolanus et al. who (1993), examined the the relative of function explains deficiency in Jurkat of chimeric Syk clone receptors containing Syk or ZAP-70. E6-1-derived mutants et most found (Fargnoli al., 1995), mature They that cross-linking a CDl16-Syk chimera alone T cells and lines also have low levels was sufficient generally of to induce signal transduction events, whereas Syk (Chan et for the a al., 1994b), accounting similar CD16-ZAP-70 chimera required cross-linking with requirements for CD45 and Lck in TCR transduction a chimera signal containing a Src member to family induce that have been observed several other by laboratories comparable signaling events. However, Kong et al. (1995) (Pingel and Thomas, 1989; et that and Koretzky al., 1990, 1991; suggested Syk ZAP-70 are functionally equivalent. Karnitz et al., 1992; Shiroo et al., Straus and It is that the 1992; Weiss, noteworthy latter studies involved the recon- 1992; McFarland et al., stitution of an avian 1993). B cell line, not a T cell. The functional Previous reports have suggested that both and difference between Syk Syk and ZAP-70 revealed in our T ZAP-70 are recruited to tyrosine-phosphorylated ITAMs cell mutants have been may masked in the context of the in in antigen-receptor complexes (reviewed Weiss and B cell system, perhaps because the presence of active B Littman, 1994). The requirement for the SH2 domains of cell Src kinases such as family Lyn is sufficient to activate Syk in the reconstitution of both CD45- and Lck-deficient ZAP-70. E6-1-derived lines that suggests the phosphorylation of Interestingly, ZAP-70 is phosphorylated at wild-type an ITAM is still for to levels necessary Syk function in these in the CD45-deficient JS-7 cells that contain Syk. cells, for its presumably allowing recruitment. The kinase This is an unexpected finding since in these cells, Lck responsible for the of phosphorylation ITAMs in the (the kinase thought to phosphorylate ZAP-70) is in an absence of Lck or CD45 is not known. While it is possible inactive conformation. It may be that Syk is able to that itself is Syk responsible for the ITAM phosphorylate phosphorylation, ZAP-70 in trans. In support of this model, this seems since unlikely Syk has not been we have reported observed that Syk can phosphorylate a kinase- to interact with an unphosphorylated ITAM, and the inactive version of ZAP-70 when both proteins are over- requirement for its SH2 domains would suggest that it in expressed COS-7 cells (data not shown). However, the only interacts with an ITAM which has already been specific sites of phosphorylation on ZAP-70, and what phosphorylated (Rowley et al., 1995). Moreover, 4 ITAMs effect this phosphorylation may have on the kinase activity are poor substrates for in vitro Syk (A.Weiss, unpublished of ZAP-70, have not been determined. Alternatively, Lck data). A more likely is that possibility Fyn, which appears bound to Syk may be able to phosphorylate ZAP-70. to be less dependent on the presence of CD45, may Indeed, in JS-7 cells, phosphorylated Syk and Lck are partially compensate for the loss of Lck or CD45 (Sieh found to be associated. The Lck SH2 domain has been et al., 1993). Finally, it remains possible that another, as implicated in binding Syk and ZAP-70 (Duplay et al., yet unidentified, kinase may be responsible for ITAM 1994; Aoki et al., 1995; Thome et al., 1995). Therefore, phosphorylation. This is an intriguing possibility since the the phosphorylated negative regulatory tyrosine of the Lck stable association of either Lck or Fyn with the TCR that is bound to Syk may be displaced from its SH2 complex has been observed only at low stoichiometry domain, allowing Lck to phosphorylate other substrates, (Samelson et al., 1990; Burgess et al., 1991; Duplay despite its hyperphosphorylation on the carboxy-terminal et al., 1994). tyrosine. Again, this mechanism of Lck activation would Once Syk and ZAP-70 are recruited to the phosphoryl- have to be specific for Syk and not for ZAP-70. ated ITAM, their functions appear to be regulated in Couture et al. (1994a,b) have proposed that Syk operates distinct ways. Phosphorylated but not unphosphorylated upstream of Src family members in TCR signaling by ITAM peptides from either FcERI or Ig-odf are able to phosphorylating and activating Lck. However, in those increase the catalytic activity of Syk in vitro, independent studies, signaling was only assessed as the phosphorylation of an interaction involving a Src family kinase (Rowley of a 70 kDa band (Couture et al., 1994a). Furthermore, et al., 1995; Shiue et al., 1995). This activation may occur some of those studies were performed using JCaM1.6 by a mechanism of transphosphorylation involving two cells, which have a mutation in Syk transcripts and Syk molecules bound to neighboring ITAMs. Although therefore are deficient in endogenous Syk expression Syk and ZAP-70 were reported to have similar binding (Couture et al., 1994a; Fargnoli et al., 1995). Additionally, affinities for association with the CD3£ ITAM, the catalytic Syk is not a critical upstream activator of Lck or ZAP-70 activity of ZAP-70 is not increased when it binds to an in TCR signaling because most mature T cells and lines, 6258 Differences in Syk and ZAP-70 activation which are able to signal normally through their TCRs, described (Straus and Weiss, 1992). After normalizing for protein content using the Bio-Rad protein assay, 3.6 mg of total cell lysate were express low levels of Syk. Furthermore, T cells from mice immunoprecipitated per sample. Immunoprecipitations were carried out deficient in Syk are able to signal normally following as previously described (Qian et al., 1993). For immunoprecipitations TCR stimulation (Turner et al., 1995). On the other hand, 1 lt using ascites, of ascitic fluid was used For using per sample. those Syk may be sufficient to mediate TCR signaling in the rabbit heterosera, 4 of rabbit antisera were used For per sample. pl immunoblots of whole cell lysates, 100 of were loaded absence of ZAP-70. This is suggested by observations ,ug protein per lane. Immunoprecipitates or lysates were resolved SDS-PAGE by that a cell line made from peripheral T cells of human and transferred to polyvinylidene difluoride membranes (Immobilon) ZAP-70-deficient patients is able to signal in response to (Millipore Ltd, Bedford, MA). The membranes were blocked with 5% TCR stimulation, and these cells have an increased level dry milk and in powder 3% bovine serum albumin 10 mM Tris (BSA) (pH 7.6), 500 mM and or in NaCl 0.05% with 5% BSA of Syk expression as compared with a peripheral T cell Tween-20, phosphate-buffered saline. Blots were then incubated with primary line derived from normal patients (Gelfand et al., 1995). and washed with 10 mM Tris 500 mM NaCl and antibody (pH 7.6), Thus, the findings described here demonstrate the ability 0.05% Tween-20. After incubation of blots with secondary antibody of Syk to function independently of CD45 or Lck. Further- coupled to horseradish or alkaline results were peroxidase phosphatase, more, differential expression of Syk in various cell lines visualized by either enhanced chemiluminescence (ECL) (Amersham, Arlington Heights, IL) or alkaline detection by phosphatase is likely to explain the variety of signaling phenotypes (Zymed, South San Blots were to manufac- Francisco, CA). stripped according seen in CD45-deficient cells. Finally the fact that Syk, but turer's instructions and as described above. (Amersham) reprobed not ZAP-70, is able to function in the absence of CD45 or Lck is a clear difference between two closely related Luciferase assays family members and indicates that these two kinases have A total of 107 cells were co-transfected with 20 of NFAT-Luc reporter plasmid and amounts of or ZAP-70 significantly different requirements for activation. The varying empty vector, Syk plasmids at 250 V and 960 tF using a Bio-Rad Gene by electroporation differential activities of these two kinases suggest that Pulser Transfected cells were (Bio-Rad Laboratories, Hercules, CA). they may play distinct, rather than completely redundant, fetal bovine cultured for 24 h in RPMI-1640 with 10% supplemented roles in lymphocyte signaling. were then counted serum, penicillin, and Cells streptomycin L-glutamine. bottom and 105 live cells were well in 96-well round plated per plates or stimulated with in 100 Cells were left unstimulated (Coming) ,ul. Materials and methods of ascitic or C305 ascites (1:1000 dilution fluid), phorbol myristate for 6 h. were then acetate (50 ng/ml) and ionomycin (1.0 ltM) Samples Cells and antibodies of 100 mM 5.0 mM lysed in a 100 volume KPO4 (pH 7.8), pl The human leukemic Jurkat T cell lines J.E6-1 (Weiss et al., 1984), 1% Triton and this was mixed with dithiothreitol and X-100, lysate J45.01 (Koretzky et al., 1991), JCaMl.6 (Straus and Weiss, 1992), J.D id mM 10 mM 20 mM 100 of assay buffer [200 ATP, KPO4 (pH 7.8), (Peyron et al., 1991) and JS-7 (Peyron et al., 1991) were maintained in of 1.0 mM luciferin. Luciferase MgCl2] followed 100 by ,ul activity, RPMI-1640 medium supplemented with 10% fetal calf serum, penicillin, was determined in for each expressed in units, arbitrary duplicate streptomycin and glutamine (Irvine Scientific, Irvine, CA). The J.D cell Fold induction was calculated as the ratio of experimental condition. line is the wild-type parental Jurkat line described by Peyron et al. stimulation divided the in the luciferase activity following by activity (1991). JS-7 refers to 'Jurkat sublcone #7,' a subclone of the CD45- unstimulated state for each condition. deficient Jurkat clone #25 described in the same paper. Note that J.D is a wild-type Jurkat line that is distinct from the wild-type J.E6-1 Jurkat line (Weiss et al., 1984) that has been described previously. Murine Acknowledgements monoclonal antibodies (mAbs) and their specificities include: C305, Jurkat Ti n-chain (Weiss and Stobo, 1984); 6B10.2, 4 (van Oers et al., NFAT-Luc D.Cantrell We thank G.Crabtree for the reporter plasmid; 1995); 2F3.2, ZAP-70 (Iwashima et al., 1994); 4GI0, phosphotyrosine and Drs A.DeFranco and N.van for for the Oers pEF-BOS plasmid; (Upstate Biotechnology, Incorporated, Lake Placid, NY); 1F6, Lck critical of the This work discussions and helpful reading manuscript. (Burkhardt et al., 1994); 9.4, CD45 (HB 10508, American Type Culture the Medical Scientist was in (D.H.C.) supported part by Training Program Collection, Rockville, MD); and 12CA5, hemagglutinin epitope Institutes of Health Grant GM39553 and National (to A.W.). by (Boehringer-Mannheim, Indianapolis, IN). Rabbit antisera and their specificities are 1373, Syk (van Oers et al., 1995) and 1222, ZAP-70 (Chan et al., 1992). The 4G10 mAb was covalently coupled to protein References A-Sepharose CL-4B (Pharmacia LKB, Piscataway, NJ) using dimethyl- The and Pillai,S. pimelimidate (Harlow and Lane, 1988). Kim,T.J. (1995) SH2 Aoki,Y., Kim,Y.-T., Stillwell,R., with Biol. kinases associate Clhem., domains of Src J. family Syk. 15658-15663. Plasmids 270, and Levin,S.D., cDNAs encoding wild-type human ZAP-70, wild-type rat Syk or mutants Gross,J.A., Cooke,M.P., Qian,X. Appleby,M.W., T cell in mice Defective of Syk were subcloned into the mammalian expression vector pEF-BOS Perlmutter,R.M. (1992) receptor signaling of 751-763. the isoform Cell, 70, (Mizushima and Nagata, 1990) for transient transfections. Mutants of lacking thymic p59f"". and Roifman,C.M. (1994) Cohen,A. HA epitope-tagged rat Syk (Rowley et al., 1995) were constructed by Arpaia,E., Shahar,M., Dadi,H., and CD8+ selection in T cell site-directed mutagenesis using the p-Alter system (Promega, Madison, Defective thymic receptor signaling kinase. 947-958. WI). The kinase-inactive mutant was generated by mutating Lys395 to humans ZAP-70 76, lacking Cell, of the interaction and Arg. The double SH2 mutant has been described (Rowley et al., 1995). Bu,J.-Y., Shaw,A.S. Chan,A.C. (1995) Analysis kinases with the T-cell For the YYFF mutant, point mutations were introduced to both of ZAP-70 and antigen change syk protein-tyrosine Proc. Natl Acad. Sci. USA, 92, resonance. of the Tyr residues at positions 518 and 519 to Phe. The XbaI fragment receptor by plasmon 5106-5110. of the C-terminal epitope-tagged ZAP-70 cDNA in pSV7d was subcloned Drucker,B., Zalvan,C., into pEF-BOS. For the N-terminal epitope-tagged Syk constructs, the Anderson,P., Burgess,K.E., Odysseos,A.D.. Biochemical identification of and the HA-Syk sequence in the Rudd,C.E. (1991) SalI-EcoRI fragment containing pMEX Schlossman,S.F. and between the Ti(TCR)/ interaction vector was subcloned into pEF-BOS. The NF-AT reporter construct a direct CD4:pS61ck physical 1663-1668. CD3 Eur Immunol., 21, (NFAT-Luc) was a generous gift from G.Crabtree. J. complexes. Prendergast,M., Burkhardt,A.L., Mahajan,S., Stealey,B., Rowley,R.B., of non- and (1994) regulation Fargnoli,J. Bolen,J.B. Temporal Cell stimulations, immunoprecipitations, electrophoresis kinase T following transmembrane activity and immunoblots enzyme protein tyrosine Biol. Chern., 269, 23642-23647. dilution cell J. cells were stimulated with anti-TCR antibodies (1:1000 antigen receptor Jurkat engagement. and (1992) ZAP-70: a Iwashima,M., Turck,C.W. Weiss,A. of C30537IC ascitic fluid) at for 2 mi. 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(1984) Requirement for the coexpression of T3 and the T cell on a human T cell line. antigen receptor malignant J. Exp. Med., 160, 1284-1299. The role of T3 surface Weiss,A., Wiskocil,R. and Stobo,J.D. (1984) T cells: a two stimulus molecules in the activation of human events at a requirement for IL-2 production reflects occurring pre- 123-128. translational level. J. Immunol., 133, revised on 1996 Received on January July 5, 2, 1996;

Journal

The EMBO Journal – Springer Journals

Published: Nov 1, 1996

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