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Proc. Natl. Acad. Sci. USA Vol. 93, pp. 12165-12170, October 1996

Mechanism of activation for Zap-70 catalytic activity PHILIP V. LoGRASSO*, JULIO HAWKINS, LORI J. FRANK, DOUGLAS WISNIEWSKI, AND ALICE MARCY Department of Molecular Design and Diversity, Merck Research Laboratories, P.O. Box 2000, Rahway, NJ 07065 Communicated by C. Thomas Caskey, Merck Research Laboratories, West Point, PA, August 15, 1996 (received for review March 12, 1996)

ABSTRACT There is a growing body of evidence, includ- determining the biochemical and enzymatic role Zap-70 plays ing data from human genetic and T- function in these processes. studies, which implicate a {-associated of Mr 70,000 The current study was designed to investigate the mecha- (Zap-70) as a critical protein in T-cell acti- nism of activation for Zap-70 by utilizing purified vation and development. During T-cell activation, Zap-70 components of the TCR signaling pathway, including mono- becomes associated via its src homology type 2 (SH2) domains meric and dimeric forms of the cytoplasmic C chain (as with tyrosine-phosphorylated immune-receptor tyrosine ac- determined by mass spectrometry and size exclusion chroma- tivating motif (ITAM) sequences in the cytoplasmic C chain of tography), bisphosphorylated C peptides, and Flag epitope- the T-cell receptor. An intriguing conundrum is how Zap-70 tagged Zap-70. Capture of 33P-labeled product on phospho- is catalytically activated for downstream cellulose paper and autoradiography were used to monitor events. To address this question, we have used purified product formation. To date, most previous published reports Zap-70, tyrosine phosphorylated glutathione S- on the activation of Syk-family PTKs (i.e., Syk and Zap-70) (GST)-Zeta, and GST-Zeta-1 cytoplasmic domains, and var- describe studies utilizing immunoprecipitates of cell lysates or ious forms of ITAM-containing peptides to see what effect permeabilized cells (15-19). binding of ; had upon Zap-70 activity. The The major finding of our study is that the catalytic activity catalytic activity of Zap-70 with respect to autophosphoryla- of purified Flag-Zap-70 with regard to tion increased -5-fold in the presence of 125 nM phosphor- and phosphorylation of an exogenous substrate, gastrin117, is ylated GST-Zeta or GST-Zeta-1 cytoplasmic domain. A 20- increased -5- and 20-fold, respectively, in the presence of 125 fold activity increase was observed for phosphorylation of an nM phosphorylated glutathione S-transferase (GST)-Zeta or exogenous substrate. Both activity increases showed a GST- GST-Zeta-1 cytoplasmic domain, but not by various forms of Zeta concentration dependence. The increase in activity was monomeric ; chain and bisphosphorylated C peptides. Taken not produced with nonphosphorylated GST-Zeta, phosphor- together our data suggest an activation mechanism for Zap-70 ylated C, or phosphorylated ITAM-containing peptides. The catalysis which occurs by an intermolecular trans-phosphory- increase in Zap-70 activity was SH2 mediated and was inhib- lation reaction. ited by phenylphosphate, Zap-70 SH2, and an spe- cific for Zap-70 SH2 domains. Since GST-Zeta and GST- Zeta-1 exist as dimers, the data suggest Zap-70 is activated MATERIALS AND METHODS upon binding a dimeric form of phosphorylated C and not by Expression of Flag-Zap-70. Human Zap-70 was expressed peptide fragments containing a single phosphorylated ITAM. as a fusion with the Flag epitope at its N terminus (residues Taken together, these data indicate that the catalytic activity MDYKDDDDKH) in baculovirus infected Sf9 cells. Infected of Zap-70 is most likely activated by a trans-phosphorylation cells were harvested after 48 hr and cell lysates were prepared. mechanism. Flag-Zap-70 was purified from Sf9 lysates by affinity chroma- tography utilizing anti-Flag M2 affinity gel (Kodak). A final Many of the details describing the re- concentration of 500 mM NaCl was added to the cell lysate and sponsible for T-cell receptor (TCR) activation and develop- is essential to prevent protein aggregation in this purification ment have recently been elucidated [for a detailed review, see step. Cell lysate was applied to the column and washed with Chan et al. (1)]. The most widely accepted model includes data four column volumes of 50 mM Tris (pH 8) containing 500 mM from studies with chimeric molecules and reconstituted recep- NaCl, 1 mM DTT, 2 mM EDTA, 10% glycerol, 50 ,uM tors (2-4) which supports engagement of the TCR followed by Na3VO4, 50 ,uM Na2MoO4, and inhibitors. Flag- phosphorylation of the immune-receptor tyrosine activating Zap-70 was eluted from the column in the above buffer motifs (ITAMs) in the cytoplasmic domains of the CD3 and ; containing 100 ,ug/ml Flag peptide (Kodak). Fractions con- chains by src-family protein tyrosine (PTKs) (3, 5). taining Flag-Zap-70 were further purified by size exclusion This phosphorylation in turn is followed by recruitment of a chromatography on a Superdex 200 (16/1OHR) column (Phar- c-associated protein of Mr 70,000 (Zap-70) to ; in a src macia). The sample was eluted at 0.7 ml/min in 50 mM Tris homology type 2 domain (SH2)-phosphotyrosine-dependent (pH 7.8) containing 500 mM NaCl, 10% glycerol, and 1 mM manner (5-9). Less clear are the details of the biochemical DTT. Peak fractions were identified by Western blot analysis events which follow recruitment of Zap-70 to the TCR. For using monoclonal mouse-anti-Zap-70 antibody ( example, little is known about the nature of the downstream Laboratories, Lexington, KY). These fractions were pooled, substrate(s) for Zap-70 kinase function, or by what mechanism and protein concentration was determined by a quantitative Zap-70 is activated for catalysis of these substrate(s). Bradford assay. Flag-Zap-70 appeared as a single band on Recent evidence, the most compelling of which comes from SDS/PAGE as detected by Coomassie blue staining. human genetic studies (10-14), reports patients with severe combined immunodeficiency due to Zap-70 deficiency. These Abbreviations: Zap-70, c-associated protein of Mr 70,000; GST, glu- data immediately implicated Zap-70 as a critical in tathione S-transferase; ITAM, immune-receptor tyrosine activating T-cell function and development and heightened interest in motif; SH2, src homology type 2 domain; TCR, T-cell receptor; PTK, protein tyrosine kinase; PVDF, poly(vinylidene difluoride); IRK, receptor kinase. The publication costs of this article were defrayed in part by page charge *To whom reprint requests should be addressed at: Merck Research payment. This article must therefore be hereby marked "advertisement" in Laboratories, P.O. Box 2000, Building R50A-300, Rahway, NJ 07065. accordance with 18 U.S.C. §1734 solely to indicate this fact. e-mail: [email protected]. 12165 Downloaded by guest on September 28, 2021 12166 Biochemistry: LoGrasso et al. Proc. Natl. Acad. Sci. USA 93 (1996)

Expression of GST-Zeta-1. DNA sequences specifying 7.0), 30 mM MgCl2 (Sigma); 1 mM DTT (Sigma); 200 ,ug/ml amino acids 69-88 of human Zeta-1 ITAM (20, 21) were bovine (Sigma), 10 ,tM Na3VO4 (Sigma), 125 ,uM synthesized with complementary oligonucleotides which in- ATP (Pharmacia), 1.5 ,uCi [y-33P]ATP (2000 Ci/mmol; 1 Ci = cluded sequences for BamHI and EcoRI restriction sites. The 37 GBq) (DuPont/NEN); 50 ,uM gastrinl17, and 100 nM coding strand sequence was 5'-GGATCCAACCAGCTGT- Flag-Zap-70. Reactions were incubated at 30°C for 40 min. ACAACGAACTGAACCTGGGTCGTCGTGAAGAATA- Aliquots (10 ,tl) of the reaction were terminated on ice with CGACGTTCTGGACAAGTAAGAATTC. Equimolar 5 ,ul 50 mM EDTA (Sigma). Flag-Zap-70 autophosphoryla- amounts of the two oligonucleotides were annealed and PCR- tion reactions were carried out as described above, excluding amplified using flanking primers. The PCR product was the addition of 50 ,uM gastrinl17 in the reaction mixture. restricted with BamHI and EcoRI and ligated to BamHI and Fifteen microliters of the stopped reaction was applied to P-81 EcoRI-treated pGEX-2T (Pharmacia). Recombinant GST- phosphocellulose paper (Whatman), and samples were washed Zeta-1 clones were identified and the DNA sequence verified. each three times with 7 ml of 75 mM H3PO4 to remove excess To express the GST-Zeta-1 in Escherichia coli BL21(DE3), 3 [y-33P]ATP. After washing, the P-81 paper was added to 8 ml ml of a 2 hr culture was diluted 1:166 into 500 ml of Luria broth of Packard Ultima Gold liquid scintillation cocktail and with 100 ,ug/ml ampicillin. Cells were grown at 37°C until the counted using a Packard Tricarb 2500-TR liquid scintillation A600 reached -0.6 and induced with 0.1 mM isopropyl P-D- counter. thiogalactoside for 2 hr. Cultures were harvested by centrifu- Activation and Inhibition. The activation of Flag-Zap-70 gation at 4000 x g for 10 min. The cell pellet was washed in 100 catalysis of gastrinl17 phosphorylation was carried out as ml PBS and resuspended in 16 ml of 100 mM Tris (pH 8.0) described above with the addition of one of the following: containing protease inhibitors (bestatin, aprotinin, leupeptin, 12.5-500 nM phosphorylated GST-Zeta and GST-Zeta-1; 25 and pepstatin A, each at a final concentration of 1 jig/ml) and nM to 13 ,tM phosphorylated C; and 25 nM to 25 ,uM frozen at -70°C. bisphosphorylated Zeta-1, Zeta-2, and CD3 £ peptides (Cal- Expression of GST-Zeta. The entire coding sequence for ifornia Peptide Research, Napa, CA). human ; was recovered from Jurkat, MOLT-4, and peripheral Inhibition of the phosphorylated GST-Zeta activation of blood cDNAs (CLONTECH) using flanking Flag-Zap-70 gastrin117 catalysis reactions was carried out as primers based on regions within ; (20). DNA of ; described for nonactivated reactions and contained 125 nM from all three sources revealed that the predicted phosphorylated GST-Zeta and one of the following: 1 mM or residues differed at positions 60 and 61 from that of ref. 20 but 100 ,AM phenylphosphate (Sigma); 50 ,ug/ml or 5 ,ug/ml mouse agreed with that of Exley et al. (22). The cytoplasmic portion anti-human Zap-701_254 (Upstate Biotechnology, Lake Placid, of ; (residues 55-109) was PCR-cloned using the following NY); or 27 ,uM, 2.7 ,tM, or 270 nM Zap-70 SH2. primers: 5'-CCATGGGATCCAGCAGGAGCGCAGACG Autoradiograms. Unactivated and activated Flag-Zap-70 and 5'-CCATGGATCCATTAGCGAGGGGGCAGG and catalysis of gastrin1i7 was monitored by 16% tricine SDS/ the MOLT-4-derived PCR product. The PCR product was PAGE (NOVEX, San Diego). Aliquots (10 ,l) from a 25 ,lI digested with BamHI and ligated to BamHI-treated pGEX-2T reaction containing 1.5 ,tCi 33P-labeled ATP, 100 nM Flag- (Pharmacia). E. coli BL21 cells transformed with pGEX-2T-C Zap-70, and 50 ,tM gastrinl_17 were removed at 0 and 40 min were induced with 0.1 mM isopropyl ,B-D-thiogalactoside for 4 and analyzed by 16% tricine SDS/PAGE. Radioactive signal hr at 37°C. Cells were harvested, resuspended in PBS buffer, was transferred to a Phosphorlmager screen (Molecular De- lysed with a French press at 19,000 psi, and centrifuged at vices) for 5 days. Unactivated and activated Flag-Zap-70 100,000 x g for 1 hr. autophosphorylation was monitored by 10% Tris-glycine SDS/ Purification of GST-Zeta, GST-Zeta-1, and Zap-70 SH2. PAGE (NOVEX). Radioactive signal was transferred to a The GST-Zeta and Zeta-1 were purified from bac- PhosphorImager screen for 8 days. terial lysates using glutathione sepharose 4B (Pharmacia) and Immunoblotting. Western blot analysis (25) on the unacti- eluted with 10 mM glutathione using the manufacturer's vated and phosphorylated GST-Zeta activated Flag-Zap-70 procedures. Essentially all of the purified GST-Zeta and autophosphorylation reactions using a mouse antiphosphoty- GST-Zeta-1 migrated as dimers when analyzed by size exclu- rosine monoclonal antibody (PY20; Transduction Laborato- sion HPLC. Many GST fusions proteins have been shown to be ries) was carried out. Aliquots (10 pkl) from a 25 pl reaction dimers by size exclusion chromatography (23). Zap-70 SH2 containing 100 nM Flag-Zap-70 and either 125 nM phosphor- domains (residues 1-263) were expressed in E. coli utilizing the ylated GST-Zeta or 125 nM nonphosphorylated GST-Zeta pET3a vector (Novagen) and purified on an Zeta-1 ITAM were removed after 0 and 40 min reaction time and stopped affinity column. with 10 pul SDS/PAGE buffer. Samples were separated by 10% Kinase Reaction Conditions. Ick phosphorylation of GST- SDS/PAGE (NOVEX) and transferred to poly(vinylidene Zeta and GST-Zeta-1. Phosphorylation of fusion proteins was difluoride) (PVDF) membranes (NOVEX) followed by de- performed on the glutathione sepharose 4B bound GST-Zeta tection with alkaline coupled to antiphosphoty- or GST-Zeta-1 using purified truncated human -32 (32 kDa rosine. Western blotting of the phosphorylated fusion proteins catalytic domain, without the SH3 and SH2 domains) tyrosine was performed using either antiphosphotyrosine monoclonal kinase in a buffer containing 50 mM Mops (Sigma) (pH 7.0); antibody PY20 (Transduction Laboratories) or anti-human Ick 30 mM MgCl2 (Sigma), 1 mM DTT (Sigma), 200 ,ug/ml bovine rabbit polyclonal antibody (Upstate Biotechnology) followed serum albumin (Sigma), 200 ,uM ATP (Sigma), and 10% by detection with alkaline phosphatase-coupled secondary glycerol (Fisher) for 2 hr at room temperature. Next, the beads antibody. were washed three times each with PBS and PBS containing 1% octyl glucopyranoside and eluted with glutathione as described (24). RESULTS Flag-Zap-70 phosphorylation of gastrin_117 and autophos- Our experiments were designed to determine if the interaction phorylation. A peptide corresponding to residues 1-17 of between different forms of purified cytoplasmic C chain and gastrin and also containing a tetra-lysine C terminus (EGP- Zap-70 could affect the catalytic activity of this enzyme. Fig. WLEEEEEAYGWMDFKKKK-NH2) was synthesized. The 1 presents the amino acid sequences of GST-Zeta and GST- tetra-lysine carboxy terminus was added to the peptide to Zeta-i cytoplasmic domains, as well as the individual Zeta-i, increase binding to P-81 phosphocellulose paper. Kinase re- Zeta-2, and CD3 £ bisphosphorylated ITAM sequences. To actions were carried out in 25 Al volumes containing final test whether ; containing multiple phosphorylated ITAMs had concentrations of the following: 50 mM Mops (Sigma) (pH a different effect on activation of Zap-70 than peptide frag- Downloaded by guest on September 28, 2021 Biochemistry: LoGrasso et al. Prcoc. Natl. Acad. Sci. USA 93 (1996) 12167

GST zeta We determined the detection limit of the lck-antibody to be -3 MKWKALFTAAILQAQLPITEAQSFGLLDP&CYLLDG1LF1YGVILTTA1F ng. Moreover, no in vitro kinase activity toward an Ilk-specific substrate, cdc-2 peptide was detected when we utilized up to RVKFSRSADAPAYQOGCiQLYNELNLGRREEYDVLD+RG 1 ,uM phosphorylated GST-Zeta (an 8-fold higher concentra- 2 tion than used in Table 1; see below) and no Zap-70 (data not RDPEMGG KPRRKN PI+GLYN ELCIKDKWAEAYSEIG KG shown). 3 The maximal velocity, V,,ax, measured at steady-state for the RRRGKGiGLYQGLSTATKDTYDALH LPPR conversion of gastrinl 17 to phosphotyrosine-containing gas- trinl17, is given in Table 1. Data were recorded in the absence GST zeta 1 GSNQLYNELNLGRREEYDVLDK and presence of various phosphorylated forms of cytoplasmic zeta 1 peptide GSNQLpYNELNLGRREEpYDVLDK (, or bisphosphorylated C peptide. Both 125 nM phosphory- zeta 2 peptide EGLpYNELQKDKMAEApYSEIGM lated GST-Zeta and 125 nM GST-Zeta-1 increased the epsilon peptide NPDpYEPIRKGORDLpYSGLNQ activity of Flag-Zap-70 by 15-fold (Table 1). Concentrations up to 13 ,uM for phosphorylated 4 and 25 ,uM for bisphos- FIG. 1. Amino acid sequence of GST-containing ITAM proteins phorylated Zeta-1, Zeta-2, and CD3 E had no effect on and ITAM-containing peptides. The entire amino acid sequence of ; For these comparable is presented. Solid inverted triangle signifies the beginning of the activation of Flag-Zap-70. peptides cytoplasmic domain of ; and the shaded region signifies the membrane concentrations to those reported for GST-Zeta and GST- spanining region. The Zeta-1, Zeta-2, and Zeta-3 ITAMs are boxed. Zeta-I are shown in Table 1. In addition, we were unable to Both C and Zeta-1 are expressed as GST fusions. The amino acid detect phosphorylated GST-Zeta activation of a 42-kDa cat- sequences of Zeta-1, Zeta-2, and CD3 e bisphosphorylated peptides alytically active kinase domain of Zap-70 toward gastrinl17 are listed. phosphorylation (data not shown). We were able to corroborate and extend the findings of ments containing a single phosphorylated ITAM we phos- Table 1 using a different methodology for detection of 33P- phorylated GST-Zeta and GST-Zeta-I in vitro with a consti- containing phosphopeptide and phosphoproteins. Fig. 3A tutively active Ick kinase domain (Fig. 2). Fig. 2A shows the shows an autoradiogram of the increase in incorporation of 33P anti-phosphotyrosine western blot of GST-Zeta and GST- into gastrinll7 (Fig. 3A, lane 5) when the reaction contains 125 Zeta-I after treatment with and separation from Ick kinase nM phosphorylated GST-Zeta. This effect is not seen in the domain. For GST-Zeta and GST-Zeta-1, the predominant presence of 125 nM GST-Zeta (Fig. 3A, lane 7) as compared phosphoprotein bands are seen at -40 and 32 kDa, respec- with a control reaction which did not contain any GST-Zeta tively (Fig. 2A, lanes 2 and 3). These bands are representative (Fig. 3A, lane 3). An increase relative to control (Fig. 3B, lane of the different phosphorylation states of ;. Fig. 2B shows the 3) in the intensity of an approximatc 70 kDa band (Fig. 3B, lanle corresponding anti-lck Western blot indicating no detection of 5) corresponding to Flag-Zap-70 indicates the increase in Ick in either sample after separation (Fig. 2B, lanes 2 and 3). autophosphorylation activity due to 125 nM phosphorylated GST-Zeta. Again, 125 nM GST-Zeta (Fig. 3B, lane 7) shows lane This fact was A 1 2 3 no activation relative to control (Fig. 3B, 3). further illustrated in Fig. 3C, which shows the antiphosphoty- rosine Western blot of samples as in Fig. 3B, indicating the presence of increased phosphotyrosinie-containing protein at (70 kDa for the sample treated with phosphorylated GST- kDa Zeta (Fig. 3C, lane 5). It is unclear exactly what the origin of 50 - the bands at less than 50 kDa is. These bands are seen more often with GST-Zeta and do not appear in similar experiments done with GST-Zeta-1. It is unlikely that they represent lck because Western blotting indicated Ino dctection of Ick. It may 34, be that they represent a less than 50-kDa form of Zap-70 that does not react with a monioclonal anti-Zap-70 antibody (data 28 not shown). The phosphorylatcd GST-Zeta and GST-Zeta-I conceil- B tration dependence upon the fold increase in Flag-Zap-70 1 2 3 4 catalytic activity for phosphorylation of gastrinl17 is presented in Fig. 4A. Activation of catalytic activity with increasing kDa phosphorylated GST-Zeta or GST-Zeta- 1 concentration was observed, reaching a maximum at -250 nM. Similar results 50 - were obtained for the effect on autophosphorylation (Fig. 4B). 34,'. Tablc 1. Activation of Flag-Zap-70 catalytic activity 28 - V,llax (gastrin), nM/min Unactivafed 20 +125 nM GST-Zctap 345 +125 nM GJST-Zcta-i1) 260 +250 nM GST--Zeta 31 FiG. 2. Western blot analysis of Ick kinase domain-treated GST- + 100 nM Zetap 22 Zeta and GST-Zeta-1. Samples from a 10% SDS/PAGE Tris-glycine +250 nM Zeta-I1p 34 gel system were transferred to PVDF membrane followed by detection +250 nM Zeta-2p 20 with an alkaline phosphatase-coupled immunoassay. (A) Antiphos- +250 nM CD3 20 photyrosine blotting. Lanes: 1, Bio-Rad low molecular weight markers; cp 2, phosphorylated GST-Zeta; 3, phosphorylated GST-Zeta-1. (B) For GST-Zeta and Zeta, p represenits the phosphoprotein form of Anti-lck blotting. Lanes: 1-3, as in A; 4, 10 ng lck-32. Arrowhead the proteini. For the peptides. p represents bisphosphorylated Zeta-il indicates position of Ick. Zeta-2 and CD3 E peptidcs (as shown in Fig. 1). Downloaded by guest on September 28, 2021 12168 Biochemistry: LoGrasso et al. Proc. Natl. Acad. Sci. USA 93 (1996)

A A 30 kDa 1 2 3 4 5 6 7 8 97- 25 66- 46' 0) / V, 20 30 o L 21.3 15 12.5r C 0 6.5/ U- 10 2.3 - 4I0 4. 5

1- -11 U.,--it ss =-Ii...;M.0 .;; zf:z::. 0 0 100 200 300 400 500 600 B 1 2 3 4 5 6 7 [GST-Z-P] nM B 15 kDa i 97 3. IHj: (h) 10 (a 66,W CD

46Ew ..Ilia LL 5

0 C 1 2 3 4 5 6 7 8 0 100 200 300 400 500 600 [GST-ZETA-PJ nM kDa FIG. 4. Phosphorylated GST-Zeta and GST-Zeta-1 increase 112 W Flag-Zap-70 kinase activity. (A) Concentration dependence of phos- phorylated GST-Zeta and GST-Zeta-1 for activation of Flag-Zap-70 84 V gastrinlb17 catalysis. 0, Phosphorylated GST-Zeta; 0, phosphorylated GST-Zeta-1. (B) Concentration dependence of phosphorylated GST- Zeta and GST-Zeta-1 for the activation of Flag-Zap-70 autophos- 53 P phorylation. a, Phosphorylated GST-Zeta; El, phosphorylated GST- Zeta-1. The concentrations of phosphorylated GST-Zeta and GST- Zeta-1 are stated for the dimeric form. Fitted curves are shown. The solid line corresponds to the circles and the dashed line with the squares. compete with ; for binding to the SH2 domains of Flag-Zap- FIG. 3. Analysis of unactivated and activated Flag-Zap-70 catalysis 70. Table 2 presents the percent inhibition of activation for of gastrinl-17 and autophosphorylation. (A) Aliquots (10 ,ul) from a 25 Flag-Zap-70 catalysis of gastrinl17. All three inhibitors ,ul reaction containing 1.5 ,uCi 33P-labeled ATP, 100 nM Flag-Zap-70, blocked activation by greater than 80% thereby reducing and 50 ,uM gastrinl-17 were removed at 0 and 40 min and analyzed by to near basal but had little effect on nonacti- 16% tricine SDS/PAGE. Radioactive signal was transferred to a catalysis levels, Phosphorlmager screen for 5 days and developed. Lanes: 1 and 8, vated Zap-70 kinase activity. 14C-labeled biotinylated molecular weight markers; 2 and 3, 0 and 40-min Flag-Zap-70 with no GST-Zeta additions; 4 and 5, 0 and DISCUSSION 40-min Flag-Zap-70 in presence of 125 nM phosphorylated GST- Our experiments were designed to determine if Zap-70 could Zeta; 6 and 7, 0 and 40-min Flag-Zap-70 in the presence of 125 nM be activated binding to the ; chain of the GST-Zeta. (B) Analysis of 10% Tris-glycine SDS/PAGE run at 30 mA catalytically upon for 75 min. Components are as in A excluding gastrinl1l7. Signal was Table 2. Inhibition of activation of Flag-Zap-70 transferred to a PhosphorImager screen for 8 days and developed. gastrinl_17 catalysis Lane order is as in A. (C) Antiphosphotyrosine Western blot analysis for activation of Flag-Zap-70 autophosphorylation. A 10% Phosphorylated Tris-glycine SDS/PAGE gel was transferred to a PVDF-membrane GST-Zeta activation, followed by detection with alkaline phosphatase coupled to antiphos- % inhibition Lanes: molecular weight photyrosine. 1, Bio-Rad prestained high + 1 mM Phenylphosphate 88 markers; 2-7, order as in A; 8, Bio-Rad prestained low molecular weight markers. 100 ,uM Phenylphosphate 50 50 ,ug/ml a-Zap-70 (SH2)1_254 90 5 jig/ml a-Zap-70 (SH2)1_254 35 To establish that the activation of Flag-Zap-70 was medi- 27 ,uM Zap-70 SH2 100 ated through the binding of its SH2 domains to dimeric, 2.7 ,uM Zap-70 SH2 100 phosphorylated ;, we investigated the effect of inhibiting the 270 nM Zap-70 SH2 100 binding of Flag-Zap-70 to C. To do this we utilized an antibody Percent inhibition is relative to 125 nM phosphorylated GST-Zeta directed against the tandem SH2 domains of Zap-70, purified activated Flag-Zap-70. One hundred percent inhibition is equal to no Zap-70 SH2 domains that should block Flag-Zap-70 associa- activation. Similar results were obtained for inhibition of phosphory- tion with C, and phenylphosphate, a compound which should lated GST-Zeta-1 activated Flag-Zap-70. Downloaded by guest on September 28, 2021 Biochemistry: LoGrasso et al. Proc. Natl. Acad. Sci. USA 93 (1996) 12169 TCR and to investigate the mechanism of this activation stoichiometry used in our experiments. Indeed, van Oers et al. process. To most closely match in vitro the dimeric nature of (31) have shown that additional Zap-70 may be recruited to the the ; chain, as it has been reported to exist (1), we utilized GST TCR following engagement. fusions of cytoplasmic ITAM-containing C chain. A distin- The literature is less clear on the effects that binding of guishing feature of this study is that we utilized purified bisphosphorylated ITAMs have upon the activation of Syk components of the c-chain and Zap-70. This afforded us the catalysis. For example, Johnson et al. (15) reported no effect opportunity to directly investigate the C-chain/Zap-70 inter- on the catalytic activity of Syk in the presence of 1 mM action without the complications associated with immunopre- bisphosphorylated CD3 e. However, Shiue et al. (18) and cipitates from cell lysates. To date, most published reports Rowely et al. (17) report -10-fold activation of Syk kinase investigating the activation of Syk-family PTKs (15-19) revolve activity in the presence of greater than 10 ,tM FcsRI-y-ITAM around immunoprecipitation experiments. and the Ig ,B-ITAM. It is unclear why such high concentrations Analysis of our data shows a clear trend in terms of which of bisphosphorylated ITAM are needed for Syk activation-in forms of ; are capable of activating Zap-70 autophosphoryla- the later two reports leaving the interpretation of these tion and catalysis of gastrin1i7 (Table 1 and Figs. 3 and 4). discrepant sets of data rather murky. Without exception, dimeric forms of phosphorylated cytoplas- The results of our present work are consistent with those of mic C [i.e., GST fusions; the crystal structure of GST has been Neumeister et al. (16) in that under our experimental condi- reported to be a dimer (26)] are capable of activating Flag- tions we were only able to demonstrate activation of Zap-70 Zap-70 autophosphorylation and gastrin117 catalysis. Con- when Zap-70 was bound to multiple phosphorylated ITAMs. versely, all monomeric forms of C, whether they contain In our experiments, an additional requirement for activation multiple or singly phosphorylated ITAMs, had no effect on the was a dimeric form of ;. However, since Neumeister et al. (16) activation of Zap-70 activity. These findings are consistent observed activation of Zap-70 using fusions of ; and a tandem with a trans activation mechanism for Zap-70, which is de- s coexpressed with fyn (multiple phosphorylated ITAMs on a pendent on the dimeric nature of the r chain. For example, the single chain), they reasoned that Zap-70 may serve as a presence of bisphosphorylated Zeta-1, Zeta-2, and CD3 s substrate for an adjacent Zap-70 molecule. We were not able peptides which are recognized by the SH2 domain of Zap-70 to observe activation of Zap-70 using phosphorylated C, which (8, 9, 21) had no effect on Flag-Zap-70 kinase activity sug- should presumably contain adjacent Zap-70 molecules on a gesting that mere binding is insufficient to increase the rate of single chain. One potential explanation for the lack of activa- autophosphorylation and gastrinli17 catalysis. This observa- tion could be incomplete phosphorylation of {, thereby not tion is consistent with that of Neumeister et al. (16), who affording the presentation of adjacent Zap-70 molecules. Or determined in immunoprecipitation experiments no enhanced rather, it is likely that activation is occurring through a Zap-70 autophosphorylation when bound to CD3 s or y. Also trans-phosphorylation reaction from one Zap-70 molecule on consistent with our results are those of Wange et al. (19) which one C chain to another Zap-70 molecule on a second ; chain. show that inhibition of Zap-70 binding to the TCR prevents This interpretation for Zap-70 is consistent with several other Zap-70 from activation. protein tyrosine kinases including src (33, 34), Lyn (35), and In addition, just as the phosphorylated ITAM analogue the kinase (IRK) (36-38), where a trans- F2(PMP)2-Tam C3 was an inhibitor of Zap-70 binding to the phosphorylation activation mechanism has been shown. In TCR and subsequent catalytic activation, our results on inhi- fact, Kurosaki et al. (29) speculated that activation of syk may bition by anti-Zap (SH2) (1-254), phenylphosphate, and iso- occur by a trans-phosphorylation process upon syk recruitment lated Zap-70 (SH2) indicate the activation of Zap-70 kinase to phosphorylated Ig-a/Ig-f3. It should be noted that other activity upon binding a dimeric form of the ; chain is mediated reports have suggested that IRK is activated by an intramo- through the SH2 domains. Supporting this notion is the data lecular process (39). from the 42-kDa catalytically active kinase domain of Zap-70, Recent evidence from the x-ray crystal structure of the which does not contain the SH2 domains and is not activated kinase domain for IRK (36) suggests a trans-phosphorylation by phosphorylated GST-Zeta or GST-Zeta-1. The result also mechanism in which Y1162, the IRK autophosphorylation site, suggests that contaminating Ick kinase activation of Zap-70 is precludes binding of MgATP; therefore, movement of the loop unlikely because one would expect a similar 15-fold activation containing Y1162 is required for trans-phosphorylation of this of the kinase domain if Ick were responsible for activation site by the other (-subunit of IRK. Barker et al. (33) have rather than dimeric phosphorylated GST-Zeta or GST- suggested the trans-phosphorylation of one 13-subunit to an- Zeta-1. It is also unlikely that an Ick species devoid of its SH2 other in IRK is analogous to intermolecular phosphorylation and SH3 domains would have a high affinity for Zap-70, by two monomers. One might envision a similar role for Y493 further decreasing the chance for Ick activation of Zap-70. in Zap-70 as compared with Y1162 in IRK, whereby the Finally, it has been shown that both fyn (27, 28) and tyn (28) position of sequences containlng Y493 and consequently the exhibit enzymatic activation upon binding Ig-a in an SH2- phosphorylation state of Y493 controls trans-phosphorylation dependent manner. A recent report (29) presents data showing between two Zap-70 molecules. Indeed, Chan et al. (40) and that syk is activated upon binding the B-cell receptor. Coupled Wange et al. (41) have shown that phosphorylation of Y493 is with our results, these data suggest binding ofsrc and syk family essential for Zap-70 activation. PTKs to phosphorylated ITAMs via their SH2 domains may be Finally, the notion that activation of Zap-70 upon binding to a common mechanism for activation of kinase function. the ; chain relieves an allosteric inhibition of the kinase In apparent contrast to our in vitro results, Madrenas et al. domain by the tandem SH2 domains cannot be categorically (30) and van Oers et al. (31) have shown that in thymocytes dismissed. However, it is likely that the contribution from Zap-70 is constitutively associated with phosphorylated ; and allosteric interaction is minor, given that the binding to CD3 s chains, yet Zap-70 and T cells are not always in an bisphosphorylated ITAMs and phosphorylated ; have no activated state. One explanation for this may be that in the T significant activation effect on Zap-70 catalysis. If anything, cell there are that regulate the phosphorylation relief of inhibition of kinase activity by the tandem SH2 state, and consequently the activation state of Zap-70. This is domains may be making a small contribution to the overall supported by a recent finding by Plas et al. (32) where they have activation of Zap-70. shown that in the the protein tyrosine phosphatase The system we describe is unique in that we assembled in SHP-1 binds to Zap-70 and decreases Zap-70 kinase activity. vitro purified components of the TCR which allow us to Alternatively, it could be that the stoichiometry of phosphor- investigate the biochemical mechanism for kinase activation of ylated C and Zap-70 in the T cell is different than the 1:1 Zap-70. Our results suggest catalytic activation of Zap-70 by Downloaded by guest on September 28, 2021 12170 Biochemistry: LoGrasso et al. Proc. Natl. Acad. Sci. USA 93 (1996)

trans-phosphorylation from one Zap-70 bound to one mono- 16. Neumeister, E. N., Zhu, Y., Richard, S., Terhorst, C., Chan, A. C. mer of the ; chain to a second Zap-70 bound to the second & Shaw, A. S. (1995) Mo. Cell. Biol. 15, 3171-3178. monomer ofthe dimeric chain. It has been shown (40, 41) that 17. Rowely, R. B., Burkhardt, A. L., Chao, H.-G., Matsueda, G. R. & Bolen, J. B. (1995) J. Biol. Chem. 270, 11590-11594. mutation ofY493 to Phe renders Zap-70 unable to be activated 18. Shiue, L., Zoller, M. J. & Brugge, J. S. (1995) J. Bio. Chem. 270, by lck. It is interesting to speculate that activation of Zap-70 10498-10502. kinase function may be controlled by two separate, or perhaps 19. Wange, R. L., Isakov, N., Burke, T. R., Otaka, A., Rollers, P. P., coordinated, processes. Our data suggest activation of Zap-70 Watts, J. D., Aebersold, R & Samelson, L. E. (1995) J. Biol. upon binding a dimeric form of C. The Y493 mutation studies Chem. 270, 944-948. are indicative of a role for lck in Zap-70 activation. It will be 20. Weissman, A. M., Hou, D., Orloff, D. G., Modi, W. S., Seuanez, interesting to see if there is additive or synergistic activation of H., O'Brien, S. J. & Klausner, R. D. (1988) Proc. Natl. Acad. Sci. Zap-70 by these two mechanisms, or if Zap-70 is activated by USA 85, 9709-9713. 21. Bu, J.-Y., Shaw, A. S. & Chan, A. C. (1995) Proc. Natl. Acad. Sci. two separate, perhaps compensatory mechanisms. Integration USA 92, 5106-5110. of Ick into our experimental system may help to further map 22. Exley, M., Varticovski, L., Peter, M., Sancho, J. & Terhorst, C. out the role Zap-70 plays and detail the sequence of events (1994) J. Biol. Chem. 269, 15140-15146. which occur in T-cell . 23. Panayotou, G., Gish, G., End, P., Truong, O., Gout, I., Dhand, R., Fry, M. J., Hiles, I., Pawson, T. & Waterfield, M. D. (1993) We are grateful to Dr. Richard Cummings and Ms. Song Zheng for Mol. 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