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the Activated TCRtoCoupling Hemopoietic Progenitor Kinase-1 inAdaptor Downstream of Shc) Is Implicated

The Adaptor Protein Gads (Grb2-Related

Friedemann Kiefer and C. Jane McGladeStanley K. Liu, Christian A. Smith, Ruediger Arnold,

http://www.jimmunol.org/content/165/3/1417doi: 10.4049/jimmunol.165.3.1417

2000; 165:1417-1426; ;J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2000 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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The Adaptor Protein Gads (Grb2-Related AdaptorDownstream of Shc) Is Implicated in Coupling HemopoieticProgenitor Kinase-1 to the Activated TCR1

Stanley K. Liu,* 2 Christian A. Smith,2* Ruediger Arnold, † Friedemann Kiefer,† andC. Jane McGlade3*

The hemopoietic-specific Gads (Grb2-related adaptor downstream of Shc) adaptor protein possesses amino- and carboxyl-termi-nal Src homology 3 (SH3) domains flanking a central SH2 domain and a unique region rich in glutamine and proline residues.Gads functions to couple the activated TCR to distal signaling events through its interactions with the leukocyte-specific signalingproteins SLP-76 (SH2 domain-containing leukocyte protein of 76 kDa) and LAT (linker for activated T cells). Expression libraryscreening for additional Gads-interacting molecules identified the hemopoietic progenitor kinase-1 (HPK1), and we investigatedthe HPK1-Gads interaction within the DO11.10 murine T cell hybridoma system. Our results demonstrate that HPK1 induciblyassociates with Gads and becomes tyrosine phosphorylated following TCR activation. HPK1 kinase activity is up-regulated inresponse to activation of the TCR and requires the presence of its proline-rich motifs. Mapping experiments have revealed thatthe carboxyl-terminal SH3 domain of Gads and the fourth proline-rich region of HPK1 are essential for their interaction. Deletionof the fourth proline-rich region of HPK1 or expression of a Gads SH2 mutant in T cells inhibits TCR-induced HPK1 tyrosinephosphorylation. Together, these data suggest that HPK1 is involved in signaling downstream from the TCR, and that SH2/SH3domain-containing adaptor proteins, such as Gads, may function to recruit HPK1 to the activated TCR complex.The Journalof Immunology,2000, 165: 1417–1426.

T he activation of tyrosine kinases is central to the initiationof signaling pathways from diverse extracellular recep-tors. Stimulation of Ag receptors, such as the TCR, results

in rapid activation of protein tyrosine kinases such as Lck, Fyn,and Zap-70 and phosphorylation of the cytoplasmic domain ofreceptor accessory molecules, creating binding sites for Src ho-mology 2 (SH2)4 and phosphotyrosine-binding domain-containingsignaling proteins (1–6). Recruitment of cytoplasmic signalingmolecules to the activated TCR couple receptor activation withmultiple downstream pathways that initiate signaling events re-quired for the transcription of lymphokine genes (2, 5–7). The

critical substrates of TCR-activated protein tyrosine kinases in-clude the SH2 domain-containing leukocyte protein of 76 kDa(SLP-76) (8), the guanine nucleotide exchange factor Vav (9),phospholipase Cg1 (PLC-g1) (10), c-Cbl (11), and the linker foractivation of T cells (LAT) (12–15).

The three members of the Grb2 family of adaptor proteins,Grb2, Grap, and Gads, have also been implicated in the formationof protein complexes at the TCR and are important for linkingspecific pathways, such as Ras to TCR activation. The Grb2 familyof adaptors possess amino- and carboxyl-terminal SH3 domainsthat flank a central SH2 domain (7, 16–21). Gads also has a 120-aaunique region between the SH2 domain and carboxyl-terminalSH3 domain (20). This family of proteins couples tyrosine-phos-phorylated membrane-associated proteins bound by the SH2domain with cytoplasmic proteins associated through the SH3 do-mains (7, 16–22). Grb2 is ubiquitously expressed, while expres-sion of both Grap and Gads is restricted to hemopoietic cells (18–20, 23–25). It has been proposed that Grb2 and Grap may serveoverlapping functions, since their SH2 and SH3 domains appear tobind to similar proteins in T cells and since Grap null mice displayno apparent phenotype (26). In contrast, Gads is likely to have aunique function because it binds to distinct SH3 domain targets(20, 22).

The function of the Grb2 adaptor is best defined in the contextof growth factor receptor signaling, where it couples activated re-ceptors with downstream effector pathways, such as the evolution-arily conserved pathway involved in Ras activation (16, 26–34).The SH2 domain of Grb2 binds to activated growth factor recep-tors either directly or indirectly via the docking protein Shc, lo-calizing Grb2 and the stably associated Ras guanine nucleotideexchange factor, Sos, to the membrane, resulting in the activationof Ras (28–32, 34, 35). Within T cells, Grb2 is proposed to coupleTCR engagement to Ras activation through the recruitment of

*Department of Medical Biophysics and The Arthur and Sonia Labatt Brain TumorResearch Center, Hospital for Sick Children Research Institute, University of To-ronto, Toronto, Ontario, Canada; and†Max Planck Institute for Physiological andClinical Research W. G. Kerckhoff Institute, Bad Nauheim, Germany

Received for publication January 28, 2000. Accepted for publication May 19, 2000.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby markedadvertisementin accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by the Cancer Research Society, Inc., Canada, and theMedical Research Council of Canada. S.K.L. is a research student of the NationalCancer Institute of Canada supported with funds provided by the Terry Fox Run.C.J.M. is a Research Scientist of the National Cancer Institute of Canada supportedwith funds from the Canadian Cancer Society.2 S.K.L. and C.A.S. contributed equally to this work.3 Address correspondence and reprint requests to Dr. C. Jane McGlade, The Arthurand Sonia Labatt Brain tumor Research Center, Hospital for Sick Children, 555 Uni-versity Avenue, Toronto, Ontario, Canada M5G 1X8. E-mail address: jmcglade@sickkids.on.ca4 Abbreviations used in this paper: SH, Src homology; PLC, phospholipase C; Gads,Grb2-related adaptor downstream of Shc; Grap, Grb2-related adaptor protein; SAPK,stress-activated protein kinase; GCK, germinal center kinase; GLK, GCK-like kinase;GCKR/KHS, GCK-related kinase/kinase homologous to SPS1/Ste20; SLP-76, SH2domain-containing leukocyte protein of 76 kDa; LAT, linker of activated T cells;HPK1, hemopoietic progenitor kinase 1; mHPK1, murine HPK1; pY, phosphoty-rosine; HA, hemagglutinin; PVDF, polyvinylidene difluoride; MBP, myelin basicprotein.

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Grb2-Sos complexes to tyrosine-phosphorylated LAT, a mem-brane-bound linker protein (14, 15, 36). By analogy, Grap mayfunction to amplify TCR activation of Ras through recruitment ofadditional pools of Sos to membrane-associated LAT.

Gads also inducibly associates with LAT in T cells, but unlikeGrb2 and Grap, it does not form complexes with Sos (20, 22).Instead, Gads is bound to the SLP-76 adaptor protein, which playsa critical role in signaling from both pre-TCR and mature TCR (8,37–39). Gads synergizes with SLP-76 to augment both NF-AT andIL-2 activation, and this augmentation is dependent on a functionalGads SH2 domain (22). Hence, through its association withSLP-76 and LAT, Gads appears to play a central role in signalingfrom the activated TCR (22, 24, 25).

To date, SLP-76 is the only Gads SH3 domain-binding proteinthat has been identified. In an effort to identify additional signalingeffectors downstream of Gads, we used an expression screeningstrategy and identified hemopoietic progenitor kinase-1 (HPK1) asa Gads-binding protein. HPK1 belongs to a family of Ste20 ho-mologous serine/threonine kinases that includes germinal centerkinase (GCK), GCKR/KHS (GCK-related kinase/kinase homolo-gous to SPS1/Ste20), and GLK (GCK-like kinase) (40). The fam-ily is defined by their N-terminally located kinase domain and theregulatory C-terminal domain, and all have been implicated asupstream effectors in activation of the stress-activated protein ki-nase (SAPK/c-Jun NH2-terminal kinase) pathway (40–44). Herewe show that HPK1 is activated by the TCR, that Gads and HPK1interact in vivo, and that Gads can function to link HPK1 to sig-naling from the activated TCR complex.

Materials and MethodsExpression library screen

A 16-day mouse embryo expression library (Novagen, Madison, WI) wasplated and protein expression induced according to the manufacturer’s di-rections. The nitrocellulose filters were washed in TBST (20 mM Tris-base(pH 7.5), 150 mM NaCl, and 0.05% (v/v) Tween 20) and blocked at 4°Cin blocking buffer (1% nonfat milk powder (w/v) and 1 mM DTT in TBST)for 2 h. Radiolabeled GST-Gads fusion protein was prepared by incubating15 mg of GST-Gads fusion protein bound to glutathione-Sepharose beadswith heart muscle kinase (0.9 U/ml; Sigma, St. Louis, MO) and [g-32P]ATP(0.7mCi/ml) in 170ml of kinase buffer (20 mM Tris-HCl (pH 7.5), 100 mMNaCl, 12 mM MgCl2, and 1 mM DTT) at 4°C for 40 min. The beads werewashed five times with PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mMNa2HPO4, and 1.4 mM KH2PO4, pH 7.4), and the radiolabeled GST-Gadsfusion protein was eluted with 300ml of elution buffer (100 mM Tris-HCl(pH 8.0), 120 mM NaCl, and 20 mM reduced glutathione (Sigma)), fol-lowed by an additional elution with 400ml of elution buffer. The filterswere incubated overnight with radiolabeled GST-Gads fusion protein inblocking buffer at 4°C and washed four times for 10 min each time inTBST, and the filters were exposed to film.

Plasmids and Abs

The pEF-FLAG wild-type Gads and mutant Gads (N*/C, N/C*, N*/C*,and SH2*) constructs were previously described (22). The GST-Gads fu-sion constructs and polyclonal anti-Gads Ab (H1-A) were used as previ-ously described (20). The pcDNA3-HPK1:HA and pcDNA3-KDHPK1:HA contain the full-length murine wild-type and kinase-dead HPK1cDNA, respectively, with an in-frame fusion of a triple HA epitope tag.Deletion mutants lacking the proline-rich regions (P1, P2, and P4), indi-vidually or in combination, were generated as follows. AKpnI fragmentcomprising 1233 bp of murine HPK1 (mHPK1) cDNA containing the pro-line-rich motifs P1 to P4 was subcloned into pBluescript, giving rise to theplasmid pB-mHPK1:KpnI. Deletion mutants of aa 309–311 (DP1), aa 392–401 (DP2), and aa 467–471 (DP4) were generated by PCR. PCR productscontaining the desired mutations were reinserted into pB-mHPK1:KpnI,and then reintroduced into pcDNA3-HPK1:HA by swapping the corre-spondingKpnI fragments.

Synthetic peptides representing the N-terminus (MALVDPDIFNKDPREHYD) and C-terminus (TRPTDDPTAPSNLYIQE) of mHPK1 werecoupled to keyhole limpet hemocyanin via a carboxyl-terminal cysteineresidue and used to immunize rabbits and generate antisera 2 (N-terminus)

and 7 (C terminus). The anti-HPK1 antiserum 5 has been previously de-scribed (41). For anti-HPK1 immunoprecipitations, a mixture of 2.5ml ofHPK1 2 antisera and 2.5ml of HPK1 5 antisera was used. A 1/500 dilutionof HPK1 7 antisera was used for immunoblotting. Monoclonal anti-phosphotyrosine Ab (4G10; Upstate Biotechnology, Lake Placid, NY) wasused at a dilution of 1/1000 for immunoblotting. Four micrograms of anti-HA mAb (12CA5; Roche, Indianapolis, IN) was used for immunoprecipi-tations. Ten micrograms of soluble monoclonal anti-mouse CD3 (145–2C11; PharMingen, San Diego, CA) was used for stimulations.

Cell culture and transient transfections

COS1 cells were maintained in DMEM supplemented with 10% (v/v) FBS,200 mM L-glutamine, 5 U/ml penicillin C, and 5 mg/ml streptomycin sul-fate. Ten micrograms of wild-type or proline-deficient HPK1 plasmidswere transfected into a 100-mm dish of 50% confluent COS1 cells withlipofectin reagent (Life Technologies, Gaithersburg, MD) according to themanufacturer’s instructions, and cell lysates were prepared for in vitrobinding experiments as detailed below. The murine T cell hybridoma,DO11.10, was maintained in RPMI 1640 supplemented with 10% FBS(v/v), 200 mM L-glutamine, 5 U/ml penicillin C, 5 mg/ml streptomycinsulfate, and 55mM 2-ME. For electroporations, 20 million cells were re-suspended in 400ml of RPMI 1640 and electroporated with a Gene Pulser(Bio-Rad, Hercules, CA) set at 250 V and 960mF. Optimal expression oftransfected constructs was obtained with 15mg of pcDNA3-HPK1:HA and45 mg of pEF-FLAG-Gads constructs. Primary murine T cells were iso-lated from dissected lymph nodes and maintained in RPMI for 4 h beforestimulation with anti-CD3.

In vitro binding experiments

GST-Gads wild-type and mutant fusion proteins were prepared as previ-ously described (20), and purified proteins were quantified by Coomassiestaining. Wild-type or proline mutant pcDNA3-HPK1:HA-transfectedCOS1 cells were lysed in 1 ml of PLC lysis buffer (50 mM HEPES (pH7.4), 150 mM NaCl, 10% (v/v) glycerol, 1% (v/v) Triton X-100, 1.5 mMMgCl2, 1 mM EGTA, 10 mM NaPPi, 100 mM NaF, 1 mM Na3VO4, andComplete protease inhibitors (Roche)), and the cellular lysates were clar-ified by centrifugation at 14,000 rpm for 10 min at 4°C. Five hundredmicroliters of lysate was mixed with 2mg of GST and either wild-typeGST-Gads or GST-Gads mutants fusion protein conjugated to glutathione-Sepharose beads for 1 h at 4°C. The beads were washed five times withNonidet P-40 lysis buffer (50 mM HEPES (pH 7.4), 150 mM NaCl, 2 mMEDTA, 100 mM ZnCl2, 1% (v/v) Nonidet-P40, 10% (v/v) glycerol, andComplete protease inhibitors (Roche)) and 1 mM DTT, resuspended inSDS-Laemmli sample buffer, resolved on a 10% SDS-PAGE gel (NOVEX,San Diego, CA), and electrophoretically transferred to an Immobilon-Pmembrane (Millipore, Bedford, MA). The membranes were then immuno-blotted with anti-HPK1 7 antiserum.

Immunoprecipitations and Western blotting analysis

DO11.10 or lymph node-derived T cells were prewarmed at 37°C for 10min, left unstimulated, or stimulated with soluble anti-CD3 Ab for 2 minat 37°C, followed by the addition of 700ml of an ice-cold phosphataseinhibitor mix (100 mM NaF, 10 mM NaPPi, and 1 mM Na3VO4 in PBS,pH 7.4). Cells were collected by centrifugation and lysed in 1 ml of PLClysis buffer. The clarified lysates were incubated with the indicated Ab and50 ml of a 20% (v/v) protein A-Sepharose bead slurry (Sigma) for 90 minat 4°C. The immune complexes were washed five times with Nonidet P-40lysis buffer, eluted in SDS-Laemmli sample buffer, and separated on a 10%SDS-PAGE gel. Proteins were electrophoretically transferred to an Immo-bilon-P membrane and incubated in BLOTTO (5% nonfat milk powder(w/v) in TBST) or 1% (w/v) BSA in TBST (for anti-phosphotyrosine blot-ting), for 30 min before the addition of primary Ab for 1 h at room tem-perature. The membranes were washed three times for 10 min each time inTBST and were incubated at room temperature for 1 h with the appropriatesecondary Ab conjugated to HRP. Membranes were washed three times inTBST and developed using enhanced chemiluminescence (Amersham-Pharmacia Biotech, Piscataway, NJ). Where necessary, the membraneswere stripped (according to the manufacturer’s instructions) and reblotted.

In vitro kinase assays

DO11.10 cells or lymph node-derived T cells (53 106 cells/sample) wereleft unstimulated or were stimulated with soluble anti-CD3 Ab for 5 min at37°C and lysed, and anti-HPK1 immunoprecipitates were performed asoutlined above. The immunoprecipitates were washed three times with 140mM NaCl, 50 mM Tris-HCl (pH 8.0), 5 mM EDTA, and 1% Nonidet P-40(v/v), twice with 140 mM NaCl, 50 mM Tris-HCl (pH 8.0), and 5 mM

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EDTA, and once with 50 mM Tris-HCl (pH 7.5), 8 mM MgCl2, 2 mM MnCl2,and 1 mM DTT, and kinase reactions were performed in 20ml of 50 mMTris-HCl (pH 7.5), 10 mM MgCl2, and 1 mM EDTA at 30°C for 15 min in thepresence of 10mCi of [g-32P]ATP and 20mg of myelin basic protein (MBP)as outlined previously (41). The kinase reactions were stopped by adding 23SDS-Laemmli sample buffer and boiled for 5 min, and proteins were resolvedby SDS-PAGE. The proteins were electrophoretically transferred to Immo-bilon-P membrane, and the incorporated radiolabeled phosphate was assessedby PhosphorImager analysis using the ImageQuant program (Molecular Dy-namics, Sunnyvale, CA). To confirm that equivalent levels of HPK1 werepresent in each kinase assay, the membrane was subsequently immunoblottedwith anti-HPK1 antisera as described above.

ResultsIdentification of HPK1 as a Gads-binding protein in activatedT cells

To identify Gads-binding proteins, radiolabeled GST-Gads fusionprotein was used to screen an embryonic day 16 mouse expressionlibrary. A 2.6-kb cDNA clone was isolated from this screen thatencoded the full-length murine hemopoietic progenitor kinase-1(HPK1, GenBank accession no. Y09010).

Since both Gads and HPK1 proteins are expressed in T cells,(22, 41, 45), we examined whether Gads binds to HPK1 in theDO11.10 murine T cell hybridoma. DO11.10 cells were left un-stimulated or were stimulated with anti-CD3 Ab for 2 min, lysateswere prepared, and immunoprecipitations were performed withRaM IgG, anti-Gads, and anti-HPK1 Abs. Anti-phosphotyrosineimmunoblotting revealed the presence of a tyrosine-phosphory-lated protein of;100 kDa in the anti-HPK1 immunoprecipitatefollowing TCR activation (Fig. 1). Tyrosine-phosphorylatedSLP-76 (76 kDa) and LAT (36 kDa), and weaker tyrosine-phos-phorylated proteins of;100, 130, and 180 kDa were detected in

anti-Gads immunoprecipitates from anti-CD3-stimulated DO11.10lysates. The membranes were stripped and reprobed with anti-HPK1 Ab, identifying the 100-kDa phosphoprotein in both anti-HPK1 and anti-Gads immunoprecipitates as HPK1 and demon-strating that HPK1 is inducibly tyrosine-phosphorylated andassociated with Gads in vivo following TCR ligation (Fig. 1). Fol-lowing prolonged exposure of the blot to film, only a very smallamount of HPK1 was detected in Gads immunoprecipitates beforestimulation (data not shown). The identities of the 130- and 180-kDa phosphoproteins present in the anti-Gads immunoprecipitatefrom anti-CD3-stimulated lysate are unknown at this time. Gadswas not detected in anti-HPK1 immunoprecipitates, suggestingthat the anti-HPK1 Abs do not recognize HPK1 when complexedwith Gads. HPK1 was also detected in Gads immunoprecipitatesfrom primary lymph node-derived murine T cells following anti-CD3 stimulation (data not shown).

HPK1 kinase activity is up-regulated by TCR activation andrequires intact proline-rich regions

It has been previously reported that activation of the TGF-b and eryth-ropoietin receptors leads to an increase in HPK1 kinase activity (46,47). In addition, Ling et al. have previously provided evidence thatHPK1 may be involved in regulating IL-2 activation in Jurkat T cells(48). To determine whether HPK1 kinase activity is regulated by TCRengagement, in vitro kinase assays were performed on anti-HPK1immunoprecipitates from unstimulated and anti-CD3-stimulatedDO11.10 hybridoma and primary lymph node-derived T cells, usingMBP as a substrate (Fig. 2A). The level of phosphorylated MBP in-creased by 2-fold, on the average, following TCR activation in bothDO11.10 and lymph node-derived T cells, indicating that HPK1 ki-nase activity is up-regulated by TCR activation.

HPK1 possesses four proline-rich regions (P1, P2, P3, and P4),and three of these regions (P1, P2, and P4) have been shown tobind to SH3 domains in vitro (41, 48, 49). It has been demonstratedthat SH2/SH3 domain-containing adaptor proteins can associatewith HPK1, and therefore may serve to couple activated receptorswith the downstream activation of HPK1 (48, 49). To examine theeffect of loss of the proline-rich regions on TCR-dependent acti-vation of HPK1 kinase activity, an HPK1 mutant was generated inwhich the first (P1; aa 309–311), second (P2; aa 392–401), andfourth (P4; aa 467–471) SH3 domain-binding consensus motifswere deleted in combination (DP1/2/4; Fig. 4A). The third (P3; aa430–441) motif was not included in the deletion because of itspoor fit to the SH3 domain-binding consensus motif, and sinceAnafi et al. (49) previously demonstrated that a P3 peptide wasunable to compete recombinant SH3 domain binding to HPK1.HA-epitope tagged wild-type, kinase-dead, andDP1/2/4 HPK1 con-structs were transiently expressed in DO11.10 cells, the cells were leftunstimulated or were stimulated with anti-CD3 and lysed, and anti-HA immunoprecipitation was performed, followed by kinase assaysusing MBP as substrate. Following TCR activation, an;2-fold in-crease in HPK1 kinase activity was observed with the wild-typeHPK1, while a marginal increase was observed for the kinase-deadHPK1 (Fig. 2B). The induced kinase activity of theDP1/2/4 HPK1mutant was markedly reduced following TCR ligation compared withthat of wild-type HPK1. The defect in activation ofDP1/2/4 HPK1 invivo is probably due to its inability to couple to the activated TCRrather than to a structural defect that affects the kinase domain, sincethe basal in vitro kinase activity of this mutant is equivalent to that ofwild-type HPK1.5 Therefore, the proline-rich regions of HPK1 and

5 R. Arnold and F. Kiefer. Hematopoietic progenitor kinase 1 (HPK1) activatesNF-kB and promotes apoptosis after growth factor withdrawal.Submitted for publi-cation.

FIGURE 1. HPK1 is tyrosine phosphorylated and inducibly associateswith Gads following TCR activation. DO11.10 murine T cells (5.03 107

cell equivalents/condition) were left unstimulated or stimulated with sol-uble anti-CD3 Ab for 2 min, lysed, and immunoprecipitated with a nega-tive control rabbit anti-mouse Ab (RaM), anti-HPK1 (HPK1), and anti-Gads Ab (Gads). Following SDS-PAGE separation and electroblotting to aPVDF membrane, phosphorylated proteins were detected in Western blotanalysis using anti-phosphotyrosine Ab (a-pY; upper panel). The coim-munoprecipitating phosphoproteins p100, p76 (SLP-76), and p36 (LAT)are indicated with arrowheads. The blot was stripped, the top half wasblotted with anti-HPK1 Ab (a-HPK1; middle panel), and the lower halfwith anti-Gads Ab (a-Gads;lower panel). Bands corresponding to HPK1and Gads are denoted by an arrow.

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probably SH3 domain-containing adaptors are important in couplingregulation of HPK1 activity to TCR activation.

The carboxyl-terminal SH3 domain of Gads mediates binding tothe fourth proline-rich region of HPK1

To map the domain of Gads responsible for interacting with HPK1,recombinant full-length GST-Gads fusion proteins with inacti-vated SH3 or SH2 domains were assayed for their ability to pre-cipitate HPK1 from transfected COS1 cells. Inactivation of bothSH3 domains (N*/C*) or inactivation of the carboxyl-terminalSH3 domain (N/C*) eliminated the interaction between Gads andHPK1, whereas mutation of the SH2 (SH2*) domain had no effecton the ability of Gads to precipitate HPK1 in vitro (Fig. 3). Similarexperiments performed in DO11.10 cells also show that the car-boxyl-terminal SH3 domain is required for binding to endogenousHPK1 following activation of the TCR (data not shown).

Since the Gads-HPK1 association was an SH3 domain-mediatedinteraction, we reasoned that the Gads binding site was probablywithin one of the four proline-rich regions (P1–P4) of HPK1. Ad-ditional HPK1 mutants were generated in which the core SH3domain-binding consensus motifs were deleted individually or incombination (Fig. 4A). The binding of GST-Gads fusion protein toHPK1 mutants that possessed individual or combined deletions ofthe first, second, and fourth proline-rich regions was assayed; thethird proline-rich region was not removed due to reasons describedabove (Fig. 4B). Deletion of the first or second proline-rich regionof HPK1, either individually or together, did not eliminate theGads-HPK1 interaction. However, all HPK1 mutants that lackedthe fourth proline-rich region of HPK1 were no longer precipitatedby GST-Gads, suggesting that the major Gads binding site onHPK1 is within the fourth proline-rich region (Fig. 4B). Deletionof the second proline-rich region reduced binding to HPK1,whereas loss of the fourth proline-rich region abolished the inter-action. Together, these results suggest that the while the fourthproline-rich region is critical for association between Gads andHPK1, the second proline-rich region may partially contribute tothe interaction. To confirm the role of the Gads SH3 domains andthe HPK1 proline-rich regions in the Gads-HPK1 interaction invivo, full-length epitope-tagged Gads and HPK1 cDNAs with mu-tations in the domains important for the interaction were cotrans-fected into DO11.10 cells. Wild-type HA-HPK1 was efficientlycoimmunoprecipitated with FLAG-Gads; however, deletion of allthree proline-rich regions (P1/P2/P4) or the P4 region alone re-sulted in loss of HPK1-Gads association (Fig. 5A, left panel). Aninactivating mutation in the Gads SH2 domain (Gads-SH2*) hadno effect on the ability of FLAG-Gads to coprecipitate HA-HPK1,while mutation of the SH3 domains of Gads (Gads-N*/C*) abol-ished the interaction (Fig. 5A,right panel). Equivalent expressionof the HA-HPK1 and FLAG-Gads proteins was confirmed byWestern blots of whole cell lysates with anti-HA and anti-FLAGAbs (Fig. 5B).

Efficient HPK1 tyrosine phosphorylation requires the presenceof the second or fourth proline-rich region

Recruitment of HPK1 to the proximity of activated receptors bySH2/SH3 domain-containing adaptor proteins has been shown tofacilitate the subsequent tyrosine phosphorylation of HPK1 in re-sponse to activation of the epidermal growth factor and erythro-poietin receptors (47, 49). To test whether the Gads-binding re-gion, P4, is required for HPK1 tyrosine phosphorylation followingTCR activation, we transiently expressed HPK1 mutants lacking

FIGURE 3. The Gads carboxyl-terminal SH3 domain is required forHPK1 interaction. Lysates were prepared from pcDNA3-HPK1:HA trans-fected COS1 cells, normalized for equal protein concentrations, and incu-bated with immobilized GST or GST fusion proteins of wild-type Gads(wt) or Gads mutants containing inactivating point mutations in both SH3domains (N*/C*), the amino-terminal SH3 domain (N*/C), the carboxyl-terminal SH3 domain (N/C*), or the SH2 domain (SH2*). The proteincomplexes were separated on an SDS-PAGE gel, electroblotted ontoa PVDF membrane, and immunoblotted with anti-HPK1 Ab (a-HPK1;upper panel). HPK1 is denoted by an arrow. To confirm that equal amountsof GST and GST-Gads fusion proteins were used, the membrane wasstained with Coomassie brilliant blue (lower panel). GST-Gads wild-typeand mutant fusion proteins (GST-Gads) and GST are denoted by arrows.

FIGURE 2. A, HPK1 kinase activity is up-regulated following TCR activation. DO11.10 cells, starved overnight in 0.5% FCS containing RPMI 1640medium, or freshly isolated lymph node-derived murine T cells were left unstimulated or were stimulated (53 106 cell equivalents/condition) with solubleanti-CD3 Ab for 5 min. Cells were lysed and immunoprecipitated with anti-HPK1 Ab (IP:a-HPK1), and the immune complexes were subjected to in vitrokinase assays, using MBP as a substrate (upper panel). The proteins were resolved by SDS-PAGE, transferred to a PVDF membrane, and incorporatedradiolabeled phosphate was quantitated by PhosphorImager analysis; these values are depicted in chart format (lower panel) and are representative of resultsobtained in at least three independent experiments. The membrane was immunoblotted with anti-HPK1 Ab to confirm that equivalent levels of HPK1 werepresent (middle panel). Representative autoradiographs of the MBP phosphorylation and anti-HPK1 immunoblotting are shown. The bands correspondingto MBP and HPK1 are denoted.B, HPK1 kinase activity up-regulation requires intact proline-rich regions (PRR). DO11.10 murine T cells (2.03 107) weretransiently transfected by electroporation with 20mg of pcDNA3-HPK1:HA wild-type (wt), pcDNA3-KD HPK1:HA (KD) or pcDNA3-triple PRR(DP1/2/4):HA lacking the first, second, and fourth PRR. Twenty-four hours postelectroporation, the cells were left unstimulated or were stimulated withsoluble anti-CD3 Ab for 5 min at 37°C in RPMI 1640 medium. Cellular lysates were prepared and immunoprecipitated with anti-HA Ab (IP:a-HA), andimmune complexes were subjected to in vitro kinase assays, using MBP as a substrate (upper panel). The proteins were resolved by SDS-PAGE, transferredto a PVDF membrane and incorporated radiolabeled phosphate was quantitated by PhosphorImager analysis; these values are depicted in chart format(lower panel) and are representative of at least three independent experiments. The membrane was immunoblotted with anti-HPK1 Ab to confirm thatequivalent levels of HPK1 were present (middle panel). Representative autoradiographs of the MBP phosphorylation and anti-HPK1 immunoblotting areshown. The bands corresponding to MBP and HPK1 are denoted.

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the various proline-rich regions in DO11.10 cells and examined thetyrosine phosphorylation status of these HPK1 mutants followinganti-CD3 stimulation (Fig. 6). Loss of the second or fourth proline-rich region in HPK1 led to a substantial decrease in the tyrosine-

phosphorylation status of HPK1 following TCR ligation, whileloss of the first proline-rich region had no effect. Together, thesedata suggest that the second and fourth proline-rich regions areimportant for the efficient tyrosine phosphorylation of HPK1.

FIGURE 4. A, Structure of SH3 binding site mutants in HPK1. The cluster of four proline-rich motifs located in the central portion of HPK1 andsequences of the individual polyproline stretches are depicted. Three of these motifs (P1, P2, and P4) conform to the sequence consensus for SH3 bindingsites in class II orientation (underlining arrow). All four motifs are schematically depicted as left-handed polyproline type II helexes lining the relativelyconserved hydrophobic binding pocket of an SH3 domain. P1, P2, and P4 fit the sequence consensus very well and are shown in negative orientation, P3fits the sequence consensus poorly and is shown in positive orientation (52, 53). The binding positions were assigned as follows: motif P1, C-R(P-3) R(P-2)P(P-1) I(P0) A(P1) P(P2) P(P3)-N; motif P2, C-K(P-3) P(P-2) P(P-1) L(P0) P(P1) P(P2) P(P3)-N; motif P3, N-P(P-2) R(P-1) P(P0) G(P1) P(P2) P(P3)-C; andmotif P4, C-R(P-3) P(P-2) P(P-1) V(P0) L(P1) P(P2) P(P3)-N. Positions requiring proline residues, P-1/P2 in negative orientation and P0/P3 in positiveorientation are marked by asterisks. The SH3 domain binding sites P1, P2, and P4 were removed singly and in combination by introduction of the aminoacid deletions depicted in the diagram at thelower partof the figure.B, The fourth proline-rich region (PRR) of HPK1 is required for Gads binding. Lysateswere prepared from COS1 cells transfected with 15mg of wild-type pcDNA3-HPK1:HA (wt) or mHPK1 mutant constructs with deletions of the triple PRR(DP1/2/4), the first PRR (DP1), the second PRR (DP2), the fourth PRR (DP4), the first and second PRR (DP1/2), and the first and fourth PRR (DP1/4).Lysates were incubated with immobilized GST or GST-Gads wild-type fusion proteins. The protein complexes were separated on a SDS-PAGE gel,electroblotted onto a PVDF membrane, and immunoblotted with anti-HPK1 Ab (a-HPK1; upper panel). To confirm equivalent protein expression of themHPK1 mutants, whole cell lysates were separated on a SDS-PAGE gel in parallel, transferred to a PVDF membrane, and Western blotted with anti-HPK1Ab (lysates;middle panel). HPK1 is denoted with an arrow. The membrane from thetop panelwas stained with Coomassie brilliant blue to confirm thatequal amounts of GST or GST-Gads fusion proteins were used in the in vitro binding experiments (lower panel). GST and GST-Gads fusion proteins aredenoted with arrows.

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Expression of mutant Gads can inhibit the tyrosinephosphorylation of HPK1

Since a Gads-HPK1 complex was detected in anti-CD3-stimulatedDO11.10 cells, we examined the effect of overexpression of Gadsmutant proteins on the tyrosine phosphorylation of HPK1 follow-ing TCR activation. Gads mutant proteins that possessed inacti-vated SH3 or SH2 domains were transiently coexpressed with HA-tagged HPK1 in DO11.10 cells, and the relative tyrosine

phosphorylation level of HPK1 following anti-CD3 stimulationwas assessed (Fig. 7). Expression of wild-type Gads or mutantGads with inactivated SH3 domains (N*/C*) did not alter the ty-rosine phosphorylation level of HPK1 relative to the control level.However, the expression of mutant Gads with an inactivated SH2domain (SH2*) appreciably reduced tyrosine phosphorylation ofHPK1, suggesting that Gads may be directly involved in couplingHPK1 to tyrosine kinases that are activated by the TCR.

FIGURE 6. The second and fourth proline-rich regions of HPK1 mediate efficient tyrosine phosphorylation of HPK1 following TCR stimulation.DO11.10 murine T cells (2.03 107) were left untransfected (mock) or were transiently transfected by electroporation with 15mg of pcDNA3-HPK1:HAwild-type or mHPK1 mutant constructs with deletion of the first PRR (DP1), the second PRR (DP2), the fourth PRR (DP4), or the triple PRR (DP1/2/4).Twenty-four hours postelectroporation, the cells were left unstimulated or were stimulated with soluble anti-CD3 Ab for 2 min at 37°C in RPMI 1640medium (with or withouta-CD3). Cellular lysates were prepared and immunoprecipitated with anti-HA Ab (IP:a-HA), separated on an SDS-PAGE gel,and electroblotted onto a PVDF membrane. The level of tyrosine-phosphorylated HA-tagged mHPK1 was detected by Western blot analysis with anti-phosphotyrosine Ab (Blot:a-pY; upper panels). To confirm that equal amounts of HPK1 were immunoprecipitated, the blots were stripped and reprobedwith anti-HPK1 Ab (Blot:a-HPK1; lower panels). The bands corresponding to HPK1 are denoted with arrows. The data are representative of fourindependent experiments.

FIGURE 5. Mutation of the Gads SH3 domains or the HPK1 P4 region disrupt Gads-HPK1 binding in vivo.A, DO11.10 murine T cells (2.03 107)were transiently cotransfected by electroporation with 15mg of pcDNA3-HPK1:HA (HA HPK1) or HPK1 mutants (DP4 andDP1, 2, 4)- and 45mgpEF-FLAG-Gads wild-type (wt;left panel) or with pcDNA3-HPK1: HA (HA HPK1) and pEF-FLAG-Gads wild-type (wt) or Gads mutants (SH2* andN*/C*) as indicated. Cellular lysates were prepared, and Gads proteins were immunoprecipitated with anti-FLAG Ab (IP:a-FLAG), separated on anSDS-PAGE gel, and electroblotted onto a PVDF membrane. Coimmunoprecipitating HA-tagged mHPK1 was detected by Western blot analysis withanti-HA Ab (Blot:a-HA). B, To confirm equivalent protein expression of the mHPK1 and Gads mutant constructs, whole cell lysates were separated ona SDS-PAGE gel in parallel, transferred to a PVDF membrane, and Western blotted with anti-HA Ab (upper panel) or anti-FLAG Ab (lower panel).

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DiscussionIn this report we have identified HPK1 as a Gads-binding proteinand have demonstrated that Gads inducibly associates with HPK1following the activation of the TCR. Furthermore, we have foundthat TCR cross-linking results in HPK1 activation in both DO11.1murine hybridoma and primary murine T cells. Our studies alsoreveal that a small proportion of HPK1 becomes rapidly tyrosine-phosphorylated following TCR activation. The proline-rich motifs(P1, P2, and P4) of HPK1 are involved in facilitating both HPK1kinase activation and tyrosine phosphorylation in response to TCRengagement. The Gads-HPK1 interaction is mediated by the car-

boxyl-terminal SH3 domain of Gads and aa 467–471 (P4) ofHPK1. Expression of a Gads SH2 mutant can inhibit the tyrosinephosphorylation of HPK1, suggesting that in T cells, Gads func-tions to link HPK1 to the activated TCR complex.

Substantial research focused on elucidating the pathways down-stream of HPK1 has led to a common model that HPK1 initiates akinase cascade involving several intermediates including mixedlineage kinase 3 (41), MEKK1 (42), and TGF-b-activated kinase 1(50), which can all phosphorylate and activate SAPK/ERK kinase1 leading to the activation of SAPK. In contrast, less is knownabout signaling events upstream of HPK1 activation. Severalgroups have described the association of SH2/SH3 domain-con-taining adaptor proteins, including Crk, CrkL, and Grb2 withHPK1, and have implicated these adaptors in connecting HPK1 toactivated receptors (45, 48, 49). Additionally, it has been previ-ously demonstrated that expression of the HPK1 proline-rich re-gions in Jurkat T cells prevents association with Crk and Grb2adaptor proteins and disrupts IL-2 activation (48). We have foundthat the Gads adaptor protein inducibly associates with HPK1 in Tcells, and that HPK1 kinase activity is regulated by TCR activa-tion, which is dependent on the presence of its proline-rich regionsfor efficient activation. Taken together, this supports a model forHPK1 kinase regulation in T cells, where SH2/SH3 domain-con-taining adaptor proteins such as Grb2, Crk, CrkL, and Gads areable to recruit HPK1 to the proximity of the TCR, thus facilitatingsubsequent activation of the HPK1 kinase. These adaptor proteinsprobably provide a means of coupling the activated TCR complexto kinase cascades downstream of HPK1, which may ultimatelyregulate the induction of a downstream effector response.

HPK1 has been previously identified as a substrate for tyrosinekinases. In experiments using ectopically expressed HPK1 inCOS1 cells, the activated epidermal growth factor receptor, plate-let-derived growth factor receptor, and constitutively activated cy-toplasmic tyrosine kinases v-Src and v-Fps induce the tyrosinephosphorylation of HPK1 (49). Our studies provide the first reportthat endogenous HPK1 is tyrosine phosphorylated following TCRactivation. It is not known whether tyrosine phosphorylation ofHPK1 directly regulates its kinase activity, although based uponthe small proportion of tyrosine-phosphorylated HPK1 observed inanti-HPK1 immunoprecipitates from TCR-activated cells, thiswould appear unlikely. At this time, the functional significance ofHPK1 tyrosine phosphorylation, as observed in response to a widevariety of activated receptors and cytoplasmic tyrosine kinases,remains elusive.

Within the proline-rich region, murine HPK1 harbors three con-sensus SH3 domain binding sites, and human HPK1 potentiallyhas four. One of the motifs in mHPK1 (P2) and two in humanHPK1 conform with the binding consensus for Crk proteins,PxLPxK (where x is any amino acid) (41, 45, 49). The remainingtwo motifs in mHPK1 (P1, P4) have arginine at the P-3 specificityposition, which is usually preferred by Grb2 SH3 domains (41,49). We have demonstrated that the Gads carboxyl-terminal SH3domain preferentially interacts with the P4 motif. Furthermore, ourresults indicate that P2 and P4 are required for the efficient tyrosinephosphorylation of HPK1, supporting a model where tyrosinephosphorylation of HPK1 is dependent on the recruitment ofHPK1 to the proximity of activated tyrosine kinases via SH2/SH3domain-containing adaptor proteins. This model is strengthened byour observations that coexpression of a Gads SH2 mutant alsoattenuated the tyrosine phosphorylation of HPK1. This effectmight result from the ability of this mutant to effectively bindHPK1 through an intact SH3 domain, while sequestering it fromthe proximity of the activated TCR by preventing association withproteins such as LAT. In contrast, the Gads SH3 mutant was found

FIGURE 7. An inactivating mutation of the Gads SH2 domain exhibitsa dominant inhibitory effect on HPK1 tyrosine phosphorylation followingTCR stimulation.A, DO11.10 murine T cells (2.03 107) were transientlycotransfected by electroporation with 15mg of pcDNA3-HPK1:HA and 45mg of pEF-FLAG-Gads wild-type (wt) or Gads mutants with an inactivat-ing point mutation in both SH3 domains (N*/C*) or the SH2 domain(SH2*). As a control, 15mg of pcDNA3-HPK1:HA wild-type was trans-fected with 45mg of pEF-FLAG vector. Twenty-four hours postelectro-poration, the cells were left unstimulated or were stimulated with solubleanti-CD3 Ab for 2 min at 37°C in RPMI 1640 medium (with or withouta-CD3). Cellular lysates were prepared and immunoprecipitated with anti-HA Ab (IP:a-HA), separated on an SDS-PAGE gel, and electroblottedonto a PVDF membrane. The level of tyrosine-phosphorylated HA-taggedmHPK1 was detected by Western blot analysis with anti-phosphotyrosineAb (Blot:a-pY; upper panel). To confirm that equal amounts of HPK1were present in the immunoprecipitates, the blots were stripped and re-probed with anti-HPK1 Ab (Blot:a-HPK1; lower panel). The band corre-sponding to HPK1 is denoted by an arrow.B, To compare the expressionof HPK1 and Gads for each transfection condition, whole cell lysates wereseparated on a SDS-PAGE gel in parallel, transferred to a PVDF mem-brane, and Western blotted with anti-HPK1 Ab (Blot:a-HPK1; upperpanel) and anti-Gads (Blot:a-Gads;lower panel). The bands correspondingto HPK1 and Gads are denoted by arrows. These data are representative ofseven independent experiments.

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to have only a marginal effect on HPK1 tyrosine phosphorylation.This is not entirely unexpected, since the Gads SH3 mutant can nolonger interact with HPK1. Tyrosine phosphorylation of HPK1 isnot completely abolished in the presence of the Gads SH2 mutant,suggesting that other SH2/SH3 domain-containing adaptor pro-teins such as Crk, CrkL, and Grb2, probably serve redundant rolesin recruiting of HPK1 to the proximity of the activated TCR com-plex. This redundancy might also be perceived as a mechanism foramplifying the level of HPK1 kinase activation.

Although the interaction of Gads and HPK1 is SH3 domainmediated, we have found that in vivo the association in DO11.1 isnot constitutive, but is induced following TCR activation. The in-ducible nature of the Gads-HPK1 interaction is analogous to theinteractions reported for the Grb2 and Grap adaptor proteins withSos and for Grap with dynamin in activated T cells (13, 19, 51). Inkeeping with those reports, our results suggest that certain eventsmust be initiated by the TCR for the Gads-HPK1 interaction tooccur. For example, phosphorylation of HPK1 or engagement ofthe Gads SH2 domain could induce a conformational change re-sulting in an increased accessibility of the Gads SH3 domain to thefourth proline-rich region of HPK1. In support of the latter,Ravichandran et al. have shown that binding of the Grb2 SH2domain to a Shc-derived phosphopeptide significantly enhancesthe Grb2-Sos interaction (51). Alternatively, TCR activation mayresult in the relocalization of proteins that facilitates their interac-tion. The inducible association of Grb2-family members with Sos,dynamin, and HPK1 suggests that the formation of these signalingcomplexes is highly regulated within T cells.

We have previously described a potential role for Gads in cou-pling TCR activation to the distal activation of NF-AT and IL-2through its interactions with the SLP-76 and LAT adaptor proteins(22). We now propose that Gads also functions in an additionalpathway downstream of the activated TCR that regulates the ki-nase activity and tyrosine phosphorylation of HPK1.

Note: While this manuscript was in revision, Liou et al. (54) pub-lished similar results demonstrating that HPK1 is activated by theTCR.

AcknowledgmentsWe thank Kodi Ravichandran for the DO11.10 T cell hybridoma cells, IanClarke and Michelle French for assistance with isolating primary T cells,and Michelle French and Donna Berry for comments on the manuscript.

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1426 Gads COUPLES HPK1 TO THE ACTIVATED TCR

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