Leukocyte-Specific Adaptor Protein Grap2 Interacts with Hematopoietic

Leukocyte-speci®c adaptor protein Grap2 interacts with hematopoietic progenitor kinase 1 (HPK1) to activate JNK signaling pathway in T lymphocytes

Wenbin Ma1, Chunzhi Xia1, Pin Ling2, Mengsheng Qiu3, Ying Luo4, Tse-Hua Tan2 and Mingyao Liu*,1

1Department of Medical Biochemistry and Genetics, Center for Cancer Biology and Nutrition, Institute of Biosciences and Technology, Texas A&M University System Health Science Center, 2121 W. Holcombe Blvd., Houston, Texas, TX 77030, USA; 2Department of Immunology, Baylor College of Medicine, Houston, Texas, TX 77030, USA; 3Department of Anatomical Sciences and Neurobiology, School of Medicine, University of Louisville, Louisville, Kentucky, KY 40202, USA; 4Shanghai Genomics, Inc., Zhangjiang Hi-Tech Park, Pudong, Shangai 201204, P.R.C.

Immune cell-speci®c adaptor proteins create various Introduction combinations of multiprotein complexes and integrate signals from cell surface receptors to the nucleus, Activation of resting T cells through the T-cell antigen modulating the speci®city and selectivity of intracellular receptor triggers a cascade of intracellular signaling signal transduction. Grap2 is a newly identi®ed adaptor events that lead to enhanced gene transcription, protein speci®cally expressed in lymphoid tissues. This cellular dierentiation and proliferation (Cantrell, protein shares 40 ± 50% sequence homology in the SH3 1996; Weiss and Littman, 1994; Chan and Shaw, and the SH2 domain with Grb2 and Grap. However, the 1996; Crabtree and Clipstone, 1994; Wange and Grap2 protein has a unique 120-amino acid glutamine- Samelson, 1996). Although components of the T-cell and proline-rich domain between the SH2 and C- receptor complex have no intrinsic kinase activity, the terminal SH3 domains. The expression of Grap2 is earliest detectable biochemical events following stimu- highly restricted to lymphoid organs and T lymphocytes. lation of T cell receptor (TCR) are the activation of In order to understand the role of this speci®c adaptor several protein tyrosine kinases (PTKs), resulting in protein in immune cell signaling and activation, we transient tyrosine phosphorylation of numerous in- searched for the Grap2 interacting protein in T tracellular proteins. Two types of cytoplasmic PTKs lymphocytes. We found that Grap2 interacted with the are known to catalyze these phosphorylations. The Src hematopoietic progenitor kinase 1 (HPK1) in vitro and family PTKs, Lck and Fyn, associate with the in Jurkat T cells. The interaction was mediated by the cytoplasmic portions of the CD3/z and CD4/CD8 co- carboxyl-terminal SH3 domain of Grap2 with the second receptors, respectively, and phosphorylate the paired proline-rich motif of HPK1. Coexpression of Grap2 with tyrosine residues within the immunoreceptor tyrosine- HPK1 not only increased the kinase activity of HPK1 in based activation motifs (ITAM) of the TCRz and CD3 the cell, but also had an additive eect on HPK1 cytoplamic tails. The ZAP-70 tyrosine kinase is mediated JNK activation. Furthermore, over expression recruited to the activated complex by binding phos- of Grap2 and HPK1 induced signi®cant transcriptional phorylated ITAM motifs via its tandem SH2 domains. activation of c-Jun in the JNK signaling pathway and Sequential or combinatorial activation of Src family IL-2 gene reporter activity in stimulated Jurkat T cells. PTKs and the ZAP-70 leads to phosphorylation of Therefore, our data suggest that the hematopoietic many intracellular proteins, including the SH2 domain- speci®c proteins Grap2 and HPK1 form a signaling containing leukocyte protein of 76 kDa (SLP-76), the complex to mediate the c-Jun NH2-terminal kinase linker for activation of T cells (LAT), and the guanine (JNK) signaling pathway in T cells. Oncogene (2001) nucleotide exchange factor Vav, phospholipase C-g1 20, 1703 ± 1714. (PLC-g1), and the protooncoproteins c-Cbl (Jackman et al., 1995; Bustelo, 2000; Park et al., 1991; Weiss et Keywords: adaptor protein Grap2; HPK1; JNK; T- al., 1991; Clements et al., 1998b; Donovan et al., 1994; cells Weber et al., 1998; Zhang et al., 1998). Two direct consequences of protein tyrosine phosphorylation have been suggested in TCR-mediated signal tranduction. First, phosphorylation of tyrosine residues can alter the catalytic activity of enzymes. Examples are ZAP-70, which must be phosphorylated for full kinase activity (Chan et al., 1995; Wange et al., 1995) and PLC-g, *Correspondence: M Liu Received 12 September 2000; revised 21 December 2000; accepted 4 which hydrolyzes phosphoinositides and generating January 2001 second messengers that elevate intracellular Ca2+ and Interaction of Grap2 and HPK1 in T-cells WMaet al 1704 activate protein kinase C upon phosphorylation and activation. An appreciation of the importance of these activation. Second, tyrosine phosphorylation creates adaptor proteins was derived from the studies of the sites for binding by proteins with SH2 domains growth factor receptor kinase signaling pathway, where (Pawson, 1995), providing a mechanism for the initial binding and phosphorylation of the Shc adaptor assembly of protein complexes that participate in protein creates a binding site for a second adaptor, transducing antigen receptor signals to downstream Grb2. Then, Grb2 directs Sos, a guanine nucleotide cellular targets. However, a detailed explication of the exchange factor, to the membrane, and activate the Ras machinery that links ligand occupancy of the TCR to signaling pathway (Rozakis-Adcock et al., 1992; Cheng changes in cell behavior and responses remain unclear. et al., 1998). In T cells, Grb2 recruitment also occurs Hematopoietic progenitor kinase 1 (HPK1) is a via its SH2 domain, by binding T-cell receptor ITAM mammalian Ste20-related protein kinase (Hu et al., motifs directly or indirectly through Shc or p36/38. 1996; Kiefer et al., 1996). HPK1 is predominantly However, reconstitution of lymphoid receptors and expressed in hematopoietic organs and cells. It has kinases in nonlymphoid cells fails to activate these been demonstrated that transient expression of HPK1 signaling pathways, suggesting that additional lym- activates the c-Jun N-terminal kinase (JNK) signaling phoid-speci®c proteins are involved in the signal pathway, but does not stimulate ERK or p38 kinase transduction and activation of T cells. pathways (Hu et al., 1996; Kiefer et al., 1996). HPK1 is Most recently, we have identi®ed a novel human found in the signaling complex formed by the JNK leukocyte-speci®c Grb2-related adaptor protein, Grap2, interacting protein (JIP-1) and by the adaptor proteins from human bone marrow (Qiu et al., 1998). Grap2 Crk and CrkL (Whitmarsh and Davis, 1998; Ling et was also independently identi®ed and named as Gads, al., 1999). The JNK/stress-activated protein kinase GrapL, Mona, Grf40, Grid (Asada et al., 1999; Liu signaling cascade is critical for cells to transmit and McGlade, 1998; Law et al., 1999; Ellis et al., 2000). extracellular stimuli into the nucleus, thereby leading The Grap2 protein is a member of the Grb2 family of to appropriate cellular responses such as proliferation, adaptor proteins that contain amino- and carboxy- dierentiation, and apoptosis (Ip and Davis, 1998; terminal SH3 domain and a central SH2 domain Minden and Karin, 1997; Whitmarsh et al., 1998; Chen (Lowenstein et al., 1992; Rudd, 1999; Feng et al., 1996; and Tan, 2000). JNK signaling is activated by a Trub et al., 1997; Qiu et al., 1998). Grap2 shares 40 ± number of extracellular stimuli, including mitogens, 50% sequence homology in the SH3 domains and 57% epidermal and transforming growth factors (EGF and homology in the SH2 domain with Grb2 and Grap. TGF-b), cytokines, and environmental stresses. In However, unlike Grb2 and Grap, Grap2 contains a lymphocytes, HPK1 has been reported to be activated unique 120-amino acid glutamine- and proline-rich by antigen receptors in T and B cells (Liou et al., domain between the SH2- and C-terminal SH3 2000). Furthermore, HPK1 was shown to interact with domains. Database search ®nds this unique 120-amino adaptor proteins Grb2 and Crk in vitro and in acid domain has no apparent homology with other transfected cell model (Ana® et al., 1997; Ling et al., proteins. Furthermore, the expression of Grap2 is 1999; Oehrl et al., 1998). In T cells, Grb2 forms highly speci®c to lymphoid organs, T cells and complexes with signaling molecules LAT, SLP-76, monocytes/macrophages, while Grb2 is ubiquitously SOS, Cbl, Shc, and the cytoplasmic tail of T-cell expressed. In T lymphocytes, Grap2/Gads has been costimulatory receptor CD28 (Schneider et al., 1995; reported to interact with SLP-76 and LAT to regulate Clements et al., 1999). However, the signaling nuclear factor of activated T cell activation (NF-AT) speci®city mediated by Grb2 in T cells and the (Liu et al., 1999; Law et al., 1999; Zhang et al., 2000). pathways that link extracellular stimuli to JNK In addition, Grap2 also interacts with the macrophage activation have not been fully understood. colony-stimulating factor receptor and the activated T One of the outstanding issues upon TCR activation is cell costimulatory receptor CD28 (Bourette et al., 1998; the identity of signaling proteins that couple proximal Ellis et al., 2000). Therefore, Grap2 is likely to have protein kinases with downstream pathways, such as the distinct functions and serve as an important adaptor activation of the small GTP-binding proteins Ras/Rac/ protein in T cell signaling and activation, although Rho, the activation of the PLC-Ca2+-dependent Grb2 and Grap have been proposed to serve over- calcineurin pathway, and the activation of the JNK lapping functions in the cell (Cheng et al., 1998). signaling pathway. The SH3/SH2 adaptor proteins play In this report, we demonstrate that Grap2 directly essential roles in the formation of intracellular signaling interacted with the hematopoietic progenitor kinase 1 complexes, relaying extracellular signals from the (HPK1) in vitro and in mammalian cells. The plasma membrane to the nucleus of the cell. The SH2 interaction was mediated by the carboxyl-terminal domains can bind tightly to phosphotyrosine residue, SH3 domain of Grap2 and the second proline-rich and the SH3 domains can interact with proline-rich motif of HPK1. Coexpression of Grap2 and HPK1 not sequences (Songyang et al., 1994; Pawson, 1995). With only increased HPK1 kinase activity, but also had an multiple binding sites and the potential to create additive eect on HPK1-mediated activation of c-Jun various combinations of multi-protein complexes, these N-terminal kinase (JNK). In addition, cotransfection adaptor proteins are well suited to integrate signals of Grap2 and HPK1 in mammalian cells induced from cell surface receptors to the nucleus, determining transcriptional activation of c-Jun in the JNK signaling the speci®city of signaling pathways in immune cell pathway and IL-2 gene reporter activity in stimulated

Oncogene Interaction of Grap2 and HPK1 in T-cells WMaet al 1705 Jurkat T cells. Together, our data suggest that hematopoietic proteins Grap2 and HPK1 can form a signaling complex to mediate the JNK signaling pathway in T lymphocytes.

Results

Grap2 interacts with HPK1 in vitro and in mammalian cells To identify proteins that interact with Grap2 in T lymphocytes, we used an in vitro GST-fusion protein `pull-down' assay in Jurkat T cells. GST-Grap2 fusion proteins were expressed in E. coli and puri®ed by glutathione agarose column. Jurkat T cells were stimulated with anti-CD3 antibody (OKT3). Cell lysates from unstimulated or stimulated cells were prepared and incubated with GST-Grap2 fusion protein. The protein complexes were separated on SDS ± PAGE and Western blotted with either an anti-phosphotyrosine antibody (4G10) or an anti-HPK1 antibody. As shown in Figure 1a, a number of tyrosine phosphorylated proteins were detected in GST-Grap2 complex following stimulation by anti-CD3 antibodies. Among those proteins, a 100 kDa HPK1 protein was detected by an anti-HPK1 antibody (Figure 1b), indicating that Grap2 interacts with HPK1 in Jurkat T cells. To test whether the interaction is dependent on the phosphorylation status of HPK1, we stimulated the Jurkat T cells with an anti-CD3 antibody (OKT3) and examined the tyrosine phosphorylation status of the HPK1 protein. As shown in Figure 1c, HPK1 phosphorylation increased approximately twofold in Jurkat cells stimulated with OKT3 antibodies, indicating that HPK1 is phosphorylated upon T cell stimulation. We then compared the binding of the HPK1 to GST-Grap2 in stimulated or unstimulated cells. As shown in Figure 1b, GST-Grap2 bound HPK1 regardless of TCR stimulation, although a slight increase of binding was Figure 1 Interaction of Grap2 with HPK1 in vitro and in mammalian cells. (a) Jurkat T cells were either left unstimulated observed upon T-cell stimulation. These data suggest or stimulated with an anti-CD3 antibody (OKT3) and lysed. that activation of TCR stimulated the phosphorylation Lysates were precipitated using GST-Grap2 fusion protein. GST of HPK1 and increased the binding anity of HPK1 to alone was used as a negative control and did not show any Grap2 in stimulated Jurkat T cells. signi®cant binding anity to any phosphotyrosine proteins. The To con®rm that Grap2 indeed interact with HPK1 in precipitates were blotted with anti-phosphotyrosine antibody 4G10, followed by chemiluminescent detection. (b) HPK1 was mammalian cells, Grap2 was cotransfected with Flag- detected in the GST-Grap2 precipitates by anti-HPK1 antibody in tagged HPK1 in COS-7 cells. Protein extracts from the both stimulated or unstimulated Jurkat cells. GST alone did not transfected cells were immunoprecipitated using poly- bind to HPK1. (c) Treatment of Jurkat cells with OKT3 or clonal rabbit anti-Grap2 antibodies. The interaction of pervanadate increased the tyrosine phosphorylation of HPK1. Cell lysates were immunoprecipitated (IP) using an anti-HPK1 HPK1 with Grap2 was detected by Western blot using antibody. Phosphorylated HPK1 was detected by the anti- an anti-HPK1 antibody or an anti-Flag monoclonal phosphotyrosine antibody 4G10. Bottom panel: Western blot antibody (M2) in the Grap2 immuno-complex. Figure shows the amount of HPK1 protein is similar in the assay. A 1d shows that HPK1 coprecipitated with Grap2 complex preimmune rabbit serum was used as negative control and did not in cells cotransfected Grap2 and HPK1, indicating that show any binding to either HPK1 or Grap2. (d) Association of Grap2 with HPK1 in mammalian cells. COS-7 cells were the two proteins can form a complex in the cell. transfected with cDNAs encoding Grap2 or Grap2 and HPK1. Cell lysates were immunoprecipitated using a polyclonal rabbit anti-Grap2 antibody, followed by a goat anti-rabbit antibody The carboxyl-terminal SH3 domain of Grap2 interacts conjugated to agarose beads. The immunoprecipitates were with HPK1 blotted by an anti-HPK1 antibody and detected by chemiluminescent method. Similar binding of Grap2 with endogenous To determine which domain(s) of Grap2 is responsible HPK1 in Jurkat T cells were obtained (data not shown). For each for its interaction with HPK1, we constructed dierent ®gure, at least 2 ± 3 independent experiments were performed

Oncogene Interaction of Grap2 and HPK1 in T-cells WMaet al 1706 domains of Grap2 in the yeast Gal4 DNA-binding plasmid (pAS2), including the N-terminal SH3 and SH2 domain (N-SH3-SH2), the carboxyl-terminal unique domain and SH3 domain (QC), the glutamine-/proline-rich (Q/P-) domain, and the C-terminal SH3 domain (SH3-C). Each domain of Grap2 was fused in-frame into Gal4-BD in pAS2-1 expression vector by homologous recombination in yeast cells (Hua et al., 1997). To test which domain of Grap2 interacts with HPK1, yeast cells were cotransformed with expression vectors containing individual domains of Grap2 and the C-terminal domain of HPK1 (HPK1- CD) in Gal4-AD plasmid, pACT-2. Interaction of the Grap2 domain with HPK1 was monitored by the growth of yeast cells on SD plates with selective medium (-Trp/-Leu/-His) and by the activation of the LacZ reporter gene. As shown in Figure 2a, yeast cells cotransformed with either the QC domain or the SH3- C domain of Grap2 together with HPK1-CD grew on selective medium. Cotransformation of other Grap2 domains (NSH3-SH2 and Q/P domain) did not confer the growth of yeast cells. Since the QC domain contains the C-terminal unique domain and the SH3- C domain, these data suggest that the C-terminal SH3 domain is responsible for the interaction of Grap2 with HPK1 in yeast cells. To quantitate the interaction of Grap2 with HPK1, we measured the activation of the LacZ reporter gene under the control of the Gal4 promoter. As shown in Figure 2b, cotransformation of the Grap2-QC or Grap2 SH3-C domain with HPK1- CD strongly activated the LacZ activity, while other cotransformations had no signi®cant LacZ activity. P53 and large T antigen were used as a positive control in our yeast two-hybrid assays. These results indicate that the interaction of Grap2 with HPK1 is mediated by the C-terminal SH3 domain of Grap2. To con®rm our observation in yeast cells, we also performed an in vitro binding assay with GST-fusion proteins corresponding to dierent domains of Grap2 (Figure 3a). Cell lysates from COS-7 cells transfected Figure 2 Grap2 carboxyl SH3 (SH3-C) domain interacts with with Flag-tagged HPK1 were incubated with the GST- HPK1 in yeast cells. (a) Interaction of the QC domain and the Grap2 fusion proteins. The association of HPK1 with SH3-C domain of Grap2 with HPK1 in yeast cells. The QC individual Grap2 domains was detected by Western domain of Grap2 contains the glutamine-/proline-rich domain and the C-terminal SH3 domain. Cotransformation of expression blot using an anti-Flag monoclonal antibody (M2). plasmids encoding the QC domain or the SH3-C domain of Figure 3b shows that only the C-terminal SH3 domain Grap2 with HPK1-CD allows the yeast cells to grow on selection of Grap2 interacted with HPK1. GST-fusion proteins medium plates (SD/-Trp/-Leu/-His). However, coexpression of that contain the N-terminal SH3 and the SH2 domains Grap2-SH3(N)-SH2 or Grap2-Q/P domain with HPK1 failed to confer yeast cell growth on the same selective plates. Plasmids did not associate with the HPK1 protein in our in vitro encoding p53 and large T antigen were used as the positive assay. Similar data were obtained when Jurkat T cell control. (b) Activation of lacZ reporter gene activity in yeast cells lysates containing endogenous HPK1 were used in the cotransformed with Grap2-QC domain or Grap2-SH3-C domain binding assays (data not shown). Together, these with HPK1. Like p53 and large T antigen, cotransformation of results suggest that the C-terminal SH3 domain of Grap2-QC or Grap2-SH3-C domain with HPK1-CD domain signi®cantly increased lacZ gene expression, and therefore, the b- Grap2 interacts speci®cally with the HPK1 protein in galactosidase activity. For simplicity, HPK1-CD is labeled as the cell. HPK1 in the ®gures

Grap2 interacts with the second proline-rich motif of constructs were generated. Figure 4a shows the domain HPK1 structures of ®ve Flag-tagged HPK1 expression con- To determine which region(s) of HPK1 is involved in structs, including the wild-type HPK1, the kinase- the interaction with the Grap2 protein, Flag-tagged de®cient mutant HPK1 (HPK1-M46), the HPK1 wild-type HPK1 and four mutant HPK1 expression kinase domain (HPK1-KD), the HPK1 carboxyl

Oncogene Interaction of Grap2 and HPK1 in T-cells WMaet al 1707

Figure 3 Mapping the domain of Grap2 responsible for the interaction with HPK1 in vitro.(a) GST-Grap2 fusion proteins, including GST-Grap2 full-length protein, GST-Grap2-SH3(N)-SH2, GST-Grap2-Q/P-rich domain, and GST-Grap2-SH3-C domain. The proteins were constructed in pGEX-4T (Pharmacia) and expressed in BL21 cells. Proteins were puri®ed to 95% homology by glutathione-agarose column as assayed on SDS ± PAGE. (b) in vitro binding of GST-Grap2 and Grap2-CSH3 domain with HPK1. COS-7 cells were transfected with Flag-tagged HPK1. GST-Grap2 fusion proteins were incubated with the cell lysates. The protein complexes were precipitated by glutathione-agarose beads and washed extensively. HPK1 was detected by the anti-Flag M2 monoclonal antibody. The same amount of GST-fusion proteins (2 mg) was used in the binding assays. Bottom panel: Western blot analysis of HPK1 shows similar amount of proteins used in the assay domain (HPK1-CD), and the HPK1 proline-rich the HPK1 kinase domain did not bind Grap2, domain (HPK1-PR). Proteins were generated by in suggesting that the proline-rich region of HPK1 is vitro transcription and translation method and labeled essential for the interaction with Grap2. with [35S]methionine. The translation of HPK1 and It has been known that the SH3 domain binds to the HPK1 mutant proteins were resolved on SDS ± PAGE proline-rich region with consensus motif (PXXPXK/R) and detected by autoradiography (Figure 4b). To in the protein (Songyang et al., 1994; Pawson, 1995). examine which domain(s) of HPK1 is involved in the Examination of the HPK1 sequence reveals that the interaction with Grap2, we incubated the GST-Grap2- protein contains four proline-rich regions, therefore, SH3(C) domain with the in vitro translated HPK1 four putative Grap2-SH3 domain-binding motifs, PR1, proteins. Proteins bound to Grap2 were washed PR2, PR3, and PR4 (Figure 4a). To investigate extensively and separated by SDS ± PAGE. As shown whether the four individual HPK1 proline-rich motifs in Figure 4c, the full-length HPK1, the HPK1-M46, are involved in the interaction with Grap2, we the HPK1-CD domain, and the HPK1-PR domain synthesized four peptides corresponding to the putative bound with the Grap2 C-terminal SH3 domain while SH3-binding motifs in HPK1, termed PR1, PR2, PR3,

Oncogene Interaction of Grap2 and HPK1 in T-cells WMaet al 1708

Figure 4 The proline-rich domain of HPK1 is responsible for the interaction with Grap2. (a) A schematic diagram showing dierent HPK1 constructs and four proline-rich peptides. Wild-type HPK1 and four HPK1 mutant constructs, including HPK1- K46M, HPK1-KD (kinase domain), HPK1-CD (the carboxyl domain, from amino acids 292 to 833), and HPK1-PR (proline-rich domain, amino acids 288 ± 482), were used in the assays. Shown on the right are the sequences for the four proline-rich peptides synthesized (PR1, PR2, PR3, and PR4). (b) in vitro transcription and translation of HPK1 full-length and mutant proteins in the presence of [35S]methionine. Proteins were separated by SDS ± PAGE and detected by autoradiography. (c) Binding of HPK1 domains to Grap2 protein. [35S]methionine-labeled wild-type HPK1 and its mutant proteins (10 ml) were incubated with immobilized GST-Grap2-QC fusion protein. The interacting complexes were washed extensively and resolved by 10% SDS ± PAGE. HPK1 and mutants were detected by autoradiography. (d) HPK1 proline-rich peptides dierentially block the interaction of HPK1 with Grap2- SH3-C domain. Four HPK1 proline-rich peptides were added to block the interaction of HPK1 with Grap2 in competition binding assays. The amount of HPK1 bound to GST-Grap2 beads was detected by Western blot using an anti-HPK1 antibody

Oncogene Interaction of Grap2 and HPK1 in T-cells WMaet al 1709 and PR4 (Figure 4a). Using the four proline-rich peptides, we performed competition-binding assays by adding individual HPK1 peptides to the Grap2-SH3(C) and HPK1 complexes. As shown in Figure 4d, the HPK1 second proline-rich peptide (PR2) completely blocked the formation of Grap2-HPK1 complexes. The ®rst and the fourth proline-rich peptides (PR1 and PR4) of HPK1 also showed some inhibitory eect on the complex, whereas the third proline-rich peptide (PR3) had no eect on the formation of Grap2-HPK1 complex. These data suggest that the second proline- rich peptide of HPK1 is the key site for interaction with the Grap2-SH3(C) domain, while the role of the fourth proline-rich motif in the interaction with Grap2 is not certain since the fourth proline-rich domain is not conserved between human and mouse. The ®rst proline-rich domain of HPK1 may also play some role in facilitating the formation of the Grap2-HPK1 complex.

Grap2 activates HPK1 and has additive effects with HPK1 in the activation of JNK To determine the functional signi®cance of this interaction, we examined whether Grap2 could regulate HPK1 activity in the cell. The kinase activity of HPK1 was measured by an immunocomplex kinase assay in the presence or absence of Grap2. As shown in Figure 5a, cotransfection of Grap2 increased the kinase activity of HPK1 by approximately 2 ± 3-fold, suggesting that the hematopoietic adaptor protein Grap2 may recruit HPK1 to the proximity of other HPK1 activators in the cell. It has been reported that HPK1 is involved in the Figure 5 Grap2 activates HPK1 and potentiates the HPK1- JNK signaling cascade (Hu et al., 1996; Kiefer et al., mediated JNK activation. (a) Coexpression of Grap2 with HPK1 activated the kinase activity of HPK1 by approximately twofold 1996; Ling et al., 1999; Zhou et al., 1999). Over- using MBP as a substrate. COS-7 cells were transfected with expression of HPK1 activates JNK kinase activity in HPK1 alone or with HPK1 plus Grap2. HPK1 was immunopre- transfected cells (Ling et al., 1999). To examine cipitated with the anti-Flag M2 monoclonal antibody, and an whether coexpression of Grap2 and HPK1 has any equal amount of HPK1 was used for immunocomplex kinase assays. (b) Cotransfection of Grap2 with HPK1 activated JNK eect on the JNK signaling pathway, we assayed the activity synergistically. HA-tagged JNK was transfected into JNK activity in the presence of Grap2, HPK1, or both. COS-7 cells alone or with 1 mg of expression plasmids as Cotransfection of HPK1 and HA-tagged JNK activates indicated. Proteins were immunoprecipitated by an anti-HA JNK activity by about threefold and is inconsistent monoclonal antibody. JNK activity was determined by immuno- with previous reports (Ling et al., 1999). Overexpres- complex kinase assays using GST-c-Jun (amino acids 1 ± 79) as a substrate for HA-JNK1. Coexpression of Grap2 and HPK1 has sion of Grap2 with JNK slightly increased the synergistic eect on the activation of JNK. The expression level of activation of JNK activity compared to HA-tagged JNK in transfected cells was detected by Western blot with anti- JNK alone. However, in the presence of Grap2 and HA antibodies HPK1, JNK activity was increased approximately 4 ± 5- fold (Figure 5b), indicating that coexpression of Grap2 with HPK1 in the cell can increase HPK1-mediated and a reporter vector containing luciferase (pFR-Luc). activation of the JNK pathway. As shown in Figure 6a, cotransfection of JNK1, a known activator of the JNK signaling pathway, increased the c-Jun coupled luciferase activity by about Grap2 and HPK1 have an additive effect on the activation 10-fold. Overexpression of either Grap2 or HPK1 of c-Jun in the JNK signaling pathway increased c-Jun transcriptional activity by 3 ± 4-fold, To determine the eect of the Grap2-HPK1 interaction respectively. However, cotransfection of Grap2 and on the stimulation of the JNK signaling pathway, we HPK1 with the c-Jun reporter system increased the employed a PathDetect trans-reporting system with the transcriptional activity of c-Jun by at least eightfold, activation domain of c-Jun fused to the DNA-binding similar to the activation by overexpression of JNK1 in domain of Gal4. Cells were cotransfected with the the cell. These data indicate that coexpression of Grap2 pathway-speci®c c-Jun transcription activator plasmid and HPK1 has an additive eect on the activation of

Oncogene Interaction of Grap2 and HPK1 in T-cells WMaet al 1710 the JNK signaling pathway. To further con®rm the speci®c eect of Grap2-HPK1 interaction, two deletion mutants, Grap2-dSH3C (C-terminal SH3 deleted) and Grap2-dSH2 (SH2 domain deleted), were cotransfected pFR-Luc together with HPK1 in the cell, respectively. Both mutants failed to activate the JNK signaling pathway like the wild-type Grap2 protein (Figure 6a), suggesting that the additive eect is mediated by the speci®c Grap2-HPK1 interaction.

Grap2 and HPK1 potentiate IL-2 activation by the TCR and PMA TCR ligation initiates distinct signaling cascades that ultimately combine to activate transcription of the interleukin-2 (IL-2) gene, a key growth and dierentiation factor for T cells (Weiss and Littman, 1994; Karin and Hunter, 1995). IL-2 induction involves the simultaneous activation of the nuclear factor of activated T cells (NF-AT) by the calcium/calmodulin- dependent phosphatase calcineurin, and of the AP-1 complex (Verweij et al., 1990; Clipstone and Crabtree, 1992; Crabtree and Clipstone, 1994). It has been shown that cotransfection of HPK1 with a reporter construct (56TRE-CAT) increased AP-1 activity dramatically (Hu et al., 1996). To examine whether Grap2 and HPK1 are directly or indirectly involved in the IL-2 activation, we cotransfected Grap2 and HPK1 with an IL-2-driven luciferase reporter and subsequently stimulated with an anti-CD3 antibody and PMA. As shown in Figure 6b, Grap2 increased TCR-stimulated IL-2 activity by about 2 ± 3-fold, while HPK1 had limited eects on the IL-2 activation. Cotransfection of Grap2 and HPK1 together with the IL-2 reporter increased the IL-2 activity by approximately fourfold, suggesting Grap2-HPK1 may directly or indirectly participate in the activation of IL-2 gene in T-cell activation.

Discussion Figure 6 Cooperation between Grap2 and HPK1 in the The selectivity and speci®city of intracellular signaling transcriptional activation of c-Jun and IL-2. (a) Activation of c- Jun transcription in the JNK signaling pathway by cotransfection are achieved by compartmentalization and regional of Grap2 and HPK1. The activation of c-Jun was assayed by the organization of signaling components via molecular PathDetect c-Jun trans-reporting system (Stratagene, CA, USA). scaold and adaptor proteins (Whitmarsh et al., 1998; cDNAs encoding c-Jun/Gal4 fusion protein and the luciferase Rudd, 1999; Clements et al., 1999; Zhang and reporter pFR-Luc were cotransfected into COS-7 cells with individual expression vectors indicated. HA-JNK1 was used as Samelson, 2000). In the past few years, a number of positive controls in the assay. The activation of luciferase activity scaold and adaptor proteins have been identi®ed to in the cell was performed according to the manufacturer's form signaling complexes and play an important role manual. Triplicates were performed for the same transfection in determining the speci®city and selectivity of signal assay. At least three transfection experiments were performed. (b) transduction in the cell. These protein complexes may Coexpression of Grap2 and HPK1 increased the IL-2 luciferase reporter activity. Jurkat T cells were transfected with a control result from physical interaction between components plasmid encoding lacZ, or expression plasmids encoding Grap2, of particular signaling pathways or by the assembly of Grap2 mutants, HPK1, and Grap2 plus HPK1 as indicated. The signaling molecules on anchor or scaold proteins that activation of IL-2 luciferase activity was assayed in unstimulated localize their binding partners to speci®c subcellular cells or cells stimulated with an anti-CD3 antibody plus PMA (dark bars). The expression level of Grap2, Grap2 mutants, and compartments or to speci®c substrates. Examples HPK1 were similar in each transfection assay as estimated by include the MAP kinase complexes coordinated by Western blot using respective antibodies the scaold proteins Ste5p and Pbs2p in the yeast Saccharomyces cerevisiae (Choi et al., 1994), and the JNK kinase signaling complexes by the scaold cells, a variety of adaptor molecules play critical protein, JIP-1 (Whitmarsh et al., 1998). In immune functions in linking receptor proximal signal transduc-

Oncogene Interaction of Grap2 and HPK1 in T-cells WMaet al 1711 tion events such as the activation of receptor- activated by lymphocyte antigen receptors, TGF-b,or associated kinases to downstream eector molecules by erythropoietin (Zhou et al., 1999; Ana® et al., 1997; to promote the transcriptional induction of multiple Nagata et al., 1999; Liou et al., 2000). Both the genes. A number of lymphocyte speci®c adaptor receptor tyrosine kinases (EGF, PDGF) and the Src proteins, such as LAT, SLP-76, FYB/SLAP, SKAP55, family of cytoplasmic tyrosine kinases has been shown 3BP2, and BLNK, have been identi®ed to interact with to induce tyrosine phosphorylation of HPK1 (Ana® et signaling molecules, regulating lymphocyte activation al., 1997; Wang et al., 1997). It is tempting to speculate and development (Weber et al., 1998; Clements et al., that upon stimulation of TCR, Grap2 may recruit 1998a; Zhang et al., 1998; Pivniouk et al., 1998; da HPK1 to the proximity of the receptors, facilitate the Silva et al., 1997; Deckert et al., 1998; Fu et al., 1998; phosphorylation and activation of HPK1 by protein Pappu et al., 1999). Therefore, these adaptor proteins tyrosine kinases, and subsequently transmit these regulate immune cell signaling and activation by signals to downstream targets, leading to the activation selectively forming signaling complexes with dierent of the JNK signaling pathway. molecules (Clements et al., 1999; Rudd, 1999). Grap2 In T-cell activation, optimal IL-2 induction requires is a new member of the Grb2 family of adaptor two major signaling pathways triggered by costimula- proteins (Qiu et al., 1998; Law et al., 1999; Xia et al., tion of the T-cell receptor (TCR) and CD28 or 2000). Grap2 shares similar structural organization as mimicked by incubating T cells with phorbol ester Grb2 and Grap with the N- and C-terminal SH3 (i.e., PMA) and Ca2+ ionophore (Su et al., 1994). Early domains and a central SH2 domain. However, unlike studies indicate that AP-1 binds to the IL-2 promoter Grb2 and Grap, Grap2 contains a unique 120-amino (Muegge et al., 1989) and forms a complex with NF- acid glutamine-/proline-rich domain, and the expres- AT to induce IL-2 activation (Northrop et al., 1993; sion of the Grap2 protein is highly speci®c in Ullman et al., 1993). JNK is involved in the integration hematopoietic tissues and in T cells (Qiu et al., of the costimulatory signals CD3 plus CD28 or PMA 1998). In this report, we demonstrated that the Grap2 plus Ca2+ ionophore in T cells (Su et al., 1994). As a protein interacted with the hematopoietic progenitor hematopoietic upstream activator of JNK, HPK1 may kinase 1 (HPK1) to form signaling complex both in be involved in IL-2 induction in T cells. Using IL-2 vitro and in mammalian cells. The Grap2-HPK1 luciferase reporter assays, we found that coexpression interaction is mediated by the carboxy-SH3 domain of Grap2 and HPK1 enhanced IL-2 activation by T- of Grap2 and the proline-rich regions of HPK1. cell stimuli (anti-CD3 antibody and PMA) in Jurkat T Among the four proline-rich regions of HPK1, the cells (Figure 6b). Like some of the well-known adaptor second proline-rich motif was identi®ed to be critical proteins, Grap2 may form signaling complexes with for the binding of HPK1 to Grap2. Although our data distinct signaling molecules in T cells to mediate cell show that peptides from the ®rst and the fourth signaling and activation. Grap2 may couple the TCR proline-rich motifs also partially blocked the interac- and CD28 with protein tyrosine kinases, such as Src tion of HPK1 and Grap2, the role of these two family of kinases, to HPK1 to activate HPK1-mediated proline-rich motifs in the functional interaction of JNK signaling pathway during T-cell activation. HPK1 and Grap2 is uncertain and is under investiga- During the preparation of this study for publication, tion. The interaction between Grap2 and HPK1 is a study describing the interaction of Gads and HPK1 independent of the stimulation status of the cells was independently reported (Liu et al., 2000). Our although we observed that HPK1 has high anity for studies not only independently con®rm the ®nding that interaction with Grap2 upon T-cell stimulation. There- Grap2 interacts with HPK1 in T cells, but also fore, we cannot rule out the possibility that Grap2- demonstrate that the interaction modulates the activa- HPK1 form a functional signaling complex only upon tion of HPK1 and HPK1-mediated JNK signaling stimulation of T cells. pathway. While the fourth proline-rich domain of Cotransfection of Grap2 and HPK1 directly acti- HPK1 may participate in the interaction with Grap2, vated HPK1 in our kinase assays and Grap2 also the second proline-rich domain is identi®ed to play cooperated with HPK1 to activate JNK (Figure 5). more critical role in the interaction of HPK1 and These results indicate that the Grap2 protein not only Grap2 in our study. Taken together, our data suggest physically interacts with HPK1 but also functionally that the hematopoietic adaptor protein Grap2 forms a modulates HPK1-mediated JNK activation. Further- signaling complex with HPK1 to activate the JNK more, overexpression of Grap2 and HPK1 activates signaling pathway in TCR-mediated signal transduc- the JNK-mediated c-Jun transcriptional activity, tion and activation. Since Grap2 contains multiple suggesting that the Grap2-HPK1 complex plays an domains for protein ± protein interaction, Grap2 may important role in JNK-mediated signal transduction in function as a central linker protein to interact with T lymphocytes. HPK1 receives the upstream signals multiple molecules and form dierent signaling com- through its interaction with the SH2/SH3 adapter plexes during T-cell activation. Further experiments are proteins and subsequently transmits these signals to under investigation to test the hypothesis that dierent downstream targets (MEKK1 and TAK1) via its domains of Grap2 may be involved in the interactions kinase domain or distal regulatory region (Ling et with various signaling molecules to determine the al., 1999; Ana® et al., 1997). HPK1 has been identi®ed signaling speci®city and selectivity in T-cell activation as a substrate for protein tyrosine kinases and is and development.

Oncogene Interaction of Grap2 and HPK1 in T-cells WMaet al 1712 phate, 1% Triton X-100, 10% glycerol, 500 mM phenyl- Materials and methods methylsulfonyl ¯uoride [PMSF], 5 mg/ml leupeptin, 3 mg/ml aprotinin). Cell lysates were used for binding assays and Plasmid constructs kinase assays. To generate HA- or Flag-Tagged Grap2 expression plasmids, Jurkat cells in log-phase were washed with serum-free PCR was used to amplify dierent domains of Grap2 using RPMI 1640 once and electroporated in triplicate with a total the full-length of human Grap2 cDNA as a template. Pfu of 30 mg of plasmid at 240 V, 960 mF using a Gene Pulser (Strategene) was used in all PCR subcloning, and the (Bio-Rad) at 107 cells/400 ml serum-free RPMI 1640. sequences of dierent Grap2 constructs were con®rmed by Sonicated salmon sperm DNA (Sigma Chemical Co.) was DNA sequencing. To generate the GST (glutathione S- used to maintain the total DNA at 30 mg whenever necessary. transferase)-Grap2 fusion constructs, cDNA fragments After electroporation, cells were incubated in fully supple- corresponding to dierent domains of Grap2 were subcloned mented RPMI 1640 for 14 ± 18 h at 378C, 5% CO2. Cells into pGEX-2TK or pGEX-4T1 (Pharmacia Biotech, Inc.) for were then transferred into 96-well plates at 2 ± 56105 cells/ expression as a fusion protein of GST and various lengths of well in a total of 100 ml of medium. Cells were stimulated in Grap2. pCIneo-FLAG-tagged vectors of wild-type HPK1 triplicate OKT3 antibodies for 6 ± 8 h at 378C. Control cells and various HPK1 mutants, including a kinase-dead mutant were maintained in culture medium. After stimulation, cells (HPK1-K46M), the HPK1 kinase domain alone (HPK1-KD; were lysed in 50 ml Reporter Lysis buer (Promega) for amino acids 1 ± 291), the HPK1 carboxyl domain (HPK1-CD; 30 min at room temperature. Luciferase activity was amino acids 292 ± 833), and the HPK1 proline-rich domain quanti®ed by adding 50 ml of the lysate to 25 ml of luciferase (amino acids 288 ± 482) were described previously (Hu et al., assay substrate (Promega) and immediately measured with a 1996; Ling et al., 1999). Hemagglutin (HA)-tagged JNK and GenProbe LeaderTM 1 luminometer (Wallac Inc.). To GST-cJun (1 ± 79) were described previously (Diener et al., normalize for variations in transfection eciency, luciferase 1997; Hu et al., 1996). activities in dierent transfection conditions were expressed To generate Grap2 domain speci®c expression vectors in as fold increase compared to the control experiments. yeast cells, sequences corresponding to the SH3(N)-SH2, the SH2, the SH3(C), and the unique Q/P domains were Peptides, antibodies, immunoprecipitation and immunoblot ampli®ed by PCR and fused in-frame to the Gal4 BD of analysis the pAS-2 vector by homologous recombination in yeast cells. The carboxyl-terminal domain of HPK1 (HPK1-CD, Four wild-type HPK1 proline-rich peptides were synthesized amino acids 292 ± 833) was subcloned into the Gal4-AD using Fmoc (N(9-¯uorenyl)methoxycarbonyl) chemistry and plasmid, pACT-2 using the PCR technique. The luciferase puri®ed by high pressure liquid chromatography (HPLC). reporter constructs (IL-2 luciferase and NF-AT luciferase) Polyclonal antibodies to Grap2 were prepared by immuniza- were obtained from Dr G Crabtree (Stanford University). tion of rabbits with GST-Grap2-QC domain fusion protein. The c-Jun Path-Detect Assay system was purchased from Grap2-speci®c antibodies were anity-puri®ed by passage of Stratagene (CA, USA). the antisera over immobilized GST-agarose beads. Anti- HPK1 antibodies were generated against the HPK1 peptide (amino acids 341 ± 366). Other antibodies used in these Recombinant proteins studies include: anti-CD3 mAb OKT3 (American Type The pGEX plasmids encoding various GST-fusion proteins Culture Collection); anti-phosphotyrosine mAb 4G10 (Up- were used to transform E. coli strain BL21 (DE3) together state Biotechnology); anti-phosphotyrosine mAb PY-20 with 2tRNA plasmid. After induction of protein expression (Sigma); anti-Flag mAb, M2 (Sigma); anti-HA monoclonal with 1 mM IPTG (isopropyl-1-thio-D-galactopyranoside) for antibodies (12CA5; Boehringer Mannheim). 3 ± 4 h, the bacteria were resuspended in lysis buer (50 mM Immunoprecipitation and immunoblot analysis were car- Tris/HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA, 1 mM ried out as described previously (Xia et al., 2000). In brief, phenylmethylsulfonyl ¯uoride, leupeptin, 0.4 mg/ml lyso- cells were lysed with a cell extraction buer (1% NP-40, zyme) and sonicated brie¯y on ice. Triton X-100 (1%) was 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM added to lysates before centrifugation at 10 000 g for 30 min. Na3VO4,2mM PMSF and 20 mg/ml aprotinin, 3 mg/ml In certain cases, proteins in inclusion bodies were solubilized leupeptin, and 3 mg/ml aprotinin), and immunoprecipitated with RIPA buer (16PBS, 1% Igepal CA-630, 0.5% sodium with the indicated antibodies or GST-fusion proteins. Lysates deoxycholate, 0.1% SDS, pH 7.4). The GST-Grap2 fusion were rocked 30 min at 48C, centrifuged (10 000 g, 15 min), proteins were bound to glutathione agarose (Sigma) beads, and precleared with washed protein A-Sepharose 4B and non-speci®c proteins were washed away with buer (Pharmacia) or GST-coated glutathione-agarose. Anti- containing 25 mM Tris/HCl, pH 7.5, 100 mM NaCl, 1.0 mM HPK1 and anti-HA immunocomplexes were recovered by EDTA, 1.0 mM dithiothreitol and protease inhibitors. Protein using protein-A beads (Sigma). Anti-FLAG immunocom- purity and concentrations were assessed on SDS ± PAGE by plexes were recovered by using protein-G beads (Santa Cruz Coomassie blue staining. Biotechnology). All immunoprecipitates were washed four times with lysis buer and were separated by SDS ± PAGE and then transferred to polyvinylidene di¯uoride (PVDF) Cell culture, transfection, and luciferase assays membranes (Millipore). After incubation in TBST containing COS-7 cells were grown in Dulbecco modi®ed Eagle's 2% BSA or 5% dry milk powder, the membranes were medium with 10% fetal bovine serum (FBS). The day before probed with the indicated antibodies and visualized using the transfection, cells (26105 cells per well) were plated in a six- SuperSignal West Pico detection system (Pierce, IL, USA). well plate. Transfection was then performed by the GenePorter transfection reagents (Gene Therapy, Inc.) with Immunocomplex kinase assay various plasmids as indicated in the ®gures. After 48 h, the cells were harvested and lysed in lysis buer (150 mM NaCl, Assays for JNK activity were performed as described 20 mM HEPES, pH 7.4, 2 mM EGTA, 50 mM glycerophoso- previously (Hu et al., 1996) Brie¯y, HA-tagged JNK1 from

Oncogene Interaction of Grap2 and HPK1 in T-cells WMaet al 1713 50 mg of transfected COS-7 cell lysates was immunoprecipi- Plasmids (1 ± 2 mg/reaction) were added to a reaction mixture, tated by using an anti-HA monoclonal antibody and protein including 25 ml of rabbit reticulocyte lysate, 2 ml of reaction A-agarose beads in 500 ml of lysis buer. After being washed buer, 1 ml of T7 RNA polymerase, 1 ml of amino acid twice with lysis buer, twice with LiCl buer (500 mM LiCl, mixture minus methionine (1 mM), 4 mlof[35S]methionine 100 mM Tris-Cl, pH 7.6, 0.1% Triton X-100), and twice with (10 mCi/ml), ribonuclease inhibitor (40 U/ml), and nuclease- kinase buer (20 mM MOPS [morpholine propanesulfonic free H2O to a ®nal volume of 50 ml. The reactions were acid], pH 7.2, 2 mM EGTA, 10 mM MgCl2, 0.1% Triton X- incubated at 308C for 90 min. The in vitro translated proteins 100), the immunoprecipitates were incubated with 30 mlof were then used for in vitro binding assays. The interactions of kinase buer containing 2 mg of GST-c-Jun (amino acids 1 ± in vitro translated proteins with GST fusion proteins were 79), 20 mM unlabeled ATP, and 20 mCi of [32P]ATP. The directly examined by autoradiography. The ability of reactions were incubated at 308C for 30 min and terminated individual HPK1 proline-rich peptides to block the interac- by boiling the samples in 46SDS sample buer. Proteins tion of HPK1 with Grap2 was performed as previously were separated by SDS ± PAGE (12%). Similar kinase assays described (Ling et al., 1999) or detected by Western blot were performed for HPK1 activity. Jurkat T cells and COS-7 using anti-HPK1 antibodies. cells transiently expressing HPK1 were immunoprecipitated by using an anti-HPK1 antibody or an anti-FLAG monoclonal antibody, and kinase assays were performed with myelin basic protein (MBP). Acknowledgments This work was supported in part by a Scientist Develop- In vitro translation and binding assays ment Grant 0030160N from the American Heart Associa- In vitro transcription and translation of proteins were tion National and the Basil O'Connor Starter Scholar performed essentially according to the manufacturer's Research Award from March of Dimes Foundation (to M instructions (Promega Biotech, Inc.). Proteins were labeled Liu) and NIH grants RO1-AI42532 and RO1-AI38649 (to and detected by the incorporation of [35S]methionine. T-H Tan).

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Oncogene