Involvement of Hematopoietic Progenitor Kinase 1 in T Cell Receptor Signaling*

Hematopoietic progenitor kinase 1 (HPK1), a mammalian Ste20-related serine/threonine protein kinase, is a hematopoietic-specific upstream activator of the c-Jun N-terminal kinase. Here, we provide evidence to demonstrate the involvement of HPK1 in T cell receptor (TCR) signaling. HPK1 was activated and tyrosine-phosphoryl-ated with similar kinetics following TCR/CD3 or pervanadate stimulation. Co-expression of protein-tyrosine kinases, Lck and Zap70, with HPK1 led to HPK1 activation and tyrosine phosphorylation in transfected mammalian cells. Upon TCR/CD3 stimulation, HPK1 formed inducible complexes with the adapters Nck and Crk with different kinetics, whereas it constitutively interacted with the adapters Grb2 and CrkL in Jurkat T cells. Interestingly, HPK1 also inducibly associated with linker for activation of T cells (LAT) through its proline-rich motif and translocated into glycolipid-enriched microdomains (also called lipid rafts) following TCR/CD3

A key event in the regulation of immune responses is the activation of T cells. Optimal T cell activation requires two signals. A primary signal is delivered by the engagement of the T cell antigen receptor (TCR) 1 with the major histocompatibility complex-antigen complex on antigen-presenting cells. Ligation of CD28 on T cells with the B7 proteins (B7-1 and B7-2) on antigen-presenting cells provides a costimulatory signal. Engagement of the TCR initiates tyrosine phosphorylation of im-mune receptor tyrosine-based activation motifs within the TCR-associated CD3 subunits by the Src family protein-tyrosine kinases, Lck and Fyn (1,2). This leads to the subsequent recruitment and activation of the cytoplasmic tyrosine kinases, Zap70 and Syk (1,2). These protein-tyrosine kinases further activate several signaling molecules to transmit the TCR-induced proximal signals to downstream effectors. One of these critical signaling molecules is the linker for activation of T cells (LAT) (3). LAT is a 36 -38-kDa palmitoylated transmembrane protein that localizes to specific plasma membrane compartments known as glycolipid-enriched microdomains (GEMs) or detergent-insoluble lipid rafts, which are critical for T cell signaling (4,5). Following TCR stimulation, LAT becomes heavily tyrosine-phosphorylated and serves as an anchor protein for association with a number of SH2 domain-containing signaling molecules, including Grb2, Gads, phospholipase C-␥1, and the p85 subunit of phosphatidylinositol 3-kinase (6,7). Engagement of the CD28 receptor initiates multiple signaling pathways through Itk tyrosine kinase, Grb2, and phosphatidylinositol 3-kinase to facilitate TCR signaling (8,9). Thus, costimulation of TCR and CD28 triggers a series of biochemical events, ultimately leading to the activation of downstream targets, including NF-AT, NF-B, and AP-1, which in turn mediate interleukin-2 production.
One important downstream pathway mediating T cell costimulatory events is the c-Jun N-terminal kinase (JNK) cascade (10). The JNK family, a group of serine/threonine protein kinases, belongs to the mitogen-activated protein kinase (MAPK) superfamily, which consists of two other groups: the extracellular signal-regulated kinase and p38 kinase families (11). Activation of these MAPKs is achieved through evolutionarily conserved signaling cascades, which comprise MAPK kinases (MAP2Ks), MAPK kinase kinases (MAP3Ks), and sometimes MAPK kinase kinase kinases (MAP4Ks) (12). Downregulation of JNK activity is correlated to the onset of T cell anergy (13). Studies of jnk1-and jnk2-null mice showed that both JNK1 and JNK2 are required for the differentiation of CD4 ϩ T cells into effector Th1 cells (14,15). Although the importance of JNK in T cells has been established, the links between receptor engagement and JNK activation remain poorly defined. Recently, several mammalian Ste20-related MAP4Ks have been identified as upstream activators of JNK (12). They include germinal center kinase (GCK) (16), hematopoietic progenitor kinase 1 (HPK1) (17,18), HPK/GCK-like kinase (also referred to as NIK) (19,20), GCK-like kinase (21), and the kinase homologous to SPS1/STE20 (KHS, also referred to as GCKR) (22,23). It is likely that one or more of these MAP4Ks may mediate JNK activation during T cell costimulation.
HPK1 is a 97-kDa serine/threonine protein kinase expressed only in hematopoietic cells and tissues (17,18). HPK1 consists of an N-terminal kinase domain followed by four proline-rich motifs, and a citron homology domain at its distal C terminus (12). Previously, HPK1 was shown to interact with several SH2/SH3 adapters, including Crk, CrkL, Grb2, and Nck (24 -26). These adapters play an important role in the formation of signaling complexes following TCR/CD3 or CD28 stimulation (27). Because of HPK1's restricted tissue expression, activation of the JNK pathway, and association with important T cell signaling adapters, we explored the role of HPK1 in TCR and CD28 signaling. We found the activation and tyrosine phosphorylation of HPK1 upon TCR/CD3 stimulation in Jurkat T cells. CD28 stimulation, however, did not induce HPK1 activation and tyrosine phosphorylation. In addition, protein-tyrosine kinases Lck and Zap70 were involved in the regulation of HPK1. We found that HPK1 constitutively interacted with the adapters, Grb2 and CrkL, in Jurkat T cells and formed inducible complexes with the adapters, Nck and Crk, upon TCR/CD3 stimulation. More interestingly, HPK1 was inducibly associated with LAT and was recruited into the GEMs following TCR/CD3 stimulation. This work demonstrates the involvement of HPK1 in TCR signaling and provides possible links from the TCR to HPK1.

EXPERIMENTAL PROCEDURES
Antibodies-The anti-CD3 monoclonal antibody (OKT3) was purified from hybridoma cell supernatants on a protein G affinity column using standard protocols. The anti-CD28 mAb (clone 9.3) was kindly provided by Dr. C. June (University of Pennsylvania, Philadelphia, PA). Anti-HPK1 (N-19) and anti-CrkL (C-20) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Another anti-HPK1 antibody (Ab484) was described previously (17). The anti-phosphotyrosine mAb (4G10), anti-Grb2, anti-LAT and anti-Nck antibodies were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). The anti-Crk and anti-Nck mAbs were purchased from Transduction Laboratories (Lexington, KY). The anti-LAT antibody was a gift from Drs. W. Zhang and L. E. Samelson (National Institutes of Health, Bethesda, MD). The anti-HA (12CA5) and anti-FLAG (M2) mAbs were purchased from Roche Molecular Biochemicals and Sigma, respectively.
Immunocomplex Kinase Assays, Immunoprecipitation, and Immunoblotting-For the HPK1 immunocomplex kinase assays, cell lysates (100 g) were immunoprecipitated with an anti-HPK1 antibody (Ab484) or anti-Flag mAb (M2). Kinase assays were performed as described previously (24). Myelin basic protein (MBP) and GST-Crk were used as substrates where indicated. For the JNK kinase assays, cell lysates were immunoprecipitated with an anti-HA mAb (12CA5). Kinase assays were performed as described previously (17). For immunoprecipitation and immunoblotting, cell lysates were immunoprecipitated by antibodies as indicated in figure legends. Immunoprecipitates were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes. The membranes were blocked with either 5% milk in Tris-buffered saline/Tween buffer (20 mM Tris (pH 7.5), 135 mM NaCl, 0.1% Tween 20) or 2% bovine serum albumin buffer (10 mM Tris-Cl, (pH 8.0), 150 mM NaCl, 2% bovine serum albumin fraction V) for anti-phosphotyrosine immunoblotting. The membranes were probed with antibodies as described in the figure legends, and then incubated with horseradish peroxidaseconjugated secondary antibody according to the primary antibody used.
Preparation of GEM Fractions-GEM fractions were prepared as described previously (3). In brief, Jurkat T cells (5 ϫ 10 7 ) were either unstimulated or stimulated with an anti-CD3 mAb (OKT3, 10 g/ml) for 2 min. Cells were harvested and lysed in 1 ml of 1% Trition X-100 lysis buffer. Cell lysates were mixed well with 1 ml of 80% sucrose and transferred to ultracentrifuge tubes. These mixtures were overlaid with 2 ml of 30% sucrose and 1 ml of 5% sucrose sequentially, and then subjected to ultracentrifugation for 16 -18 h at 4°C. Twelve 0.4-ml fractions were collected from the top of the sucrose gradient. These fractions were subjected to immunoblotting.

TCR/CD3 Stimulation Activates HPK1 in Jurkat T
Cells-To investigate whether HPK1 is regulated by TCR or CD28 signaling, we first performed an immunocomplex kinase assay to examine HPK1 kinase activity in Jurkat T cells upon TCR/CD3 stimulation, CD28 stimulation, or both in combination. We found that TCR/CD3 stimulation alone was able to induce HPK1 activation 2 min after stimulation, and that this activation decreased after 10 min. A similar pattern of HPK1 activation was also observed in Jurkat T cells with TCR/CD3 plus CD28 costimulation (Fig. 1A). However, CD28 stimulation alone did not result in significant HPK1 activation (data not shown). We also observed TCR/CD3-induced HPK1 activation in purified human peripheral blood T cells (Fig. 1B). To confirm the specificity of HPK1 activation by TCR/CD3 stimulation, we transiently expressed the Flag-tagged wild-type HPK1 (Flag-HPK1) or its kinase-dead mutant (Flag-HPK1(M46)) into Jurkat TAg cells to examine their kinase activity following TCR/ CD3 stimulation. By an immunocomplex kinase assay using an anti-Flag antibody, we found that only Flag-HPK1, but not Flag-HPK1(M46), was activated upon TCR/CD3 stimulation (Fig. 1C). These results demonstrate that the engagement of TCR/CD3 elicits a signal to activate HPK1.
Tyrosine Phosphorylation of HPK1 upon TCR/CD3 Stimulation-Early events in TCR signaling include protein-protein interactions and tyrosine phosphorylation of signaling molecules. Previous studies showed that transfected HPK1 in COS-1 cells is tyrosine-phosphorylated upon EGF stimulation and is also recruited to the EGF receptor tail through the adapters Grb2 and Crk (24,25). Recently, adapters Crk and CrkL were also shown to form inducible complexes with tyrosine kinases Zap70 and Fyn, respectively, upon TCR/CD3 stim-ulation (34,35). These observations provide a possible link between protein-tyrosine kinases and HPK1 during T cell activation. We therefore examined whether TCR signaling could induce tyrosine phosphorylation of HPK1. HPK1 was immunoprecipitated from the TCR/CD3-stimulated Jurkat cell lysates by an anti-HPK1 antibody. The immunoprecipitates were then subjected to anti-phosphotyrosine immunoblot analyses. Our results revealed that HPK1 was tyrosine-phosphorylated as early as 2 min after TCR/CD3 stimulation, and that this signal sustained to 10 min (Fig. 2, top panel). Subsequent stripping and reprobing of the blots with another anti-HPK1 antibody confirmed that the position of HPK1 corresponded exactly to that of the phosphotyrosine band. A similar result was observed in Jurkat T cells treated with TCR/CD3 plus CD28 costimulation but not CD28 stimulation alone (Fig. 2, bottom and middle panels). Interestingly, the kinetics of HPK1 tyrosine phosphorylation was similar to that of its kinase induction, suggesting a correlation between these two biochemical events.
Activation and Tyrosine Phosphorylation of HPK1 in Pervanadate-treated Jurkat T Cells-The similar kinetics between HPK1 tyrosine phosphorylation and its kinase induction led us to further explore the possible link between these two biochemical events. To that purpose, we examined the HPK1 response in Jurkat T cells stimulated by pervanadate, a potent tyrosine phosphatase inhibitor capable of activating protein-tyrosine kinases and mimicking T cell activation (36). Our results showed that pervanadate treatment induced substantial and prolonged tyrosine phosphorylation of HPK1 in Jurkat T cells (Fig. 3A). The same pervanadate-treated Jurkat lysates were further analyzed by immunocomplex kinase assays using anti-HPK1 antibodies. HPK1 kinase activity was potently stimulated after pervanadate treatment (Fig. 3B). These results strongly suggest that tyrosine phosphorylation of HPK1 is important for regulating its kinase activity during T cell activation.
Tyrosine Kinases Lck and Zap70 Are Involved in Regulation of HPK1-We next attempted to determine which protein-tyrosine kinase(s) could regulate HPK1 during TCR stimulation. We compared HPK1 tyrosine phosphorylation in JCaM 1.6 (Lck-deficient Jurkat), P116 (Zap70-deficient Jurkat) cells, and wild-type Jurkat T cells. HPK1 tyrosine phosphorylation in-FIG. 1. Activation of HPK1 following TCR/CD3 stimulation in Jurkat T cells. A, Jurkat T cells were either untreated or treated with an anti-CD3 mAb (OKT3, 10 g/ml) or in combination with an anti-CD28 mAb (9.3, 1 g/ml). Cells were collected and lysed in 1% Triton X-100 lysis buffer at the times indicated. Cell lysates (100 g/sample) were used for anti-HPK1 immunocomplex kinase assays using MBP as a substrate. Phosphorylation of MBP was detected by autoradiograghy (top panel). Comparable HPK1 protein levels in individual lanes were confirmed by immunoblotting (bottom panel). IP, immunoprecipitation; WB, Western blot. B, human peripheral blood T cells were purified from healthy donors, then treated and analyzed similarly as described in panel A. Results were representative of five independent donors. C, Jurkat TAg cells (2 ϫ 10 7 ) were transfected with 10 g of empty vector (pCI-neo), Flag-tagged wild-type HPK1 (Flag-HPK1), or kinase-dead HPK1 mutant (Flag-HPK1-M46). 40 h after transfection, cells were left untreated or treated with an anti-CD3 mAb (OKT3, 10 g/ml) for 5 min, and then lysed in 1% Triton X-100 lysis buffer. Cell lysates (100 g) were subjected to immunocomplex kinase assays using an anti-Flag  duced by costimulation of TCR/CD3 and CD28 was significantly decreased in JCaM 1.6 and P116 cells, whereas pervanadate-induced HPK1 tyrosine phosphorylation still remained at a substantial level in these two mutant cell lines (Fig. 4A). This suggests that T cell-specific tyrosine kinases Lck and Zap70 are critical for HPK1 tyrosine phosphorylation during T cell activation. However, pervanadate treatment could still induce HPK1 tyrosine phosphorylation through activating other tyrosine kinases in addition to Lck and Zap70. By cotransfection studies, we observed that ectopic expression of Lck and Zap70 with the kinase-dead mutant HPK1(M46) led to the HPK1 tyrosine phosphorylation in COS-1 cells (Fig. 4B). Moreover, wild-type HPK1, but not the kinase-dead mutant HPK1(M46), was activated in the presence of Lck and Zap70 in transfected HEK293T cells (Fig. 4C). These results suggest that proteintyrosine kinases Lck and Zap70 mediate TCR-induced HPK1 activation.
Differential Inducible Association of HPK1 with Adapters Nck and Crk upon TCR/CD3 Stimulation-HPK1-interacting SH2/SH3 adapters (e.g. Grb2, Nck, Crk, and CrkL) may function to couple HPK1 to protein-tyrosine kinases or tyrosine phosphorylated proteins. However, whether HPK1 interacts with these adapters in T cells was unclear. Therefore, we were interested in studying complex formation between HPK1 and these adapters before and after TCR/CD3 stimulation. By a series of co-immunoprecipitation analyses, we first observed that HPK1 formed an inducible complex with Nck in Jurkat T cells after 1 min of TCR/CD3 stimulation, and this complex sustained until 10 min of TCR/CD3 stimulation (Fig. 5A). The kinetics of HPK1 interaction with Nck correlated with that of HPK1 activation, suggesting the possible involvement of Nck in the regulation of HPK1 during TCR activation. In addition, we found that the adapter Crk, unlike Nck, inducibly bound to HPK1 after 10 min of TCR/CD3 stimulation (Fig. 5B).

Constitutive Association of HPK1 with Adapters Grb2 and CrkL in Jurkat T Cells-By similar co-immunoprecipitation FIG. 3. Pervanadate treatment induces tyrosine phosphorylation and kinase induction of HPK1 in Jurkat T cells.
A, Jurkat T cells were treated with 100 M pervanadate for the indicated times. Cells were collected, lysed in 1% Nonidet P-40 lysis buffer, and subjected to immunoprecipitation with an anti-HPK1 antibody (Ab484). The immunoprecipitates were analyzed by immunoblotting with an anti-phosphotyrosine mAb (4G10). The same membrane was reprobed with an anti-HPK1 antibody (N-19). B, Jurkat T cells were treated by pervanadate as described for panel A. Cell lysates (100 g/sample) were subjected to anti-HPK1 immunocomplex kinase assay using GST-Crk as a substrate. The equal amount of HPK1 for kinase assays was demonstrated by immunoblotting with an anti-HPK1 antibody (N-19). IP, immunoprecipitation; WB, Western blot. approaches, we noticed that HPK1 formed a constitutive complex with Grb2 in Jurkat T cells before and after TCR/CD3 stimulation (Fig. 6A). In addition, a constitutive HPK1-CrkL complex was also observed in Jurkat T cells (Fig. 6B). Our results, shown in Figs. 5 and 6, demonstrate the differential complex formation between HPK1 and several SH2/SH3 adapters during TCR/CD3 stimulation, indicating an important role for HPK1 in T cell signaling.

Inducible Association of HPK1 with LAT upon TCR/CD3
Stimulation-It is known that the linker protein LAT plays a critical role in TCR signaling through interaction of its phosphotyrosine residues with the SH2 domains of several signaling molecules, including Grb2, Gads, phospholipase C-␥1, and the p85 subunit of phosphatidylinositol 3-kinase (6, 7). Because of the LAT-Grb2 interaction, we attempted to examine the were left untreated or treated with an anti-CD3 mAb (OKT3, 10 g/ml). Cells were collected at the times indicated, lysed by 1% Nonidet P-40 lysis buffer. A, cell lysates were subjected to immunoprecipitation with an anti-LAT antibody and followed by immunoblotting sequentially with anti-HPK1 (N-19) and then anti-LAT antibodies. B, Flag-HPK1 (10 g) and LAT (5 g) were transfected into COS-1 cells separately or in combination. Cells were harvested and lysed 40 h after transfection. Lysates were immunoprecipitated with an anti-Flag antibody and then subjected to the immunoblot analyses using anti-LAT and anti-HPK1 (N-19) antibodies sequentially. C, Jurkat TAg cells (2 ϫ 10 7 ) were transfected with 5 g of HA-HPK1-PR. 24 h after transfection, the cells were left untreated or treated with an anti-CD3 mAb for 1 min. Cells were harvested, lysed in 1% Nonidet P-40 lysis buffer, and subjected to immunoprecipitation using an anti-HA mAb. The immunoprecipitates and direct Jurkat lysates were resolved by 10% SDS-PAGE and followed by immunoblotting using an anti-LAT antibody. Expression of HA-HPK1-PR was examined by immunoblotting with an anti-HA mAb. D, Jurkat TAg cells (2 ϫ 10 7 ) were transfected with 10 g of HA-JNK alone, or together with 30 g of HPK1-PR, MKK7-K76E, or MKK4-AL. 24 h after transfection, cells were left untreated or treated with a combination of anti-CD3 and anti-CD28 mAbs for 20 min. Cells were then harvested, lysed in 1% Triton X-100 lysis buffer, and subjected to immunocomplex kinase assays using an anti-HA mAb and GST-c-Jun as a substrate. Phosphorylation of GST-c-Jun was detected by autoradiography. The result is representative of three independent experiments. IP, immunoprecipitation; WB, Western blot. potential association between LAT and HPK1 in T cells. We performed the co-immunoprecipitation analyses and found that LAT formed an inducible complex with HPK1 as early as 1 min after TCR/CD3 stimulation (Fig. 7A). To confirm this interaction, we also utilized a cotransfection system to demonstrate that overexpression of LAT and HPK1 led to the LAT-HPK1 complex formation in transfected COS-1 cells (Fig. 7B). We further determined the region(s) of HPK1 for LAT association. Two HPK1 truncated mutants, the HPK1 kinase domain (Flagtagged HPK1-KD) and proline-rich region (HA-tagged HPK1-PR), were transfected into Jurkat-Tag cells to test their ability to interact with LAT. Interestingly, our data showed that only HPK1-PR inducibly associated with LAT upon TCR/CD3 stimulation (Fig. 7C). HPK1-KD did not associate with LAT (data not shown).
After exploring the regulation of HPK1 by TCR signaling, we next investigated whether HPK1 mediates TCR signaling for JNK activation during T cell costimulation. HPK1-PR and two MAP2K mutants, MKK4-AL and MKK7-K76E, were cotransfected with HA-JNK, respectively, into Jurkat TAg cells to examine their effect on JNK activation by TCR/CD3 and CD28 costimulation. We found that HPK1-PR failed to block JNK activation, whereas two MAP2K mutants, MKK4-AL and MKK7-K76E, blocked JNK activation effectively (Fig. 7D). Although the JNK kinase assay was very sensitive to HA-JNK activity, we were unable to detect HA-JNK protein levels in transfected Jurkat T cells due to low transfection efficiency (data not shown). We did detect HPK1-PR expression as shown in Fig. 7C. Thus, failure to block JNK activation by HPK1-PR was not due to the low level of HPK1-PR expression.
Recruitment of HPK1 into GEMs (or Lipid Rafts) upon TCR/ CD3 Stimulation-Emerging evidence has indicated that GEMs (or lipid rafts) play an important role in T cell signaling (5). Disruption of these lipid microdomains results in the downregulation of TCR signaling and consequently attenuated T cell activation. In T cells, many critical signaling molecules are enriched in GEMs constitutively or upon TCR/CD3 stimulation. They include tyrosine kinases (e.g. Lck and Syk), adapters (e.g. LAT and Grb2), and other signaling molecules (e.g. Ras and phospholipase C-␥1) (5,37). Given the evidence that HPK1 inducibly interacted with LAT, we tested whether HPK1 could be recruited into GEMs or lipid rafts upon TCR/CD3 stimulation. Our results showed that HPK1 rapidly translocated into GEMs upon TCR/CD3 stimulation (Fig. 8, middle panel). However, HPK1 from unstimulated Jurkat T cells only remained in the Triton X-100-soluble fractions (Fig. 8, top panel). We also showed the presence of LAT in GEMs or lipid rafts as a control for this analysis (Fig. 8, bottom panel). This result suggests that the inducible LAT-HPK1 complex may recruit HPK1 into GEMs, leading to activation of HPK1 by protein-tyrosine kinases. DISCUSSION Advances have been made in the characterization of signaling molecules (e.g. Lck, Zap70, and LAT) proximal to the TCR and downstream effectors (e.g. AP-1, NF-AT, NF-B, and JNK) involved in interleukin-2 production. However, which and how the intermediate modulators integrate both the TCR and CD28 signals to downstream effectors for T cell activation remain unclear. Here we have investigated the role of a hematopoieticspecific JNK activator, HPK1, in T cell signaling. We first noticed the early kinase induction (1-2 min) of HPK1 following TCR/CD3 engagement in both Jurkat and human peripheral blood T cells, suggesting a close link between HPK1 and TCR/ CD3-proximal signaling events. This idea is further supported by the observations that HPK1 was tyrosine-phosphorylated at early time points following TCR/CD3 stimulation, and that HPK1 was potently tyrosine-phosphorylated and activated by pervanadate stimulation.
By cotransfection studies and the analysis of two Jurkat mutant cell lines JCaM 1.6 (Lck-deficient) and P116 (Zap70deficient), we observed that Lck and Zap70 were essential for TCR-mediated HPK1 tyrosine phosphorylation. Moreover, we provided evidence that ectopic expression of Lck and Zap70 with HPK1 led to both HPK1 tyrosine phosphorylation and kinase induction. Our results are supported by a recent paper (39) indicating that TCR-induced HPK1 activation is abolished in both Lck-deficient and Zap70-deficient Jurkat T cells. Future studies mapping the tyrosine phosphorylation site(s) within HPK1 will provide more insight into the regulation of HPK1 by Lck and Zap70. Interestingly, our data showed that HPK1 displayed a more significant level of tyrosine phosphorylation in pervanadate-treated JCaM 1.6 (Lck-deficient) cells than pervanadate-treated P116 (Zap70-deficient) cells, implying that Zap70 plays a critical role for HPK1 tyrosine phosphorylation in T cells. It is likely that in addition to Lck and Zap70, there are other protein-tyrosine kinases capable of activating HPK1. For instance, c-Abl tyrosine kinase inducibly binds to HPK1 in Jurkat T cells in response to DNA-damaging agents, and this c-Abl-HPK1 interaction results in HPK1 phosphorylation and activation (38).
Our results showed that CD28 stimulation was unable to induce HPK1 kinase induction and tyrosine phosphorylation and also had no further effect on TCR/CD3-induced HPK1 activation. This suggests that, unlike JNK, HPK1 is mainly activated by TCR/CD3 stimulation. However, we do not exclude the possibility that CD28 may play a role in the regulation of HPK1 by other mechanisms, such as HPK1 localization and complex formation. Although HPK1 is a JNK activator and is implicated in TCR signaling, our data from transfection studies showed that the HPK1 mutant HPK1-PR, responsible for interaction with LAT and SH2/SH3 adapters, failed to block JNK FIG. 8. Localization of HPK1 to GEMs upon TCR/CD3 stimulation. Jurkat T cells (5 ϫ 10 7 ) were either left unstimulated or stimulated with an anti-CD3 mAb (OKT3, 10 g/ml) for 2 min. Cells were harvested, lysed in 1 ml of 1% Triton X-100 lysis buffer, and mixed with 1 ml of 80% sucrose. These lysates were overlaid with 2 ml of 30% sucrose and 1 ml of 5% sucrose sequentially and then subjected to ultracentrifugation overnight (16 -20 h) at 4°C. Gradient fractions were collected in 0.4-ml aliquots from the top. These aliquots were subjected to immunoblotting using anti-HPK1 (Ab484) and anti-LAT antibodies. WB, Western blot. activation by TCR plus CD28 costimulation. Recently, Liou et al. (39) also showed similar results by using a kinase-inactive HPK1 mutant. These results suggest that HPK1 is not indispensable for JNK activation during T cell costimulation. One possibility is that other MAP4Ks may play a redundant role in JNK activation in T cells. For example, other HPK1-related kinases, such as GCK and GCK-like kinase , are also expressed in T cells and contain proline-rich motifs for interaction with SH2/SH3 adapters, suggesting their potential roles in TCR or CD28 signaling.
Our result showed that HPK1 was recruited into GEMs after 2 min of TCR/CD3 stimulation. This finding further suggests that HPK1 participates in the early events of TCR signaling. In addition, the recruitment of HPK1 into GEMs is possibly involved in HPK1 activation and tyrosine phosphorylation. The underlying mechanism by which HPK1 is recruited into GEMs is still unclear. One possibility is that upon TCR/CD3 stimulation, SH2/SH3 adapters couple HPK1 to tyrosine-phosphorylated LAT or other signaling molecules localized in GEMs where HPK1 is activated and tyrosine-phosphorylated by proteintyrosine kinases. The finding of early inducible association between HPK1 and LAT upon TCR/CD3 stimulation supports this possibility.
We found that HPK1 differentially interacted with various adapters, including LAT, Nck, Crk, CrkL, and Grb2, in Jurkat T cells. HPK1 interaction with the adapters Grb2 and CrkL was found to be constitutive at all time points we tested, whereas HPK1 interaction with Nck and LAT was induced after 1 min of TCR/CD3 stimulation. The interaction of HPK1 with Crk, however, was induced at a later time point 10 min after TCR/CD3 stimulation. The early inducible association of HPK1 with Nck and LAT following TCR/CD3 stimulation suggests the potential roles for Nck and LAT in TCR-mediated HPK1 activation. Since LAT does not contain an SH3 domain for its direct binding to the HPK1 proline-rich motifs, the inducible HPK1-LAT complex formation is likely through SH2/ SH3 adapters. One possibility is that the constitutive HPK1-Grb2 complex may be recruited to tyrosine-phosphorylated LAT upon TCR/CD3 stimulation. Recently, others have shown that HPK1 interacts with adapters Grap and Gads in T cells (39,40,41). Because these two adapters are also shown to interact with LAT (7), they are potential candidates to couple HPK1 to LAT. More studies are needed to reveal the underlying mechanism of LAT-HPK1 complex formation in T cells. The constitutive HPK1-CrkL complex may participate in the TCRmediated signaling events through CrkL binding to Fyn upon TCR/CD3 stimulation (35). Unlike the previous finding that CrkL binds and activates HPK1 in an overexpression system (24), the constitutive HPK1-CrkL interaction in Jurkat T cells did not lead to HPK1 activation. The possible reason is that in transfected cells, overexpressed CrkL and HPK1 may circumvent regulation by other endogenous or tissue-specific factors to amplify the signaling pathways. In contrast, these factors may tightly regulate endogenous CrkL and HPK1 in Jurkat T cells. Thus, this could be one reason why the constitutive CrkL-HPK1 interaction did not lead to HPK1 activation in Jurkat T cells. Finally, we have reported the late inducible association between HPK1 and Crk. The functional relevance of this late interaction is unclear. One possibility is that Crk couples HPK1 to other downstream signaling events in T cells. In addition to the inducible Crk-HPK1 complex, we observed a low level of constitutive Crk-HPK1 complex in Jurkat T cells (data not shown). Therefore, Crk may play a role in coupling HPK1 to Zap70 upon TCR/CD3 stimulation. HPK1 association with multiple adapters in T cells implies an important role for HPK1 in T cell signaling. Future studies will focus on dissecting the multiple HPK1-mediated signaling pathways in T cells.