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• W1990970872 abstract "Article8 December 2005free access Activation or suppression of NFκB by HPK1 determines sensitivity to activation-induced cell death Dirk Brenner Dirk Brenner Tumor Immunology Program, German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Alexander Golks Alexander Golks Tumor Immunology Program, German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Friedemann Kiefer Friedemann Kiefer Max-Planck-Institute for Molecular Biomedicine, Münster, Germany Search for more papers by this author Peter H Krammer Peter H Krammer Tumor Immunology Program, German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Rüdiger Arnold Corresponding Author Rüdiger Arnold Tumor Immunology Program, German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Dirk Brenner Dirk Brenner Tumor Immunology Program, German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Alexander Golks Alexander Golks Tumor Immunology Program, German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Friedemann Kiefer Friedemann Kiefer Max-Planck-Institute for Molecular Biomedicine, Münster, Germany Search for more papers by this author Peter H Krammer Peter H Krammer Tumor Immunology Program, German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Rüdiger Arnold Corresponding Author Rüdiger Arnold Tumor Immunology Program, German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Author Information Dirk Brenner1, Alexander Golks1, Friedemann Kiefer2, Peter H Krammer1 and Rüdiger Arnold 1 1Tumor Immunology Program, German Cancer Research Center (DKFZ), Heidelberg, Germany 2Max-Planck-Institute for Molecular Biomedicine, Münster, Germany *Corresponding author. Tumor Immunology Program, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69112 Heidelberg, Germany. Tel.: +49 6221 423769; Fax: +49 6221 411715; E-mail: [email protected] The EMBO Journal (2005)24:4279-4290https://doi.org/10.1038/sj.emboj.7600894 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Restimulation of the T-cell receptor (TCR) in activated T cells induces CD95 (Fas/Apo-1)-mediated activation-induced cell death (AICD). The TCR-proximal mechanisms leading to AICD are elusive. Here we characterize hematopoietic progenitor kinase 1 (HPK1) as a differentially regulated TCR-proximal signaling protein involved in AICD of primary T cells. We show that HPK1 is a functional component of the endogenous IκB kinase (IKK) complex and is crucial for TCR-mediated NFκB activation. While full-length HPK1 enhances IKKβ phosphorylation, siRNA-mediated knockdown of HPK1 blunts TCR-mediated NFκB activation and increases cell death. We also demonstrate proteolytic processing of HPK1 into HPK1-C, specifically in AICD-sensitive primary T cells. The cleavage product HPK1-C sequesters the inactive IKK complex and suppresses NFκB upon TCR restimulation by binding to IKKα and IKKβ. T cells of HPK1-C transgenic mice are sensitized towards TCR-mediated AICD. Consequently, preventing HPK1-C generation in primary T cells by siRNA-mediated knockdown results in decreased AICD. Thus, these results show a novel mechanism of sensitization of T lymphocytes towards AICD by suppression of NFκB, and propose that HPK1 is a life/death switch in T lymphocytes. Introduction Life and death of peripheral lymphocytes is strictly controlled to maintain physiological levels of T and B cells. The regulation of T-cell apoptosis during the immune response is controlled by activation-induced cell death (AICD) in response to T-cell receptor (TCR) triggering (reviewed in Green, 2003; Krueger et al, 2003). While initial stimulation of primary T cells from peripheral blood leads to proliferation, restimulation of expanded T cells results in AICD (Brunner et al, 1995; Dhein et al, 1995). Therefore, primary T cells shift from AICD resistance towards AICD sensitivity. In contrast to the well-documented role for death receptors (Zheng et al, 1995; Li-Weber and Krammer, 2003; Peter and Krammer, 2003) and mitochondrial pathways (Hildeman et al, 2002) in sensitization to AICD, little is known about the contribution of TCR-proximal signaling proteins, which modulate the TCR signal in a way that it induces activation and survival or death by AICD. Regulation of lymphocyte fate and the execution of immune functions are connected to transcription factors of the NFκB family (Hildeman et al, 2002; Karin and Lin, 2002; Ruland and Mak, 2003). NFκB/Rel proteins determine life and death decisions in developing lymphocytes, immune responses and cell growth, and provide important signals in T cells to ensure cell survival (Ghosh and Karin, 2002; Kane et al, 2002; Schmitz et al, 2003). The inhibition of NFκB is considered to fulfill an important proapoptotic function (Pham et al, 2004; Kamata et al, 2005). NFκB family transcription factors are rendered inactive within the cytoplasm by interaction with IκB proteins. Upon TCR stimulation, a high-molecular-weight IκB kinase (IKK) complex is activated. The IKK complex is comprised of two enzymatic subunits IKKα and IKKβ, and the regulatory subunit IKKγ (NEMO). Activation of the IKK complex results in phosphorylation of the NFκB-inhibitory IκB proteins mediated by IKKβ and subsequent ubiquitination and degradation of IκB proteins. Derepressed NFκB dimers translocate into the nucleus and activate NFκB-regulated genes (Hayden and Ghosh, 2004). Hematopoietic progenitor kinase 1 (HPK1) is comprised of a N-terminally located kinase domain and a C-terminally located regulatory domain, called citron homology domain. By ectopic expression in epithelial cells, full-length HPK1 is rendered active and selectively activates the SAPK/JNK and the NFκB pathways (Kiefer et al, 1996; Arnold et al, 2001). In vitro, the N-terminal kinase domain of HPK1 can be separated from the citron homology domain by a caspase-3 activity, resulting in suppression of NFκB (Arnold et al, 2001). HPK1 kinase activity is strongly enhanced by TCR crosslinking (Liou et al, 2000; Liu et al, 2000) and involves phosphorylation by protein kinase D1 (Arnold et al, 2005). Here we show that HPK1 is a differentially regulated TCR-proximal signaling protein proteolytically processed into the C-terminal cleavage fragment, HPK1-C, in expanded AICD-sensitive primary T cells. HPK1-C suppresses NFκB and sensitizes T cells towards TCR-mediated cell death. For full length HPK1, we show a novel association to the endogenous IKK complex and a crucial role in TCR-mediated IKKβ activation. In contrast, HPK1-C leads to suppression of NFκB by sequestering the IKK complex. T cells of HPK1-C transgenic (tg) mice show a suppressed TCR-mediated IKK activity and are sensitized towards AICD. Furthermore, siRNA-mediated knockdown of HPK1 in Jurkat or in primary naive T cells results in enhanced AICD, whereas preventing the generation of proapoptotic HPK1-C in primary preactivated (day 6) T cells results in decreased AICD. Results AICD-sensitive primary T cells show conversion of HPK1 into the C-terminal cleavage fragment HPK1-C We performed a microarray-based screen to identify differentially expressed molecules modulating TCR signaling. We compared AICD-resistant to AICD-sensitive primary human T cells and found upregulation of HPK1 specifically in AICD-sensitive cells. Increased expression of HPK1 was confirmed by real-time RT–PCR (Figure 1A) and by Western blotting (WB) (Figure 1B, upper panel). Upon activation and expansion, cultured T cells shift from an AICD-resistant to an AICD-sensitive phenotype (Peter et al, 1997). Interestingly, we detected conversion of full-length HPK1 into the C-terminal cleavage fragment, HPK1-C, at day 6 of culture, when T cells show the highest sensitivity towards AICD (Figure 1B, lower panel). The cleavage of HPK1 therefore correlates with the sensitization of T cells towards AICD. Remarkably, HPK1-C does not result from background apoptosis of expanding cells since the viability during the culture period (day 1–6) is constantly increasing (data not shown). Figure 1.HPK1-C suppresses TCR-mediated NFκB activation and augments apoptosis. Expansion of T cells leads to increased HPK1 expression and conversion to HPK1-C. Primary human T cells isolated from peripheral blood were stimulated with PHA and expanded in vitro. Samples were taken at the indicated culture day and HPK1 was quantified by real-time RT–PCR (A) or by WB using the indicated Abs (B). Viability of the expanded T cells was higher than 90% at the time points when HPK1-C could be detected. (C) Jurkat T cell pools harboring stable integration of either empty vector or HPK1-N or HPK1-C expression vectors were analyzed for specific cell death after incubation with 10 μg/ml plate-bound anti-CD3 Abs for 24 or 48 h. Expression of HPK1 or HPK1-C or HPK1-N was analyzed in lysates from the indicated Jurkat T cell pools by WB using Abs against HPK1 or HPK1-C or MYC, respectively. (D) Jurkat T cell pools shown in C-expressing HPK1-C or control Jurkat T-cell pools (vector) were analyzed for specific cell death after incubation with 0.3 or 3 μg/ml plate-bound anti-CD3 Abs (left panel) or with dilutions of soluble CD95L at 0.3 × 10−4 or 3 × 10−4 (middle panel) or with 67 or 200 nM staurosporine (Stauro, right panel) for 18 h. (E) The Jurkat T cell pools shown in (D) were transiently transfected with an NFκB-specific reporter gene system and stimulated by plate-bound anti-CD3 or left nonstimulated (Control) for 8 h. Equal expression of CD3 and comparable stimulation by CD3 was controlled by flow cytometry or WB using anti-phospho-JNK1/2-specific Abs. (F) Primary human T cells at day 1 or day 6 of culture were stimulated by anti-CD3 Abs (CD3) for 1 or 2 h or left nonstimulated (0). Nuclear extracts were subjected to EMSA using 32P-labeled oligonucleotides containing an NFκB (left panel) or an NF-Y (right panel)-binding site. Equal binding of the constitutively active transcription factor NF-Y to its binding site serves as a loading control. Longer exposure (top middle panel) and specificity control for the NFκB EMSA (bottom middle panel). EMSA reactions were coincubated with 25- or 100-fold excess of unlabeled NFκB-specific (spec.) or NFκB-unspecific NF-Y-binding site (unsp.). Values given depict the average and standard deviation of triplicate measurements. Download figure Download PowerPoint HPK1-C suppresses TCR-mediated NFκB activation and augments apoptosis To investigate whether HPK1 cleavage products have an impact on TCR-induced apoptosis, we generated stably transfected Jurkat T cell pools expressing HPK1-N or HPK1-C. HPK1-C- but not HPK1-N-expressing Jurkat T cells showed strongly enhanced TCR-mediated cell death (Figure 1C). This HPK1-C-mediated increase in cell death was only seen after TCR stimulation, while treatment with CD95L or staurosporine did not enhance cell death in HPK1-C-expressing Jurkat T cells (Figure 1D). As previously reported for non-T cells (Arnold et al, 2001), HPK1-C expressing Jurkat T cells showed suppressed TCR-mediated NFκB activation (Figure 1E). Therefore, sensitization towards cell death by HPK1-C correlates with suppression of NFκB. As reported previously, we did not find any influence of HPK1-C on the SAPK/JNK pathway upon stimulation of the TCR (Schulze-Luehrmann et al (2002) and Figure 6E). To further substantiate the involvement of HPK1 in TCR-mediated NFκB induction, we compared nuclear lysates of day 1–6 T cells by electrophoretic mobility shift assay (EMSA) (Figure 1F). While NFκB induction in day 1 T cells was clearly visible, NFκB induction, but not constitutive NF-Y activity, was significantly decreased in the HPK1-C containing day 6 T cells and could only detected upon longer exposure. In accordance with the prosurvival role for NFκB in T cells, the lack of NFκB activation in HPK1-C-expressing Jurkat T cells or primary day 6 T cells clearly correlates with enhanced TCR-mediated cell death. This result suggests a role for HPK1-C in sensitization towards AICD by suppression of the NFκB pathway. Figure 2.Full-length HPK1 interacts specifically with IKKβ, while HPK1-C can associate with IKKα and IKKβ. COS1 cells were transiently transfected with plasmids encoding FLAG-tagged versions of IKKα or IKKβ or IKKγ alone or in combination with HA-tagged full-length HPK1 (A, B) or MYC-tagged HPK1-N (C) or T7-tagged HPK1-C (D). Following anti-FLAG immunoprecipitation, the presence of coimmunoprecipitated HPK1 variants was analyzed by WB using tag-specific Abs. Download figure Download PowerPoint Full-length HPK1 interacts specifically with IKKβ, while HPK1-C can associate with IKKα and IKKβ While several studies demonstrated that HPK1 activates NFκB transcriptional activity (Arnold et al, 2001; Schulze-Luehrmann et al, 2002), the exact mechanism of the HPK1-mediated NFκB activation is not known. To delineate this mechanism, we tested for direct interaction of HPK1 with IKKs IKKα and IKKβ. COS-1 cells were transfected with IKKα or IKKβ alone or in combination with HPK1. Coimmunoprecipitation revealed a specific interaction of full-length HPK1 with IKKβ (Figure 2A). We did not detect interaction of full-length HPK1 with IKKα or IKKγ (Figure 2B). While HPK1-N neither interacts with IKKα nor with IKKβ (Figure 2C), HPK1-C does associate with IKKα and IKKβ (Figure 2D). Here, we show for the first time that HPK1 and HPK1-C interact with components of the NFκB-activating IKK complex. In addition, this result suggests that HPK1-mediated activation and HPK1-C-mediated suppression of the NFκB pathway share the same molecular targets. Furthermore, our data support the finding that HPK1-N does not influence NFκB activation (Arnold et al, 2001). Figure 3.Preassociation of endogenous HPK1 with the IKK complex dissociates upon TCR stimulation. (A) BJAB cell pools selected for stable expression of high or low levels of FLAG-tagged IKKβ were subjected to anti-FLAG (upper panels) or anti-HPK1 (lower panels) immunoprecipitation. The presence of endogenous coimmunoprecipitated HPK1 was shown by WB using HPK1-specific Abs (upper panels). Precipitated IKKβ was analyzed by WB using a FLAG-tag specific Ab (lower panels). (B) DC27.1 T cells were stimulated with anti-CD3 Abs for the indicated time or left nonstimulated. The endogenous IKK complex was immunoprecipitated using anti-IKKγ Abs and tested for the presence of coimmunoprecipitated endogenous HPK1 by anti-HPK1 WB. (C) Primary human T cells at day 1 of culture were used to immunoprecipitate the endogenous IKK complex using anti-IKKγ Abs and tested for the presence of coimmunoprecipitated endogenous HPK1 by anti-HPK1 WB. A nonprecipitating Ab was used to control for specificity. Download figure Download PowerPoint Preassociation of endogenous HPK1 with the IKK complex is released upon TCR stimulation To test the interaction of IKKβ with endogenous HPK1, we used tg BJAB cells expressing different levels of FLAG-tagged IKKβ. Immunoprecipitation of FLAG-tagged IKKβ showed copurification of endogenous HPK1, depending on the amount of IKKβ present in the BJAB cells (Figure 3A, top panels). Furthermore, immunoprecipitation of endogenous HPK1 revealed copurification of exogenous IKKβ depending on the amount of IKKβ expressed (Figure 3A, bottom panels). This demonstrates a constitutive and specific association of endogenous HPK1 with IKKβ in lymphoid cells. Figure 4.HPK1 stimulates IKK activity by enhancing phosphorylation of IKKβ. (A) COS1 cells were transiently transfected with plasmids encoding FLAG-tagged wt IKKβ or the ATP-binding site mutant IKKβ(K44A) alone or in combination with the HA-tagged wt HPK1 or the ATP-binding site mutant HPK1(K46E). After anti-FLAG immunoprecipitation, the IKKβ proteins were tested for their ability to transphosphorylate in vitro a recombinant GST:IκBα protein using 32Pγ-ATP. Phosphorylated GST:IκBα was separated by SDS–PAGE and visualized by autoradiography (upper panel). The presence of immunoprecipitated IKKβ protein and the expression level of HPK1 were shown by WB (lower panels). (B) Purified GST:HPK1 expressed in COS 1 cells was used to set up a reconstituted in vitro kinase assay system. The kinase reaction was performed in the presence of recombinant GST:IκBα using various amounts of GST:HPK1 alone or in combination with purified FLAG-tagged IKKβ. Reaction products were separated by SDS–PAGE and visualized by autoradiography (upper panels). The presence of immunoprecipitated IKKβ protein is shown by WB (lower panel). (C) COS1 cells were transiently transfected with FLAG-tagged ATP-binding site mutant IKKβ(K44A) or the HA-tagged wt HPK1 alone or in combination. After anti-FLAG immunoprecipitation, the IKKβ proteins were tested for phosphorylation of Ser181 by WB using anti-phospho-IKKβ Abs. The presence of immunoprecipitated IKKβ protein is shown by anti-IKKβ WB. All experiments depicted show one out of three experiments with identical outcome. Download figure Download PowerPoint To further confirm the involvement of HPK1 in NFκB activation in lymphoid cells, we pulled down all components of the IKK complex by precipitation of IKKγ (NEMO) (Tegethoff et al, 2003; Quirling et al, 2004). We detected endogenous HPK1 from nonstimulated DC27.1 T cells to be part of the endogenous IKK complex (Figure 3B, first lane). Surprisingly, the association of HPK1 with the IKK complex was lost immediately after TCR stimulation, while a re-association of HPK1 could be detected after 45 min of stimulation (Figure 3B). Neither HPK1 nor IKKβ show a significant activity in nonstimulated T cells, but both proteins are activated with a fast kinetic directly following TCR ligation (Liou et al, 2000; Liu et al, 2000). Whereas HPK1 activity is reported to peak at 2 min after stimulation, IKKβ activity is maximal after 15 min. According to our data, HPK1 dissociates from the IKK complex in a stimulation-dependent manner, while the reassociation correlates with the decline of IKKβ and HPK1 activity. Furthermore, we found that HPK1 could be specifically coimmunoprecipitated with the endogenous IKK complex from primary human day 1 T cells (Figure 3C). This result further supports the physiological relevance of the HPK1–IKKβ interaction. HPK1 stimulates IKK activity by increasing phosphorylation of IKKβ So far, the molecular mechanism for HPK1-mediated NFκB activation was unknown. To elucidate this, we expressed both proteins in COS1 cells and tested the immunoprecipitated IKKβ for its ability to phosphorylate the IKKβ-specific substrate, GST:IκBα. While IKKβ expressed alone showed a basal activity (Figure 4A, first lane), the coexpression of HPK1, but not the kinase-deficient mutant HPK1(K46E), strongly stimulated IKKβ activity (Figure 4A). This result indicates that HPK1 kinase can activate IKKβ. Figure 5.Full-length HPK1 is crucial for TCR-mediated NFκB activation and survival of T cells. (A) Jurkat T cells were transiently transfected with double-stranded siRNA oligonucleotides comprising a HPK1-specific sequence (HPK1-(5)) or a nonspecific sequence (control). At 48 h after transfection, cells were stimulated with anti-CD3 Abs for the indicated time or left nonstimulated; endogenous IKKγ proteins were immunoprecipitated and the co-precipitated IKK proteins were tested for their ability to phosphorylate in vitro a recombinant GST:IκBα protein using 32Pγ-ATP. The siRNA-mediated downregulation of HPK1 was compared to actin by WB. (B) Jurkat T cells were transfected as in (A) and stimulated by anti-CD3 Abs for 2 h or left nonstimulated (0). Nuclear extracts were subjected to EMSA as described in Figure 1F. siRNA-mediated downregulation of HPK1 was compared to actin by WB. (C) Jurkat T cells were transfected as in (A) and stimulated with 30 μg/ml plate-bound anti-CD3 or 5 ng/ml soluble anti-CD95 Abs for 18 h and analyzed by flow cytometry. Values given depict the average and standard deviations of triplicate measurements. The experiment was repeated four times with similar outcomes. Download figure Download PowerPoint We further analyzed whether HPK1 directly phosphorylates IKKβ and thereby enhances its activity. Therefore, we established a reconstituted in vitro kinase assay system by combining purified GST:HPK1 and FLAG-IKKβ. GST:HPK1 purified from COS1 cells is capable of autophosphorylation, but does not show kinase activity towards the IKKβ substrate GST:IκBα (Figure 4B, first lane). In contrast, IKKβ shows only marginal autophosphorylation and weak kinase activity towards its substrate GST:IκBα (Figure 4B, second lane). Already, traces of purified GST:HPK1 added to IKKβ lead to an increase in IKKβ phosphorylation and kinase activity towards GST:IκBα (Figure 4B). To further support a direct phosphorylation of IKKβ by HPK1, we expressed increasing amounts of HPK1 with the kinase-deficient IKKβ(K44A) harboring a point mutation in the ATP-binding site (Figure 4C). Indeed, we detected phosphorylation of IKKβ(K44A) in the presence of HPK1, which is likely to be mediated by HPK1 and cannot be caused by the kinase-deficient IKKβ(K44A). Therefore, we conclude that direct IKKβ phosphorylation by HPK1 contributes to the activation of the IKK complex. However, our experimental system does not firmly rule out the existence of copurified factors, which would help to positively regulate NFκB activity. Full-length HPK1 is crucial for TCR-mediated NFκB activation and survival of T cells Jurkat T cells express full-length HPK1 only. To investigate the role of full-length HPK1 in TCR-mediated IKK activation in Jurkat T cells, we used siRNA-mediated knockdown of endogenous human HPK1. Surprisingly, TCR-mediated IKK activation was completely blocked in HPK1-deficient Jurkat T cells (Figure 5A). Furthermore, upon prolonged TCR stimulation, NFκB activation was even dropping below baseline in HPK1-deficient Jurkat T cells, while the constitutive NF-Y activity was remaining constant (Figure 5B). These results imply that HPK1 is crucial for TCR-mediated NFκB activation and that the loss of HPK1 cannot be compensated by other NFκB-activating molecules in Jurkat T cells. Consistent with the antiapoptotic, prosurvival role of NFκB, TCR stimulation leads to enhanced cell death in HPK1-deficient Jurkat T cells (Figure 5C), further supporting the role of NFκB signaling pathways in AICD. Our siRNA approach did not prevent TCR signaling in general, as TCR-induced tyrosine phosphorylation detected by antiphosphotyrosine antibodies (Abs) was not impaired (data not shown). Furthermore, the cell death sensitization was specific for TCR-induced cell death as death via CD95 stimulation was not altered (Figure 5C). In summary, we propose a prosurvival role of full-length HPK1 due to activation of NFκB. Figure 6.HPK1-C binding blocks TCR-mediated IKK activation. (A) COS1 cells were transiently transfected with plasmids encoding FLAG-tagged IKKβ with or without HA-tagged HPK1 or the T7-tagged HPK1-C. After anti-FLAG immunoprecipitation, the IKK proteins were tested for their ability to autophosphorylate and to transphosphorylate in vitro a recombinant GST:IκBα protein using 32Pγ-ATP. (B) The HPK1-C stably expressing the Jurkat T-cell pool depicted in Figure 1C or parental Jurkat T cells were stimulated with anti-CD3 Abs for the indicated time or left nonstimulated. Endogenous IKKγ proteins were immunoprecipitated and the co-precipitated IKK proteins were tested for their ability to phosphorylate in vitro a recombinant GST:IκBα protein using 32Pγ-ATP. (C) HPK1-C stably expressing Jurkat T cells were stimulated as in B, T7-tagged HPK1-C proteins were immunoprecipitated and the co-precipitated endogenous IKKβ proteins were shown by WB. Protein expression levels in the lysates were controlled by WB. Efficiency of TCR stimulation was controlled by WB using anti-phospho-JNK1/2 (P-JNK1/2) Abs. (D) HPK1-C stably expressing or parental Jurkat T cells were stimulated with TNFα or PMA/ionomycin (P/I) for the indicated time or left nonstimulated. Lysates were subjected to WB using the indicated Abs. (E) HPK1-C stably expressing or parental Jurkat T cells were stimulated as in (B) and lysates were controlled by WB using anti-phospho-JNK1/2 (P-JNK1/2) and anti-JNK1/2 Abs. Download figure Download PowerPoint HPK1-C binding blocks TCR-mediated IKK activation Full-length HPK1, capable of activating IKKβ, and the NFκB-inhibitory cleavage fragment HPK1-C are present at day 6 in primary human T cells (Figure 1B). To investigate whether HPK1-C has the capacity to competitively inhibit HPK1-mediated IKKβ activation, we transfected COS1 cells with HPK1 and IKKβ with or without HPK1-C and tested for activation of IKKβ (Figure 6A). While HPK1 led to enhanced phosphorylation of IKKβ and pronounced activation of IKKβ kinase activity, addition of HPK1-C resulted in a nearly complete suppression of IKKβ activity (Figure 6A, right lane). To elucidate the molecular mechanism of HPK1-C on modulation of the NFκB pathway, we analyzed HPK1-C-expressing Jurkat T cells (Figure 1) for association of HPK1-C with the endogenous IKK complex. As expected, the HPK1-C-expressing Jurkat T cells showed a pronounced suppression of IKK activity after TCR stimulation (Figure 6B, right panels) compared to the parental Jurkat T cells (Figure 6B, left panels). In contrast to full-length HPK1 that leaves the IKK complex upon activation (Figure 3B), HPK1-C remained bound to endogenous IKKβ after TCR stimulation (Figure 6C, top panel). As expected, the presence of HPK1-C did not interfere with the stimulation of the SAPK/JNK pathway by the TCR (Figure 6C, bottom panel). Besides a slight decrease in phosphorylated Akt, stimulation of various downstream signaling pathways by TNFα or PMA/ionomycin does not seem to be altered significantly in the presence of HPK1-C (Figure 6E). As already mentioned, TCR-mediated stimulation of the SAPK/JNK pathway in the presence of HPK1-C remained unaffected (Figure 6E). This result suggests that constant association of HPK1-C with IKKβ blocks specifically TCR-induced IKK activation and thereby directly modulates TCR-proximal signaling. We conclude that binding of HPK1-C to either IKKα or IKKβ is sufficient for suppression of the canonical, IKKβ-mediated pathway of NFκB activation. HPK1-C mediates AICD in primary T cells by inhibition of IKK activation As demonstrated for primary human T cells (Figure 1B), AICD-sensitive mouse T cells at culture day 4 (Baumann et al, 2005) also show conversion of full-length HPK1 into HPK1-C (Figure 7A). To clarify how HPK1-C would influence IKK activation and AICD of primary T cells, we generated HPK1-C tg mice and compared primary mouse T cells from these mice to their wild-type (wt) littermates. As expected, analysis of IKK activation after TCR stimulation showed a strong reduction of IKK kinase activity and NFκB activation in primary HPK1-C tg T cells (Figure 7B and C). The HPK1-C-mediated suppression of IKK activation, which results in decreased NFκB activation (Figure 7C), is reflected in decreased expression of the NFκB target gene Bcl-2A1 in tg mouse T cells (Figure 7D) upon TCR stimulation. Besides a slight decrease in phosphorylated Akt, TCR-mediated stimulation of various downstream signaling pathways does not seem to be altered in the presence of HPK1-C (Figure 7E), demonstrating that HPK1-C is specifically suppressing the NFκB signaling pathway. Suppression of NFκB activation by HPK1-C again correlated with enhanced cell death in the AICD-sensitive HPK1-C tg T cells (culture day 5) compared to their wt littermates (Figure 7E). According to the previous finding showing that the sensitization to cell death by HPK1-C does not depend on higher sensitivity towards CD95L (Figure 1), also primary HPK1-C tg T cells do not show elevated apoptosis in response to CD95L (data not shown). In conclusion, this result implies that HPK1-C enhances AICD in primary T cells by inhibition of NFκB activation. Figure 7.HPK1-C mediates AICD in primary T cells by inhibition of IKK activation. (A) HPK1 is converted to its C-terminal fragment, HPK1-C, during expansion of primary mouse T cells after concanavalin A stimulation in vitro. Samples were taken at the indicated culture day and analyzed for expression of HPK1 or HPK1-C by WB using the indicated Abs. Viability of the expanded cells was higher than 90% at the time points where HPK1-C could be detected. The experiment presented is representative of four repeats. (B) Purified T cells of wt mice or HPK1-C tg mice were stimulated by anti-CD3 Abs for 15 min (CD3) or left nonstimulated (−). Endogenous IKKγ proteins were immunoprecipitated and the co-precipitated IKK proteins were subjected to an in vitro kinase assay using recombinant GST:IκBα as substrate. The kinase reaction was separated by SDS–PAGE and quantified using a Phosphor-Imager. Values given are expressed relative to the basal activity of nonstimulated cells. (Inset) Expression of HPK1 and HPK1-C was analyzed in lysates from primary mouse T cells by anti-HPK1 and anti-T7 tag WB. (C) T cells of wt or HPK1-C (tg) mice as seen in (B) were stimulated by anti-CD3 Abs for 1 or 2 h or left nonstimulated (0). Nuclear extracts were subjected to EMSA as described in Figure 1F. (D) T cells of wt or HPK1-C (tg) mice were stimulated by anti-CD3 Abs for the indica" @default.
• W1990970872 created "2016-06-24" @default.
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• W1990970872 creator A5038297299 @default.
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• W1990970872 creator A5073198400 @default.
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• W1990970872 date "2005-12-08" @default.
• W1990970872 modified "2023-09-23" @default.
• W1990970872 title "Activation or suppression of NFκB by HPK1 determines sensitivity to activation-induced cell death" @default.
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