Hematopoietic progenitor kinase 1 negatively ...

© 2007 Nature Publishing Group http://www.nature.com/natureimmunology

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Hematopoietic progenitor kinase 1 negatively regulates T cell receptor signaling and T cell–mediated immune responses Jr-Wen Shui, Jonathan S Boomer, Jin Han, Jun Xu, Gregory A Dement, Guisheng Zhou & Tse-Hua Tan HPK1 is a Ste20-related serine-threonine kinase that inducibly associates with the adaptors SLP-76 and Gads after T cell receptor (TCR) signaling. Here, HPK1 deficiency resulted in enhanced TCR-induced phosphorylation of SLP-76, phospholipase C-c1 and the kinase Erk, more-persistent calcium flux, and increased production of cytokines and antigen-specific antibodies. Furthermore, HPK1-deficient mice were more susceptible to experimental autoimmune encephalomyelitis. Although the interaction between SLP-76 and Gads was unaffected, the inducible association of SLP-76 with 14-3-3s (a phosphorylated serine–binding protein and negative regulator of TCR signaling) was reduced in HPK1-deficient T cells after TCR stimulation. HPK1 phosphorylated SLP-76 and induced the interaction of SLP-76 with 14-3-3s. Our results indicate that HPK1 negatively regulates TCR signaling and T cell–mediated immune responses.

HPK1 (also called MAP4K1) is a hematopoietic-specific protein serine-threonine kinase and is a member of the MAP4K family of mammalian Ste20-related protein kinases1–3. HPK1 is expressed mainly in hematopoietic organs and cells1, suggesting potential involvement of HPK1 in the regulation of signaling in hematopoietic lineages, including lymphocytes. HPK1 consists of an N-terminal kinase domain followed by four proline-rich motifs capable of binding to proteins containing Src homology 3 domains. HPK1 also has a citron homology domain at its distal C terminus, which may act as a regulatory domain and may be involved in macromolecular interactions. HPK1 functions as a MAP4K in that it activates MAP3K proteins, including MEKK1, MLK3 and TAK1, and leads to activation of the MAPK Jnk1,3. Furthermore, HPK1 influences Fas-mediated apoptosis; caspase-3-mediated cleavage of HPK1 at Asp385 converts HPK1 from an activator of transcription factor NF-kB into an NF-kB inhibitor, thus favoring the induction of apoptosis4,5. The functional involvement of HPK1 in T cell receptor (TCR) signaling has been well documented6–10. After TCR stimulation, the tyrosine kinases Lck and Zap70 induce tyrosine phosphorylation and activation of HPK1 (refs. 6,7). Additionally, the adaptor proteins Lat, Gads and SLP-76 inducibly associate with HPK1 (refs. 7–9), presumably forming a regulatory complex that translocates to glycolipidenriched microdomains (also called lipid rafts)7. HPK1 also inducibly or constitutively interacts with several other adaptor proteins, including Nck7, HIP-55 (refs. 11,12), Crk7, Clnk10, Grb2 (refs. 6,7), Grap13 and CrkL7. Moreover, HPK1 suppresses prostaglandin E2–induced transcription of the osteosarcoma oncogene Fos14. In mouse erythro-

leukemia SKT6 cells, HPK1 is also involved in erythropoietin-induced cell growth and differentiation15. Those studies emphasize the diverse functions of HPK1 in cellular signaling pathways. HPK1, as a regulator of MAPK activity in T cells, may cooperate with adaptor proteins to transmit TCR signals to ‘downstream’ cellular events. However, HPK1 may be involved in the transmission of signals triggered by diverse stimuli, including growth factors, stress, inflammation and differentiation cues, in distinct cell types, such as hematopoietic progenitor cells, leukemic cells or macrophages14–16. Overexpression studies indicate that HPK1 cooperates with several signaling adaptors to regulate the activity of transcription factor AP-1 and activation of the promoter of the gene encoding interleukin 2 (IL-2)6,9,10,17,18. To gain more insight into the function of HPK1 in the regulation of T cell–mediated immune responses in vivo, we generated HPK1-deficient (Map4k1–/–) mice. Map4k1–/– T cells were hyperresponsive to TCR stimulation, as demonstrated by enhanced phosphorylation of SLP-76, phospholipase C-g1 (PLC-g1) and the kinase Erk, augmented T cell proliferation, increased cytokine production, increased humoral immune responses and enhanced susceptibility to autoimmune disease. We further found that HPK1 phosphorylated serine and threonine residues of SLP-76 and that HPK1 was essential for optimal TCR-induced interaction between SLP-76 and 14-3-3t. Given that 14-3-3t is a specific phosphorylated serine (phosphoserine)–binding protein and a negative regulator of TCR signaling19–22, our results suggest a previously unknown mechanism of HPK1 action in T cells by which HPK1, by phosphorylating SLP-76, promotes SLP76–14-3-3t interactions and, ultimately, shutdown of TCR signaling.

Department of Immunology, Baylor College of Medicine, Houston, Texas 77030, USA. Correspondence should be addressed to T.-H.T. ([email protected]). Received 15 September; accepted 26 October; published online 19 November 2006; doi:10.1038/ni1416

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RESULTS Normal lymphoid development in Map4k1–/– mice Map4k1–/– mice (Supplementary Figure 1 online) were born at the expected mendelian ratio and were phenotypically healthy and fertile. Wild-type and Map4k1–/– mice had similar numbers of thymocytes (Fig. 1a). HPK1 deficiency did not alter the ratio of CD4+ to CD8+ thymocytes or the expression pattern of CD44 and CD25 on CD4–CD8– thymocytes (Fig. 1a,b). In addition, the number of mature thymocytes (CD3hiTCRbhi), and the number of thymocytes undergoing positive selection (CD69hiTCRbhi) were similar in wild-type and Map4k1–/– mice (Fig. 1c). Thymocyte negative selection was not impaired in the absence of HPK1, as determined by endogenous Mtv-9 superantigen–mediated deletion of Vb5+ thymocytes (Fig. 1c). Wild-type and Map4k1–/– mice had similar numbers of peripheral CD4+ cells, CD8+ cells, B220+ cells, regulatory T cells (CD4+CD25+CD69– or CD4+Foxp3+) and memory T cells (CD4+CD44+; Fig. 1d–f). The T cell/B cell and CD4+ cell/CD8+ cell ratios in the spleen and lymph nodes were also unaffected by HPK1 deficiency. These data suggested that HPK1 is not essential for lymphocyte development. Proliferation and apoptosis of Map4k1–/– T cells Given that HPK1 is known to be involved in TCR signaling and T cell activation, we determined whether HPK1 deficiency affected T cell proliferation and apoptosis. Purified Map4k1–/– T cells cultured with irradiated splenocytes were hyperproliferative in response to stimulation with antibody to CD3 (anti-CD3; Fig. 2a). However, proliferation induced by phorbol 12-myristate 13-acetate (PMA) plus ionomycin, which bypasses proximal TCR signaling events and directly activates protein kinase C (PKC) and the GTPase Ras, was similar in wild-type and Map4k1–/– mice6 (Fig. 2a). This suggested that HPK1 functions ‘upstream’ of PKC and Ras activation during T cell activation. To determine whether that hyperproliferation was autonomous to Map4k1–/– T cells, we stimulated purified T cells with plate-bound anti-CD3 and soluble anti-CD28. Map4k1–/– T cells, even in the absence of costimulation from antigen-presenting cells, underwent

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WT KO WT KO WT KO Figure 1 Lymphoid development in Map4k1–/– mice. (a) Flow cytometry of 3.3 3.2 3.2 3.0 thymocyte cellularity and phenotype. DP double-positive; DN, double-negative; 2.9 2.7 SP, single-positive. (b) Flow cytometry of the expression of CD25 and CD44 on CD4–CD8– double-negative thymocytes. DN1, CD25–CD44+; DN2, CD4 CD4 CD25+CD44+; DN3, CD25+CD44–; DN4, CD25–CD44–. Numbers above and CD4 below dot plots (a,b) indicate percent cells in each quadrant. Absolute cell numbers (right; a,b) were derived from at least ten (a) or six (b) mice of each genotype. (c) Flow cytometry quantification of thymocytes expressing various surface markers (horizontal axis). (d) Flow cytometry of surface marker expression on splenocytes (left two graphs) and lymph node cells (right two graphs). CD4/CD8, CD4+ cell/CD8+ cell ratio; T/B, T cell/B cell ratio. (e) Flow cytometry visualization of regulatory T cells. Recently activated CD69+ T cells were excluded by gating. Numbers above outlined areas indicate percent CD25+CD4+ cells (left) or Foxp3+CD4+ cells (right). (f) Flow cytometry of memory T cells. Numbers in top right quadrants indicate percent CD44+CD4+ cells. WT, wild-type; KO, Map4k1–/–. All data are representative of at least three independent experiments (error bars (a–d), s.d.). CD25


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more cell divisions than did wild-type cells, indicating that Map4k1–/– T cells are intrinsically hyper-responsive to mitogenic stimuli (Fig. 2b). Experiments using cells labeled with carboxyfluorescein diacetate succinimidyl diester produced similar results (Fig. 2c). These data indicated that HPK1 is a negative regulator of T cell proliferation. As HPK1 is an established regulator of NF-kB and Jnk, which influence apoptosis, we investigated whether the apparent hyperproliferation of Map4k1–/– T cells could have been due to decreased apoptosis. To assess activation-induced cell death in vivo, we examined CD4+CD8+ thymocyte deletion induced by intraperitoneal injection of anti-CD3 (ref. 23). Anti-CD3-induced thymocyte deletion was similar in a dose-dependent way in wild-type and Map4k1–/– mice (Fig. 2d). We obtained similar results when we restimulated preactivated lymph node cells in vitro with anti-CD3, anti-Fas or dexamethasone (Fig. 2e). These results suggested that Map4k1–/– T cells undergo apoptosis in a way similar to wild-type T cells. HPK1 suppresses T cell–mediated immunity in vivo To investigate whether the enhanced in vitro proliferation of Map4k1–/– T cells correlated with in vivo activation of Map4k1–/– T cells, we immunized wild-type and Map4k1–/–mice with a T cell– dependent antigen, keyhole limpet hemocyanin (KLH). Complete Freund’s adjuvant (CFA) induces a mixed T helper type 1 and type 2 response, whereas alum induces a T helper type 2–dominated response24. After KLH restimulation, T cells from Map4k1–/– mice immunized with KLH in CFA or alum proliferated more and produced more IL-2, IL-4 and interferon-g than did cells from wild-type mice immunized the same way (Fig. 3a,b). We obtained similar results with T cells from mice immunized with another T cell– dependent antigen, chicken g-globulin (CGG) (Fig. 3c). Consistent with the KLH immunization results, Map4k1–/– T cells restimulated with CGG proliferated more and produced more IL-2 than did wild-type T cells, but restimulation with PMA plus ionomycin triggered similar proliferation and IL-2 production by wild-type and Map4k1–/– T cells.

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Next, to determine whether HPK1 activity influences T cell– dependent humoral responses, we measured antigen-specific antibody production in immunized wild-type and Map4k1–/– mice. Map4k1–/– sera contained more antigen-specific antibodies (of many isotypes) during primary and secondary immunizations (Fig. 3d). These results indicated that HPK1 deficiency resulted in a more vigorous T cell– dependent humoral response in immunized mice. Exacerbated autoimmunity in Map4k1–/– mice In vivo dysregulation of immune functions, including altered T cell activation, proliferation, cytokine production and T cell–mediated antibody production, may lead to the onset of autoimmunity. To determine whether Map4k1–/– mice are more susceptible to autoimmune disease, we induced experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis, by immunizing wildtype and Map4k1–/– mice with myelin oligodendrocyte protein (MOG) in CFA. Map4k1–/– mice were more susceptible to EAE and demonstrated severe paralysis of forelimbs and hindlimbs (Fig. 4a). More CD4+ T cells infiltrated the brain tissues of diseased Map4k1–/– mice, but similar numbers of Mac-1+ cells (microglial cells, macrophages and granulocytes) infiltrated the brain tissues of diseased wildtype and Map4k1–/– mice (Fig. 4b). To further examine the function of wild-type and Map4k1–/– MOG-specific T cells, we restimulated sensitized T cells with MOG peptides and measured T cell proliferation and cytokine production. Restimulated Map4k1–/– T cells proliferated more (Fig. 4c) and produced more interferon-g but wild-type amounts of IL-4 (Fig. 4d). Thus, HPK1 deficiency promoted the proliferation and activation of autoreactive T cells, which resulted in a more severe autoimmune phenotype in Map4k1–/– mice. Our findings suggest that HPK1 may be involved in the pathogenesis of autoimmune diseases by regulating self antigen–specific T cell activation and function.

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Figure 2 Map4k1–/– T cells are hyperproliferative in response to TCR stimulation. (a) Proliferation of purified splenic T cells stimulated with antiCD3 (a-CD3; concentration, left horizontal axis) in the presence of irradiated splenocytes (left) or with PMA plus ionomycin (P + I; right), analyzed by [3H]thymidine incorporation. (b) Proliferation of purified splenic T cells stimulated with plate-bound anti-CD3 and soluble anti-CD28 (a-CD3+CD28), analyzed by [3H]thymidine incorporation. Data (a,b) are mean ± s.d. of triplicate wells. (c) Flow cytometry of splenocytes labeled with carboxyfluorescein diacetate succinimidyl diester (CFSE) and left unstimulated or stimulated with anti-CD3; CD3+ cells were gated and analyzed. Numbers above arrows indicate percent dividing cells (left) or nondividing cells (right). (d) Flow cytometry of CD4+CD8+ thymocytes obtained 48 h after mice were injected intraperitoneally with PBS or antiCD3 (concentration, horizontal axis). (e) Viability of lymph node T cells pretreated with PMA plus ionomycin, incubated in IL-2 and stimulated with plate-bound anti-CD3 (left) or, in parallel assays, treated with anti-Fas (middle) or dexamethasone (Dex; right). Cells negative for staining with annexin V and 7-amino-actinomycin D are considered viable. Data are representative of three (a,b), or two (c,d,e) independent experiments (error bars (a,b,d), s.d.).

HPK1-mediated regulation of proximal TCR signaling The enhanced proliferation of Map4k1–/– T cells suggested that T cell activation and signaling may be augmented in the absence of HPK1. We detected similar amounts of TCRb on the surface of wild-type and Map4k1–/– T cells (Fig. 5a). In contrast, more CD25 was expressed on the surface of Map4k1–/– T cells after TCR stimulation (Fig. 5b). In addition, TCR-induced phosphorylation of several proteins was augmented in Map4k1–/– T cells (Fig. 5c). These observations indicated that TCR signaling is enhanced in the absence of HPK1. Because MAPK activation is essential in T cell proliferation and HPK1 is an upstream regulator of MAPK proteins, we investigated whether HPK1 regulates T cell proliferation and function through MAPK signaling. Given that HPK1 efficiently activates Jnk in T cells6,25 and non–T cells1,3,17, we first examined whether Jnk signaling was lower in Map4k1–/– T cells than in wild-type cells. Anti-CD3-induced Jnk activation (both phosphorylation and kinase activity) was not lower in Map4k1–/– T cells (Fig. 5d). In a parallel experiment, we noted that TCR-induced activation of the MAPK p38 was also unaffected in Map4k1–/– T cells (Fig. 5d). HPK1 also regulates Erk signaling3,6, and T cell proliferation is influenced by Erk activity. Although Map4k1–/– T cells had normal TCR-induced activation of Jnk and p38, Map4k1–/– T cells had enhanced TCR-induced activation of the kinases MEK1/2 and Erk2 (Fig. 5e and data not shown). However, Erk activation induced by PMA plus ionomycin was similar in wild-type and Map4k1–/– T cells (data not shown); this result correlated with the similar T cell proliferation induced by PMA plus ionomycin in wild-type and Map4k1–/– T cells (Fig. 2a). To confirm the specificity of the MEK1/ 2–Erk signaling defect, we treated wild-type and Map4k1–/– T cells with a MEK inhibitor (PD98059) before TCR stimulation. PD98059 treatment completely blocked Erk activation in wild-type and Map4k1–/– T cells (Fig. 5f). We also noted enhanced Erk activation

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Figure 3 Enhanced in vivo T cell activation in Map4k1–/– mice. (a,b) T cell proliferation and KLHinduced cytokine production profiles. Mice were immunized subcutaneously with KLH emulsified in CFA (a) or alum (b); sensitized draining lymph node cells were isolated 7 d later and were restimulated with KLH. IFN, interferon. (c) T cell proliferation and CGG-induced IL-2 production. Mice were immunized with CGG in CFA; sensitized draining lymph node cells were isolated 7 d later and were restimulated with CGG or PMA plus ionomycin. In a–c, proliferation was measured by [3H]thymidine incorporation and cytokine production was analyzed by ELISA. (d) Primary (left) and secondary (right) immune responses, evaluated by measurement of nitrophenol-specific antibody titers (NP-specific Ab) in sera 14 d after first and second immunization with dinitrophenol-KLH emulsified in alum. Ig, immunoglobulin. Data (a–d) are mean ± s.d. of triplicate wells and are representative of two (a,b,d), or three (c) independent experiments.

HPK1 promotes SLP-76–14-3-3s interactions SLP-76 is important in TCR signaling, as its chief function is to recruit signaling molecules to glycolipid-enriched microdomains, where the activated TCR and protein tyrosine kinases can functionally activate downstream effectors. TCR-induced SLP-76 tyrosine phosphorylation was higher in Map4k1–/– T cells and thymocytes than in wild-type cells (Fig. 7a,b). In addition, in the SLP-76–Lat–Gads complex in Map4k1–/– T cells, tyrosine phosphorylation of Gads-associated SLP-76 was higher, whereas tyrosine phosphorylation of Gadsassociated Lat was marginally higher than in wild-type T cells (Fig. 7c). Because SLP-76 associates with many signaling molecules during the transmission of TCR signals, we searched for alterations in protein-protein interactions in SLP-76 complexes that could contribute to the enhanced TCR signaling noted in Map4k1–/– T cells. We assessed Vav, Nck and Itk, which bind to phosphotyrosine residues in the N terminus of SLP-76; PLC-g1 and Gads, which bind to the

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in Map4k1–/– thymocytes stimulated with anti-CD3 and anti-CD4 (Fig. 5g), but found normal Erk activation in peritoneal macrophages stimulated with TNF (Fig. 5g). These results emphasize a unique function for HPK1 in TCR-induced Erk activation. Many T cell signaling molecules, including Lat, SLP-76 and PLC-g1, influence Erk activation26–28. Because TCR signaling stimulates the interaction of HPK1 with Lat7 and SLP76 (ref. 9), we investigated whether HPK1 inhibited Erk activation through the Lat– SLP-76–PLC-g1–Erk signaling pathway. Anti-CD3-induced PLC-g1 phosphorylation was greater in intensity and duration in Map4k1–/– T cells than in wild-type T cells (Fig. 6a); this result correlated with a more persistent TCR-induced calcium flux detected in Map4k1–/– T cells (Fig. 6b). Vav is another SLP-76-binding protein that functions upstream of PLC-g1 and Erk in T cells29,30. Tyrosine phosphorylation of Vav and Lat was enhanced in Map4k1–/– T cells (Fig. 6c,d). Thus, proximal signaling downstream of the TCR was enhanced in the absence of HPK1. After TCR stimulation, the TCR-associated protein tyrosine kinases Lck and Zap70 phosphorylate and activate several proximal signaling molecules, such as Lat, Vav, PLC-g1 and SLP-76. Therefore, increased signaling through the Lat–PLC-g1–Vav complex may result from enhanced upstream protein tyrosine kinase activity. Zap70 phosphorylation was slightly higher, whereas Lck activation was unaffected, in activated Map4k1–/– T cells (Fig. 6e,f). We found no changes in the interaction between CD3z and Zap70 in Map4k1–/– T cells relative to that in wild-type cells; this observation correlated the similar anti-CD3-induced CD3z tyrosine phosphorylation in wild-type and Map4k1–/– T cells (Fig. 6g). This result excluded the possibility that HPK1 regulates Zap70 activity by affecting its recruitment to the TCR complex.



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proline-rich region of SLP-76; Zap70, Cbl and SHP-1, which bind to unidentified regions in SLP-76; and ADAP, which binds to the SH2 domain of SLP-76. Only the SLP-76–Gads interaction was detectable in mouse primary cells; however, the interaction was not altered in Map4k1–/– T cells relative to that in wild-type cells (Fig. 7d). We then explored the involvement of SLP-76-binding suppressors of TCR signaling. Cbl binds to Zap70 (ref. 31) and SLP-76 (ref. 32), but we did not detect any changes in the Cbl–Zap70 interaction or in Cbl tyrosine phosphorylation (data not shown). SHP-1 dephosphorylates both Zap70 and SLP-76, but SHP-1–SLP-76 interactions were undetectable in mouse primary T cells with available commercial antibodies (data not shown). Because HPK1 physically interacts with SLP-76, SLP-76 itself could be the substrate of HPK1, such that serine-threonine phosphorylation of SLP-76 by HPK1 may alter SLP-76 function. HPK1 phosphorylated serine and threonine residues in SLP-76 (Fig. 7e) but not Zap70 (data

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Figure 4 Enhanced susceptibility of Map4k1–/– mice to EAE induction. Age-matched female mice were immunized with MOG peptide emulsified in CFA, followed by injection of pertussis toxin at days 0 and 2. (a) Mouse disease scores. (b) Flow cytometry of infiltrated mononuclear cells from the brain tissue of immunized mice. Numbers above bracketed lines indicate percent CD4+ cells (left) or Mac-1+ cells (right). (c) Proliferation of splenic T cells (left) and lymph node T cells (right) obtained from MOG-immunized mice, restimulated with MOG peptide and analyzed by [3H]thymidine incorporation. (d) ELISA of the production of interferon-g (left) and IL-4 (right) by splenocytes obtained from MOG-immunized mice and restimulated with MOG peptide. Data (c,d) are mean ± s.d. of triplicate wells. Data are representative of three (a) or two (b–d) independent experiments (error bars (a,b), s.d.).

The evidence reported above prompted us to further investigate the involvement of 14-3-3t in SLP-76 signaling. First, the interaction of SLP-76 with 14-3-3t was reduced in TCR-stimulated Map4k1–/– thymocytes (Fig. 7f), suggesting that HPK1 may regulate the association between SLP-76 and 14-3-3t. We confirmed the TCR-induced association of SLP-76 with 14-3-3t in Jurkat T cells (Fig. 7g). Second, we used an antibody specific for the 14-3-3 phosphoserine-binding motif to show that TCR signaling resulted in the appearance of phosphoserine motif on SLP-76 that bound 14-3-3 (Fig. 7h). Third, to determine whether HPK1 mediates the interaction between SLP-76 and 14-3-3t, we used a fusion of glutathione S-transferase (GST) and 14-3-3t (GST–14-3-3t) to precipitate SLP-76 in the presence or absence of HPK1 in HEK293T cells. SLP-76 associated with GST– 14-3-3t only in the presence of HPK1 (Fig. 7i), suggesting that HPK1

not shown). Sequence analysis showed that HPK1-mediated SLP-76 serine phosphorylation would create several potential binding sites for 14-3-3 proteins, which are known phosphoserine-binding proteins. The protein 14-3-3t is a negative regulator of TCR signaling19–22, and 14-3-3t binds and inhibits both phosphatidylinositol-3-OH kinase (PI(3)K; which triggers TCR-induced calcium signaling) and PKC-y (which promotes TCR-induced Erk phosphorylation). Calcium signaling and Erk activation were enhanced in Map4k1–/– T cells; hence, it is possible that 14-3-3t may be involved in HPK1-mediated TCR signaling. The protein 14-3-3t is also known to associate with and recruit Cbl to Zap70 in T cells. However, HPK1 did not interact with or phosphorylate Zap70 and there was no change in the Cbl–Zap70 association in Map4k1–/– T cells relative to that in wild-type T cells (data not shown).

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Figure 5 HPK1 is a negative regulator of TCR-induced Erk activation. (a) Flow cytometry of TCRb α-CD3 (min) – 5 10 – 5 10 TNF (min) – 15 30 – 15 30 α-CD4 p-Erk1 surface expression on lymph node T cells. MFI, mean fluorescence intensity. (b) Flow cytometry of p-Erk1 p-Erk2 p-Erk2 CD25 surface expression on purified splenic T cells stimulated for 3 d with plate-bound anti-CD3 Erk1 Erk1 with or without soluble anti-CD28. (c) Immunoblot of lysates of purified splenic T cells left Erk2 Erk2 unstimulated (–) or stimulated for 2 min with anti-CD3 and crosslinking antibodies (+), analyzed with a phosphotyrosine-specific antibody (4G10). p-, phosphorylated. Bottom, blot reprobed with anti-Lat to confirm equal protein loading. Right margin, molecular sizes (kDa). (d) Immunoblot of lysates of purified splenic T cells stimulated with anti-CD3, analyzed with antibody to phosphorylated Jnk (p-Jnk), Jnk, phosphorylated p38 (p-p38) or b-actin. p-GST–c-Jun, analysis of Jnk kinase activity with GST–c-Jun (1-79) as a substrate. (e) Immunoblot of lysates of purified splenic T cells stimulated with anti-CD3, analyzed with antibody to phosphorylated Erk (p-Erk) or to Erk. (f) Erk activation in lymph node T cells left untreated or pretreated for 30 min at 37 1C with PD98059 (MEK inhibitor) before stimulation, assessed as described in e. (g) Erk activation in thymocytes or peritoneal macrophages stimulated with anti-CD3 and anti-CD4 (left) or with TNF (right), respectively, assessed as described in e. Data are representative of two (a,b,f,g), or three (c–e) independent experiments (error bars (a,b), s.d.).

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Figure 6 Enhanced proximal TCR signaling in Map4k1–/– T cells. (a) Immunoprecipitation (IP) of lysates of anti-CD3-stimulated, purified splenic T cells with anti-PLC-g1, followed by immunoblot with anti-phosphotyrosine; blot was then reprobed with anti-PLC-g1. (b) Flow cytometry of purified lymph node T cells labeled with the calcium indicators Fluo-4 and Fura-red and stimulated with biotin-conjugated anti-CD3 and streptavidin, to determine the ratio of the fluorescence (Fluo-4 to Fura-red (FL1/FL4)). (c,d) Immunoprecipitation of lysates of anti-CD3-stimulated, purified splenic T cells with anti-Vav (c) or anti-Lat (d), followed by immunoblot with anti-phosphotyrosine (p-Vav (c) or p-Lat (d)); blots were then reprobed with anti-Vav (c) or anti-Lat (d). (e,f) Immunoblot analysis of lysates of anti-CD3-stimulated, purified splenic T cells with antibodies to Zap70 and phosphorylated Zap70 (e) or to Lck and phosphorylated Lck (f). (g) Immunoprecipitation of lysates of anti-CD3-stimulated, purified lymph node cells with anti-CD3z, followed by immunoblot with anti-Zap70, anti-phosphotyrosine (p-CD3z) and anti-CD3z. pp23z and pp21z, tyrosine-phosphorylated forms of CD3z. Data are representative of three (a,c,d,g), or two (b,e,f) independent experiments.

DISCUSSION Our results indicate that HPK1 is essential in regulating T cell activation. We have shown that HPK1 ablation resulted in enhanced T cell activation and immune function and that the Lat–SLP-76–PLCg1–Erk signaling pathway was involved in this process. Map4k1–/– T cells proliferated more and produced excess IL-2, suggesting that HPK1 suppresses Il2 transcription. That conclusion is supported by studies showing that HPK1 inhibits TCR-mediated activation of AP-1 (ref. 6) and that overexpression of kinase-inactive HPK1 enhances

mediates the SLP-76–14-3-3t binding. Finally, to directly demonstrate that HPK1 mediates the SLP-76–14-3-3t interaction through serine phosphorylation of SLP-76, we showed that HPK1 (but not the kinase-inactive HPK1-M46) directly phosphorylated SLP-76 in vitro, consequently creating 14-3-3-binding sites on SLP-76 (Fig. 7j, top) and inducing the binding of serine-phosphorylated SLP-76 to 14-3-3t in a GST precipitation assay (Fig. 7j, second row). These results indicate a previously unknown mechanism through which HPK1 downregulates TCR signaling (Supplementary Figure 2 online).

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Figure 7 Regulation of SLP-76 signaling by HPK1. IP: α-14-3-3 IP: α-SLP-76 binding motif (a,b) Immunoprecipitation of lysates of stimulated IP: α-SLP-76 WT KO (above lanes), purified splenic T cells (a) and thymocytes (b) α-CD3 (min) – 10 α-CD3 α-CD3 (min) – 5 10 30 (min) – 5 10 20 – 5 10 20 with anti-SLP-76, followed by immunoblot with antiα-CD4 SLP-76 phosphotyrosine (p-SLP-76); blots were then reprobed with 14-3-3τ 14-3-3τ Cell lysates anti-SLP-76. (c,d) Immunoprecipitation of lysates of stimulated, SLP-76 SLP-76 SLP-76 purified splenic T cells with anti-Gads, followed by immunoblot with anti-phosphotyrosine (p-SLP-76 and p-Lat; c) or anti-SLP-76 (d); Flag–HPK1 – – + – blots were then reprobed with anti-Gads. (e) Analysis of Flag–HPK1 – – + Flag–HPK1-M46 – – – + Flag–SLP-76 – + + – + + + Flag–SLP-76 phosphorylated proteins resulting from an in vitro kinase assay of Flag–SLP-76 mixed Flag fusion proteins; the 76-kDa phosphorylated protein Flag–SLP-76 (α-14-3-3 binding motif) band was excised for analysis. (f,g) Immunoprecipitation of lysates Flag–SLP-76 (α-Flag) GST–14-3-3τ of stimulated thymocytes (f) or Jurkat T cells (g) with anti-SLP-76, followed by immunoblot with anti-14-3-3t; blots were then GST–14-3-3τ (α-14-3-3τ) Cell lysates reprobed with anti-SLP-76. (h) Immunoprecipitation of lysates of Flag–HPK1 Flag–HPK1 (α-Flag) Flag–HPK1-M46 stimulated Jurkat T cells with an antibody specific for a 14-3-3 Flag–SLP-76 Flag–SLP-76 (α-Flag) phosphoserine-binding motif, followed by immunoblot with antiSLP-76. (i) Immunoprecipitation of lysates of Flag fusion protein– transfected HEK293T cells (above lanes) with GST–14-3-3t, followed by identification of associated proteins by immunoblot with anti-Flag (top) or anti-14-3-3t (bottom). (j) Immunoblot of phosphorylated proteins resulting from an in vitro kinase assay of mixed Flag fusion proteins, analyzed with antibody to a 14-3-3 phosphoserine-binding motif (top row); GST precipitation of phosphorylated SLP-76, eluted with excess Flag peptide and precipitated with GST–14-3-3t (second and third rows); and immunoblot of fusion proteins present in the in vitro kinase reaction, detected with anti-Flag (fourth and fifth rows). Data are representative of three (a–c,d,f,g), or two (e,h–j) independent experiments.

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ARTICLES TCR-mediated activation of the transcription factor NFAT33. However, other studies have reported that HPK1 positively regulates the activity of AP-1 and transcription factor c-Jun and transcription of Il2 in Jurkat T cells and HEK293T cells1,17,18. Those inconsistencies may be due to differences among various cell types or to the use of PMA, which bypasses proximal TCR signaling events. Distinct functions for HPK1 in Erk activation have been demonstrated in various cell types. Overexpression of HPK1 in COS cells does not suppress Erk2 activity3; in contrast, when overexpressed in Jurkat T cells, HPK1 suppresses TCR-induced Erk2 activation6. Using Map4k1–/– T cells, we have shown that HPK1 specifically inhibited Erk phosphorylation and restrained T cell activation. Although HPK1 is a potent Jnk activator, Jnk activation was unaffected by HPK1 deficiency. HPK1-independent Jnk activation in Map4k1–/– T cells may be due to the redundant activity of other Ste20-related MAP4K proteins that are also capable of activating Jnk34–37. Consistent with that idea, existing data indicate that Jurkat T cells transiently transfected with an HPK1 mutant containing the HPK1 proline-rich region show normal TCR-triggered Jnk activation7. Furthermore, negative crosstalk between Erk and Jnk has been discovered in mice lacking the phosphatase PAC-1 (ref. 38). Data have shown that mice lacking either Jnk1 or Jnk2 have compensatory adaptations and misleading phenotypes that mask a competitive relationship between Jnk1 and Jnk2 (ref. 39). Therefore, it is plausible that the phenotypes of Map4k1–/– mice may be due to alterations in the homeostatic regulation among multiple MAPK signaling pathways in Map4k1–/– T cells. We identified Zap70 as the most proximal molecule in the TCR signaling pathway affected by HPK1 deficiency. However, HPK1 did not physically bind to Zap70 and did not phosphorylate Zap70 in vitro. Therefore, it is unlikely that HPK1 directly targets Zap70 to downregulate TCR signaling. The enhanced Zap70 activation detected in Map4k1–/– T cells may be due to a negative feedback regulation of Zap70 by the SLP-76 signaling complex. Our studies have shown that HPK1 negatively regulates proximal TCR signaling, which involves the SLP-76–Gads–Lat complex. SLP-76 physically bound to HPK1 after TCR stimulation, and SLP-76 serine and threonine residues were phosphorylated by HPK1. Our results have demonstrated in vivo inducible binding of SLP-76 to 14-3-3t in primary mouse T cells and Jurkat T cells. HPK1, but not kinaseinactive HPK1-M46, phosphorylated SLP-76 in vitro, creating 14-3-3binding sites. The direct phosphorylation of SLP-76 by HPK1 induced interaction of phosphorylated SLP-76 with GST–14-3-3t. The 14-3-3 family of proteins are phosphoserine-binding molecules that ‘preferentially’ bind to RSX(pS)XP and RXX(pS)XP motifs (where ‘(pS)’ indicates phosphoserine)21,22. Based on the sequence of established 14-3-3-binding motifs and the SLP-76 protein sequence, we found multiple potential 14-3-3-binding sites on SLP-76. Among those potential 14-3-3-binding sites, RNHSPL contains Ser207 that is phosphorylated in pervanadate-treated Jutkat T cells40. After TCR stimulation, 14-3-3t associates with a 120-kilodalton (120-kDa) phosphorylated protein identified as Cbl and a 70- to 75-kDa protein, which is the approximate molecular weight of SLP-76 (ref. 41). The protein 14-3-3t binds to Cbl, PI(3)K and PKC-y and negatively regulates TCR signaling by inhibiting PI(3)K and PKC-y19,20,41. Our results suggest that the enhanced calcium signaling and Erk activation in Map4k1–/– T cells were consequences of HPK1mediated modulation of the interaction among 14-3-3t, SLP-76, PI(3)K and/or PKC-y. In summary, we have identified a previously unknown regulatory function for HPK1 in TCR signaling. HPK1 may inhibit T cell activation by modulating the interaction among the SLP-76 complexes

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and 14-3-3t or 14-3-3t-associated proteins. Many serine and threonine residues are located in the proximity of the three main phosphotyrosine sites (Tyr113, Tyr128 and Tyr145) on SLP-76. Therefore, it is plausible that in addition to generating 14-3-3-binding sites, HPK1mediated phosphorylation of SLP-76 serine and threonine residues may also change the conformation of SLP-76, resulting in decreased tyrosine phosphorylation or altered protein-protein interactions. METHODS Embryonic stem cells, antibodies and plasmids. Mouse 129/Sv/Ev embryonic stem cells (clone AB2.2) were a gift (Acknowledgments). Anti-SLP-76 (H-300), anti-Lat (FL-233), anti-PLC-g1 (1249), anti-Vav (C-14), anti-HPK1 (N-19 and C-20) and anti-Erk (C-16) were purchased from Santa Cruz Biotechnology. Anti-phosphotyrosine (4G10) and anti-Gads (06-983) were purchased from Upstate Biotechnology. Monoclonal anti-SLP-76 (AS55) was purchased from Exalpha Biologicals. Polyclonal antibody to the 14-3-3 phosphoserine-binding motif (which recognizes the R/KXX(pS)XP motif, where ‘R/K’ is arginine or lysine and ‘(pS)’ indicates phosphoserine) and monoclonal antibodies to phosphorylated Jnk, p38 and Erk were purchased from Cell Signaling Technology. Anti-Flag (M2) was purchased from Sigma. Anti-14-3-3t was purchased from Biosource. The GST–14-3-3t and Flag–SLP-76 plasmids were gifts (Acknowledgments). T cell proliferation, activation and apoptosis. T cell proliferation and stimulation were done as described11. Where indicated, cells were also treated with PMA (20 ng/ml) plus ionomycin (0.5 mM) for control experiments or were pretreated with 10 mM PD98059 before anti-CD3 stimulation. For measurement of activation-induced cell death in vivo, 6- to 8 week-old wild-type and Map4k1–/– mice were injected intraperitoneally with PBS or antiCD3e (145-2C11; BD Pharmingen). After 48 h, mice were killed and thymocytes were prepared and were analyzed by staining and flow cytometry. For measurement of activation-induced cell death in vitro, lymph node cells were stimulated for 24 h with PMA (10 ng/ml) and ionomycin (1 mg/ml) to induce equal proliferation. Cells were washed and their populations were expanded for additional 3 d with IL-2. Cells were restimulated for 48 h with plate-bound anti-CD3e (145-2C11; BD Pharmingen), and cell viability was measured by staining with annexin V and 7-amino-actinomycin D (BD Pharmingen). In vivo T cell–mediated immune responses and EAE induction. Antigenspecific (KLH or CGG) T cell proliferation and antibody production by immunized mice was measured as described11. Mice used for EAE induction were backcrossed to C57BL/6 background for nine generations. EAE was induced by subcutaneous injection of mice with 150 mg MOG peptide (amino acids 35–55: MEVGWYRSPFSRVVHLYRNGK; Genemed Synthesis) emulsified in incomplete Freund’s adjuvant (Sigma) with 250 mg Mycobacterium tuberculosis (Difco). Mice were also injected intraperitoneally with 200 ng pertussis toxin (List Biological) on days 0 and 2. All mice were monitored daily for clinical signs and were assigned scores on a scale of 0–5 as follows: 0, no overt signs of disease; 1, limp tail; 2, limp tail and partial hindlimb paralysis; 3, complete hindlimb paralysis; 4, complete hindlimb and partial forelimb paralysis; 5, moribund state or death. Mononuclear cells were prepared from brain tissues as described42. All animal experimental protocols were approved by the Institutional Animal Care and Use Committee of Baylor College of Medicine. Calcium flux, immunoprecipitation and immunoblot analysis. Lymph node T cells were labeled for 30 min at 25 1C with 2 mM Fura red and 1 mM Fluo-4 in serum-free RPMI medium containing 0.2% Pluronic F-127 (Molecular Probes). Cells were then washed twice, were allowed to ‘rest’ for 20 min in the dark and were incubated for 15 min on ice with 15 mg/ml of biotinconjugated anti-CD3e (500A2; Pharmingen). Labeled cells were washed, were resuspended with prewarmed streptavidin and were analyzed by flow cytometry to obtain the ratio of Fluo-4 to Fura-red. Immunoprecipitation and immunoblot analysis were done as described11. In vitro SLP-76 phosphorylation, phosphorylated amino acid analysis and GST precipitation assay. HEK293T cells were individually transfected with Flag–HPK1 or Flag–SLP-76. Flag–HPK1 and Flag–SLP-76 were individually

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ARTICLES immunoprecipitated with anti-Flag. Agarose beads containing either Flag–HPK1 or Flag–SLP-76 were incubated together, followed by an in vitro HPK1 kinase assay. The 76-kDa phosphorylated protein band was excised from the membrane and phosphorylated amino acids were analyzed as described34. GST precipitation assays were done as described43, with GST–14-3-3t and lysates prepared from HEK293T cells transfected with empty vector, Flag–SLP-76, or Flag–SLP-76 plus Flag–HPK1. For GST precipitation with SLP-76 phosphorylated in vitro by HPK1, HEK293T cells were individually transfected with empty vector, Flag–HPK1, Flag–HPK1-M46 or Flag–SLP-76. An in vitro kinase assay was done as described above. Proteins bound to Flag–HPK1, Flag–HPK1-M46 and Flag–SLP-76 were then eluted with 100 mg/ml of Flag peptide and were used in a GST precipitation assay with GST–14-3-3t. Statistical methods. Difference between data sets (s.d.) were analyzed with Excel software. Note: Supplementary information is available on the Nature Immunology website.

ACKNOWLEDGMENTS We thank J. Belmont for assistance with embryonic stem cell culture; F.J. DeMayo (Baylor College of Medicine Transgenic Core Facility) for embryonic stem cell microinjection; K.M. Stehling for technical assistance; and D.A. Guzman and R. Cuthbert for secretarial assistance. Mouse 129/Sv/Ev embryonic stem cells (clone AB2.2) were provided by A. Bradley (The Sanger Institute); GST–14-3-3t plasmid was provided by W.C. Lin (University of Alabama at Birmingham); and Flag–SLP-76 plasmid was provided by G. Koretzky (University of Pennsylvania). Supported by the National Institutes of Health (R01-AI42532, R01-AI066895 and R01-CA87076 to T.-H.T; and T32AI07495 to J.-W.S., J.S.B. and G.A.D) and the American Heart Association Texas Affiliate (0465456Y to G.Z.). AUTHOR CONTRIBUTIONS J.-W.S. designed and did experiments and prepared the manuscript; J.S.B. designed and did experiments and edited the manuscript; J.H., J.X. and G.A.D. did experiments; G.Z. assisted in experiments; and T.-H.T. established the initial scientific questions, designed and supervised experiments and composed the manuscript. COMPETING INTERESTS STATEMENT The authors declare that they have no competing financial interests. Published online at http://www.nature.com/natureimmunology/ Reprints and permissions information is available online at http://npg.nature.com/ reprintsandpermissions/ 1. Hu, M.C., Qiu, W.R., Wang, X., Meyer, C.F. & Tan, T.-H. Human HPK1, a novel human hematopoietic progenitor kinase that activates the JNK/SAPK kinase cascade. Genes Dev. 10, 2251–2264 (1996). 2. Boomer, J.S. & Tan, T-H. Functional interactions of HPK1 with adaptor proteins. J. Cell. Biochem. 95, 34–44 (2005). 3. Kiefer, F. et al. HPK1, a hematopoietic protein kinase activating the SAPK/JNK pathway. EMBO J. 15, 7013–7025 (1996). 4. Chen, Y-R., Meyer, C.F., Ahmed, B., Yao, Z. & Tan, T-H. Caspase-mediated cleavage and functional changes of hematopoietic progenitor kinase 1 (HPK1). Oncogene 18, 7370–7377 (1999). 5. Arnold, R., Liou, J., Drexler, H.C., Weiss, A. & Kiefer, F. Caspase-mediated cleavage of hematopoietic progenitor kinase 1 (HPK1) converts an activator of NFkB into an inhibitor of NFkB. J. Biol. Chem. 276, 14675–14684 (2001). 6. Liou, J. et al. HPK1 is activated by lymphocyte antigen receptors and negatively regulates AP-1. Immunity 12, 399–408 (2000). 7. Ling, P. et al. Involvement of hematopoietic progenitor kinase 1 in T cell receptor signaling. J. Biol. Chem. 276, 18908–18914 (2001). 8. Liu, S.K., Smith, C.A., Arnold, R., Kiefer, F. & McGlade, C.J. The adaptor protein Gads (Grb2-related adaptor downstream of Shc) is implicated in coupling hemopoietic progenitor kinase-1 to the activated TCR. J. Immunol. 165, 1417–1426 (2000). 9. Sauer, K. et al. Hematopoietic progenitor kinase 1 associates physically and functionally with the adaptor proteins B cell linker protein and SLP-76 in lymphocytes. J. Biol. Chem. 276, 45207–45216 (2001). 10. Yu, J. et al. Synergistic regulation of immunoreceptor signaling by SLP-76-related adaptor Clnk and serine/threonine protein kinase HPK1. Mol. Cell. Biol. 21, 6102–6112 (2001). 11. Han, J. et al. HIP-55 is important for T-cell proliferation, cytokine production, and immune responses. Mol. Cell. Biol. 25, 6869–6878 (2005).

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12. Ensenat, D. et al. A novel src homology 3 domain-containing adaptor protein, HIP-55, that interacts with hematopoietic progenitor kinase 1. J. Biol. Chem. 274, 33945–33950 (1999). 13. Trub, T., Frantz, J.D., Miyazaki, M., Band, H. & Shoelson, S.E. The role of a lymphoidrestricted, Grb2-like SH3–SH2-SH3 protein in T cell receptor signaling. J. Biol. Chem. 272, 894–902 (1997). 14. Sawasdikosol, S., Russo, K.M. & Burakoff, S.J. Hematopoietic progenitor kinase 1 (HPK1) negatively regulates prostaglandin E2-induced fos gene transcription. Blood 101, 3687–3689 (2003). 15. Nagata, Y., Kiefer, F., Watanabe, T. & Todokoro, K. Activation of hematopoietic progenitor kinase-1 by erythropoietin. Blood 93, 3347–3354 (1999). 16. Hu, M.C. et al. Hematopoietic progenitor kinase-1 (HPK1) stress response signaling pathway activates IkB kinases (IKK-a/b) and IKK-b is a developmentally regulated protein kinase. Oncogene 18, 5514–5524 (1999). 17. Ling, P. et al. Interaction of hematopoietic progenitor kinase 1 with adapter proteins Crk and CrkL leads to synergistic activation of c-Jun N-terminal kinase. Mol. Cell. Biol. 19, 1359–1368 (1999). 18. Ma, W. et al. Leukocyte-specific adaptor protein Grap2 interacts with hematopoietic progenitor kinase 1 (HPK1) to activate JNK signaling pathway in T cells. Oncogene 20, 1703–1714 (2001). 19. Meller, N. et al. Direct interaction between protein kinase Cy (PKCy) and 14–3-3t in T cells: 14–3-3 overexpression results in inhibition of PKCy translocation and function. Mol. Cell. Biol. 16, 5782–5791 (1996). 20. Bonnefoy-Berard, N. et al. Inhibition of phosphatidylinositol 3-kinase activity by association with 14–3-3 proteins in T cells. Proc. Natl. Acad. Sci. USA 92, 10142–10146 (1995). 21. Muslin, A.J., Tanner, J.W., Allen, P.M. & Shaw, A.S. Interaction of 14–3-3 with signaling proteins is mediated by the recognition of phosphoserine. Cell 84, 889–897 (1996). 22. Yaffe, M.B. et al. The structural basis for 14–3-3:phosphopeptide binding specificity. Cell 91, 961–971 (1997). 23. Sabapathy, K. et al. c-Jun NH2-terminal kinase (JNK)1 and JNK2 have similar and stage-dependent roles in regulating T cell apoptosis and proliferation. J. Exp. Med. 193, 317–328 (2001). 24. Dong, C. et al. ICOS co-stimulatory receptor is essential for T-cell activation and function. Nature 409, 97–101 (2001). 25. Hehner, S.P., Hofmann, T.G., Dienz, O., Droge, W. & Schmitz, M.L. Tyrosine-phosphorylated Vav1 as a point of integration for T-cell receptor- and CD28-mediated activation of JNK, p38, and interleukin-2 transcription. J. Biol. Chem. 275, 18160–18171 (2000). 26. Zhang, W., Irvin, B.J., Trible, R.P., Abraham, R.T. & Samelson, L.E. Functional analysis of LAT in TCR-mediated signaling pathways using a LAT-deficient Jurkat cell line. Int. Immunol. 11, 943–950 (1999). 27. Yablonski, D., Kuhne, M.R., Kadlecek, T. & Weiss, A. Uncoupling of nonreceptor tyrosine kinases from PLCg1 in an SLP-76-deficient T cell. Science 281, 413–416 (1998). 28. Ebinu, J.O. et al. RasGRP links T-cell receptor signaling to Ras. Blood 95, 3199–3203 (2000). 29. Reynolds, L.F. et al. Vav1 transduces T cell receptor signals to the activation of phospholipase C-g1 via phosphoinositide 3-kinase-dependent and -independent pathways. J. Exp. Med. 195, 1103–1114 (2002). 30. Reynolds, L.F. et al. Vav1 transduces T cell receptor signals to the activation of the Ras/ ERK pathway via LAT, Sos, and RasGRP1. J. Biol. Chem. 279, 18239–18246 (2004). 31. Rao, N. et al. The linker phosphorylation site Tyr292 mediates the negative regulatory effect of Cbl on ZAP-70 in T cells. J. Immunol. 164, 4616–4626 (2000). 32. Erdreich-Epstein, A. et al. Cbl functions downstream of Src kinases in FcgRI signaling in primary human macrophages. J. Leukoc. Biol. 65, 523–534 (1999). 33. Le Bras, S. et al. Recruitment of the actin-binding protein HIP-55 to the immunological synapse regulates T cell receptor signaling and endocytosis. J. Biol. Chem. 279, 15550–15560 (2004). 34. Yao, Z. et al. A novel human STE20-related protein kinase, HGK, that specifically activates the c-Jun N-terminal kinase signaling pathway. J. Biol. Chem. 274, 2118–2125 (1999). 35. Diener, K. et al. Activation of the c-Jun N-terminal kinase pathway by a novel protein kinase related to human germinal center kinase. Proc. Natl. Acad. Sci. USA 94, 9687–9692 (1997). 36. Tung, R.M. & Blenis, J. A novel human SPS1/STE20 homologue, KHS, activates Jun Nterminal kinase. Oncogene 14, 653–659 (1997). 37. Pombo, C.M. et al. Activation of the SAPK pathway by the human STE20 homologue germinal centre kinase. Nature 377, 750–754 (1995). 38. Jeffrey, K.L. et al. Positive regulation of immune cell function and inflammatory responses by phosphatase PAC-1. Nat. Immunol. 7, 274–283 (2006). 39. Jaeschke, A. et al. JNK2 is a positive regulator of the cJun transcription factor. Mol. Cell 23, 899–911 (2006). 40. Brill, L.M. et al. Robust phosphoproteomic profiling of tyrosine phosphorylation sites from human T cells using immobilized metal affinity chromatography and tandem mass spectrometry. Anal. Chem. 76, 2763–2772 (2004). 41. Liu, Y.C., Elly, C., Yoshida, H., Bonnefoy-Berard, N. & Altman, A. Activation-modulated association of 14–3-3 proteins with Cbl in T cells. J. Biol. Chem. 271, 14591–14595 (1996). 42. Okuda, Y., Okuda, M. & Bernard, C.C. Regulatory role of p53 in experimental autoimmune encephalomyelitis. J. Neuroimmunol. 135, 29–37 (2003). 43. Zhou, G. et al. Protein phosphatase 4 is involved in tumor necrosis factor-a-induced activation of c-Jun N-terminal kinase. J. Biol. Chem. 277, 6391–6398 (2002).

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