A Nck-Pak1 signaling module is required for T-cell receptor-mediated activation of NFAT, but not of JNK
1998; Springer Nature; Volume: 17; Issue: 19 Linguagem: Inglês
10.1093/emboj/17.19.5647
ISSN1460-2075
Autores Tópico(s)T-cell and B-cell Immunology
ResumoArticle1 October 1998free access A Nck-Pak1 signaling module is required for T-cell receptor-mediated activation of NFAT, but not of JNK Deborah Yablonski Deborah Yablonski Departments of Medicine, Microbiology and Immunology and the Howard Hughes Medical Institute, University of California, San Francisco, California, 94143-0795 USA Search for more papers by this author Lawrence P. Kane Lawrence P. Kane Departments of Medicine, Microbiology and Immunology and the Howard Hughes Medical Institute, University of California, San Francisco, California, 94143-0795 USA Search for more papers by this author Dapeng Qian Dapeng Qian Departments of Medicine, Microbiology and Immunology and the Howard Hughes Medical Institute, University of California, San Francisco, California, 94143-0795 USA Present address: SUGEN, Redwood City, California, 94063 USA Search for more papers by this author Arthur Weiss Arthur Weiss Departments of Medicine, Microbiology and Immunology and the Howard Hughes Medical Institute, University of California, San Francisco, California, 94143-0795 USA Division of Rheumatology, Box 0795, Howard Hughes Medical Institute, 3rd and Parnassus Avenues, University of California, San Francisco, CA, 94143-0795 USA Search for more papers by this author Deborah Yablonski Deborah Yablonski Departments of Medicine, Microbiology and Immunology and the Howard Hughes Medical Institute, University of California, San Francisco, California, 94143-0795 USA Search for more papers by this author Lawrence P. Kane Lawrence P. Kane Departments of Medicine, Microbiology and Immunology and the Howard Hughes Medical Institute, University of California, San Francisco, California, 94143-0795 USA Search for more papers by this author Dapeng Qian Dapeng Qian Departments of Medicine, Microbiology and Immunology and the Howard Hughes Medical Institute, University of California, San Francisco, California, 94143-0795 USA Present address: SUGEN, Redwood City, California, 94063 USA Search for more papers by this author Arthur Weiss Arthur Weiss Departments of Medicine, Microbiology and Immunology and the Howard Hughes Medical Institute, University of California, San Francisco, California, 94143-0795 USA Division of Rheumatology, Box 0795, Howard Hughes Medical Institute, 3rd and Parnassus Avenues, University of California, San Francisco, CA, 94143-0795 USA Search for more papers by this author Author Information Deborah Yablonski1, Lawrence P. Kane1, Dapeng Qian1,2 and Arthur Weiss1,3 1Departments of Medicine, Microbiology and Immunology and the Howard Hughes Medical Institute, University of California, San Francisco, California, 94143-0795 USA 2Present address: SUGEN, Redwood City, California, 94063 USA 3Division of Rheumatology, Box 0795, Howard Hughes Medical Institute, 3rd and Parnassus Avenues, University of California, San Francisco, CA, 94143-0795 USA The EMBO Journal (1998)17:5647-5657https://doi.org/10.1093/emboj/17.19.5647 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The T-cell antigen receptor (TCR) triggers a signaling cascade initiated by the tyrosine kinase Lck and requiring the proto-oncogene p95vav. Vav is activated by Lck and can function as a guanine nucleotide exchange factor for the Rho-family GTPases, Rac1 and Cdc42. To investigate the involvement of these GTPases in TCR signaling, we focused on their well characterized effector, Pak1. This serine/threonine kinase is activated by GTP-bound Rac1 or Cdc42. However, its role in mediating downstream signaling events is controversial. We observed rapid, TCR-dependent activation of Pak1 and TCR-inducible association of Pak1 with Nck, which was tyrosine phosphorylated following stimulation. Pak1 activation occurred independently of Ras activation or calcium flux, but was dependent on the Lck tyrosine kinase, and was downstream of Vav and Cdc42. Dominant negative Pak1 or Nck specifically inhibited TCR-mediated activation of the nuclear factor of activated T cells (NFAT) transcription factor. TCR-mediated activation of Erk2 was also inhibited by dominant negative Pak. However, Pak1 activation was neither necessary nor sufficient for TCR-dependent c-Jun N-terminal kinase (JNK) activation. Therefore, Pak1 acts downstream of Vav and is required for activation of Erk2 and NFAT by a JNK-independent pathway. This is the first demonstration of a requirement for Pak to mediate the regulation of gene expression by an extracellular ligand. Introduction The T-cell antigen receptor (TCR) mediates recognition of cell-bound foreign antigens, triggering a relatively well characterized signaling pathway (Wange and Samelson, 1996; Qian and Weiss, 1997). The earliest signaling events depend on cytoplasmic tyrosine kinases which couple TCR activation to stimulation of downstream pathways. These include Ras activation, and phospholipase C activation, which leads to an increase in the second messenger inositol 1,4,5-trisphosphate (IP3). The rise in IP3 results in a rapid and sustained calcium increase. The coordinated stimulation of Ras and calcium flux is required to activate the nuclear factor of activated T cells (NFAT), a transcription factor critical for the transcriptional regulation of lymphokines such as interleukin-2. One of the proteins critical for TCR signaling is Vav (Bustelo, 1996). vav−/− lymphocytes have defective TCR-signal transduction (Fischer et al., 1995; Tarakhovsky et al., 1995; Zhang et al., 1995a), whereas overexpression of Vav in Jurkat T cells augments both basal and TCR-stimulated NFAT activity (Wu et al., 1995). However, the mechanism by which Vav participates in NFAT activation is still unclear. Vav is a multi-domain protein containing a Dbl homology domain, characteristic of Rho-family guanine nucleotide exchange factors (GEFs) (Cerione and Zheng, 1996). Vav has recently been shown to catalyze guanine nucleotide exchange in vitro of the Rho-family G proteins (Ridley, 1996) Rac1 (Crespo et al., 1997) and Cdc42 (Han et al., 1997). This activity is dependent on its phosphorylation by Lck, a tyrosine kinase which initiates the events of the TCR-signal transduction pathway. Interestingly, Vav is tyrosine phosphorylated following TCR stimulation (Bustelo et al., 1992; Margolis et al., 1992), suggesting that its GEF activity may be activated in vivo. It is therefore possible that the essential role of Vav in the TCR pathway may include activation of Rac1 or Cdc42. Consistent with this hypothesis, a dominant negative allele of Rac1 has been shown to inhibit TCR-induced NFAT activation (Genot et al., 1996), while a dominant negative allele of Cdc42 inhibits TCR-induced repolarization of the microtubule organizing center (Stowers et al., 1995). We were intrigued by these findings, since c-Jun N-terminal kinase (JNK), which is activated by Rac and Cdc42 in some systems, is not activated by TCR stimulation alone (Su et al., 1994). To address the involvement of Rac and Cdc42 in the TCR pathway, we investigated the effect of TCR stimulation on Pak1, a well characterized effector of Rac1 and Cdc42. Pak1 belongs to a family of closely related serine/threonine kinases (Sells and Chernoff, 1997) that are activated by autophosphorylation upon binding to GTP-bound Cdc42 or Rac1 (Manser et al., 1994; Martin et al., 1995). Several Pak isoforms have been identified, including two in humans (Martin et al., 1995; Brown et al., 1996). All Pak isoforms include a C-terminal serine/threonine kinase domain, highly homologous to the kinase domain of yeast Ste20. Binding of Pak to GTP-bound Cdc42 or Rac1 is mediated by an N-terminal GTPase binding domain, known as a CRIB domain (Manser et al., 1994; Burbelo et al., 1995). In addition, the extreme N-terminus of Pak1 contains a conserved proline-rich sequence, which mediates binding of Pak1 to the second SH3 domain of Nck, an adaptor protein composed of a single SH2 domain and three SH3 domains (Bokoch et al., 1996; Galisteo et al., 1996; Lu et al., 1997). Nck may recruit Pak to the membrane by binding to tyrosine-phosphorylated growth-factor receptors, which may facilitate activation of Pak by GTP-bound Rac1 or Cdc42. A number of extracellular ligands have been reported to induce rapid activation of Pak catalytic activity, including EGF, interleukin-1α, fMLP, thrombin and insulin (Knaus et al., 1995; Teo et al., 1995; Zhang et al., 1995b; Galisteo et al., 1996; Tsakiridis et al., 1996). Nonetheless, the physiological importance of Pak activation remains controversial. Pak has been suggested to mediate several of the downstream effects of Cdc42 and Rac, including activation of JNK (Bagrodia et al., 1995; Minden et al., 1995; Brown et al., 1996) and reorganization of the actin cytoskeleton (Manser et al., 1997; Sells et al., 1997). Other researchers have disputed these conclusions, demonstrating that effector-domain mutants of Rac1 that fail to bind Pak are still capable of inducing JNK activation as well as cytoskeletal changes (Lamarche et al., 1996; Westwick et al., 1997). At present, it is clear that GTP-bound Rac1 and Cdc42 can activate Pak; however, the functional consequences of this event are not fully understood. In this work, we have found that Pak1 is activated robustly upon TCR stimulation and undergoes TCR-inducible association with the Nck adaptor, which is tyrosine phosphorylated following stimulation. Dominant negative alleles of Pak1 or Nck specifically block TCR-induced NFAT activation. Interestingly, Pak1 is required for TCR- or Vav-mediated signaling to the nucleus by a pathway which is independent of JNK. This work, for the first time to our knowledge, demonstrates a requirement for Pak1 in a signaling pathway mediating the regulation of gene expression by an extracellular ligand. Results Stimulation of Pak1 by the T-cell receptor First, we examined the effect of TCR stimulation on Pak1 catalytic activity. An in vitro kinase assay was performed on anti-Pak1 immune complexes isolated from untransfected Jurkat cells (Figure 1A). Pak1 activity was significantly increased following TCR stimulation, as visualized by phosphorylation of the exogenous substrate, histone H4. In addition, a phosphorylated band of the same mobility as Pak1 was visible on the autoradiograms (data not shown). To ensure that the activity measured is that of Pak1, a control immunoprecipitation was performed in the presence of an excess of the peptide epitope recognized by the anti-Pak1 antibody. Under these conditions, no Pak1 was detected in the immune complexes and no kinase activity was detected (Figure 1A). Furthermore, a TCR-inducible kinase activity could be immunoprecipitated using anti-hemagglutinin (HA) antibodies from cells transfected with HA-tagged Pak1, but not from untransfected cells nor from cells transfected with HA-tagged, kinase-dead Pak1 (see Figure 3B, and data not shown). We are therefore confident that the activity measured represents Pak1. Figure 1.Regulation of Pak1 catalytic activity by the T-cell receptor. (A) Jurkat T cells were stimulated for 2 min with the anti-TCR monoclonal antibody C305 (1:500) or with a buffer control, lysed, and anti-Pak1 immune complexes were assayed for kinase activity as described in Materials and methods, using histone H4 as an exogenous substrate where indicated. To control for specificity, 1 μg/ml of the anti-Pak1 epitope blocking peptide was included during immune complex formation where indicated. Reaction products were resolved on SDS–PAGE, transferred to PVDF, and phosphorylation was visualized by autoradiography (upper panel). The PVDF filter was then probed with anti-Pak1 antibodies followed by ECL (lower panel). (B) Time course of TCR-mediated Pak1 activation. Jurkat cells were stimulated with C305 for 0–15 min as indicated and anti-Pak1 immune complexes were assayed for histone H4 phosphorylation activity using a quantitative assay, as described in Materials and methods. Duplicate determinations were performed, with error bars indicating the range of the results. The data are representative of two independent experiments. Download figure Download PowerPoint Figure 2.Characterization of a dominant negative Pak1. (A) The structure of wild-type and dominant negative Pak1 (Pak1DN). (B) Effect of Pak1DN on TCR-mediated activation of wild-type Pak1. Jurkat cells were cotransfected with 10 μg/cuvette of pEFhPak1WT, encoding HA-tagged Pak1, along with 30 μg of pEF-BOS (vector), pEFPak1DN or pEFRafDN as indicated. Twenty-seven hours later, the cells were stimulated for 5 min with C305 or buffer control, lysed, and anti-HA (12CA5) immune complexes were assayed for kinase activity as in Figure 1B. The results are representative of at least two independent experiments. (C) Lysates from B were resolved on SDS–PAGE, transferred to PVDF, and probed with anti-HA antibodies to compare the expression of full length and Pak1DN. Lane 1, untransfected Jurkat cells; lane 2, Pak1 + vector; lane 3, Pak1 + Pak1DN; lane 4, Pak1 + RafDN. Download figure Download PowerPoint The time course of Pak1 activation was examined using a quantitative assay (Figure 1B). We found Pak1 activity to be increased within 2 min of TCR stimulation and to remain above basal levels for ∼10 min. Similarly, Pak1 was transiently activated following anti-TCR stimulation of mouse peripheral T cells (data not shown). However, this activation occurred 30 s after stimulation and the fold actuation was lower than that seen in Jurkat cells. This rapid time course suggests that Pak1 activation may be a relatively proximal consequence of TCR stimulation. The Lck tyrosine kinase is required for TCR-induced Pak1 activation TCR signaling in Jurkat cells is initiated by tyrosine phosphorylation of receptor ITAMs by the Src-family tyrosine kinase Lck. Subsequent recruitment of the ZAP-70 tyrosine kinase to the phosphorylated ITAMs and phosphorylation of downstream substrates leads to activation of two major pathways, a Ras-dependent pathway and a calcium-dependent pathway. To determine whether Pak1 activation is mediated by elements of the known TCR-signaling pathway, we investigated the involvement of Lck, Ras and calcium flux in Pak1 activation. The requirement for Lck was investigated using the Lck-deficient cell line JCaM1.6. This cell line fails to activate Pak1 upon TCR stimulation, whereas Pak1 was efficiently activated in JCaM1.6 cells stably transfected with wild-type Lck (Figure 2A). These results demonstrate that TCR-stimulated tyrosine phosphorylation is required for Pak1 activation. Figure 3.(A) TCR-mediated Pak1 activation is dependent on Lck. Pak activity was measured following stimulation of the TCR for 5 min, as in Figure 1B. Cells were: Jurkat (wild-type), the Lck–Jurkat derivative, JCaM1.6, or JCaM1.6 cells stably reconstituted with wild-type Lck. The results are representative of two independent experiments. (B) Pak1 is not activated by PMA or by ionomycin. Jurkat cells were stimulated for 5 min with C305, ionomycin (1 μM) and/or PMA (50 ng/ml), and Pak1 catalytic activity was measured as in Figure 1B. Download figure Download PowerPoint To test whether Pak1 activation is triggered proximally or more distally in the TCR-regulated signaling pathway, we examined the effects of the pharmacological agents PMA and ionomycin. These compounds are used to mimic experimentally downstream responses to TCR stimulation, while bypassing the requirement for proximal events such as receptor phosphorylation. PMA is sufficient to activate Ras (Downward et al., 1990), while ionomycin mimics TCR-induced calcium flux. However, neither of these stimuli, alone or in combination, activated Pak1 (Figure 2B). This result suggests that Pak1 is not activated downstream of either Ras or calcium flux, but may be activated upstream or independently of these pathways. Inhibition of TCR signaling by dominant negative Pak1 To evaluate the functional importance of TCR-induced Pak1 activation, we constructed a dominant negative allele of Pak1. It has been demonstrated previously that the truncated N-terminus of Pak2/hPak65, including the CRIB domain but lacking the kinase domain, can act as a dominant negative allele to block activation of JNK by Cdc42 (Minden et al., 1995). We constructed an analogous dominant negative allele of Pak1 by terminating the open reading frame immediately following residue 265 (Figure 3A). To assess whether this allele would inhibit TCR-mediated activation of wild-type Pak1, we transfected Jurkat cells transiently with full-length, epitope-tagged Pak1 along with the truncated Pak1 or a vector control. Cotransfection of truncated Pak1 completely blocked TCR-induced Pak1 activation (Figure 3B). In contrast, cotransfection of a dominant negative allele of Raf did not affect Pak1 activation. Western blot analysis of the immune complexes confirmed that equal amounts of full-length Pak1 were immunoprecipitated in each case (Figure 3C). This experiment demonstrates that the Pak1 N-terminus (Pak1DN) can act as a dominant negative inhibitor of TCR-induced Pak1 activation. Next, we examined the effect of Pak1DN on signaling events downstream of the TCR, using an NFAT-luciferase reporter plasmid. The NFAT transcription factor can be activated by TCR stimulation, or by a combination of PMA and ionomycin, which activates the requisite Ras and calcium pathways. Cotransfection of Jurkat cells with Pak1DN, but not wild-type Pak1, potently blocked TCR-induced NFAT activation (Figure 4A, left panel). In contrast, activation of NFAT by PMA and ionomycin was not reduced by dominant negative Pak1, but was consistently increased (Figure 4A, right panel). These results demonstrate that Pak1DN is not toxic, but specifically inhibits the TCR-signal transduction pathway at a step upstream of the points at which PMA and ionomycin act. Figure 4.Effect of Pak1DN on TCR signaling. (A) Inhibition of TCR-mediated NFAT activation by Pak1DN. Jurkat cells were transfected with 15 μg of pNFAT-Luc along with 30 μg of pEF-BOS (vector), Pak1WT or Pak1DN. Twenty-four hours later the cells were stimulated for 6 h with medium (unstimulated), C305 (1:1000), or PMA (50 ng/ml) and ionomycin (1 μM) and luciferase activity was assayed as described in Materials and methods. This experiment is representative of at least three independent experiments. (B) Dose dependence of NFAT inhibition by Pak1DN. Jurkat cells were transfected with 15 μg of pNFAT-Luc along with 0–30 μg of Pak1WT or Pak1DN. Vector DNA was added to each transfection as needed to equalize the amount of DNA. Twenty-four hours later, the cells were stimulated and assayed for luciferase activity as in (A). The results are expressed as a fraction of the activity obtained upon stimulation with PMA and ionomycin. Lysates prepared from the transfected cells were resolved on SDS–PAGE, transferred to PVDF, and probed with anti-HA antibodies to compare the expression of full-length and dominant negative Pak1 (right panel). This experiment is representative of two independent experiments. (C) The role of the Pak1DN CRIB domain in mediating inhibition of signaling. Jurkat cells were cotransfected with 20 μg of pNFAT-Luc, along with 15 μg of control vector, Pak1WT or Pak1DN, or 40 μg of Pak1DN83,86L, all in pEF-BOS, and NFAT luciferase reporter activity was measured as in (A). These amounts of plasmid produced equivalent expression of Pak1DN and Pak1DN83,86L as assessed by Western blotting (right panel). The results are expressed as a fraction of the activity obtained upon stimulation with PMA and ionomycin. This experiment is representative of five independent experiments. Download figure Download PowerPoint While overexpression of Pak1DN inhibited TCRmediated NFAT activation over a wide range of plasmid doses, overexpression of wild-type Pak1 failed to inhibit at all doses (Figure 4B, left panel). Notably, wild-type Pak1 was expressed at a higher level than the dominant negative allele at every dose tested (Figure 4B, right panel). Since both wild-type and dominant negative Pak1 include the CRIB domain, this result suggests that the inhibition of NFAT is not caused by nonspecific sequestration of Rac1 and Cdc42 by overexpression of a CRIB domain. To test further whether Pak1DN acts by sequestration of Rac1 and Cdc42, we mutated two critical histidine residues (83 and 86) required for binding of the Pak1 CRIB domain to Rac1 and Cdc42 (Manser et al., 1997; Sells et al., 1997). This construct, Pak1DN83,86L, was a less efficient inhibitor of TCR-mediated NFAT activation than Pak1DN (Figure 4C), although both were expressed at equal levels (Figure 4C, right panel). It is therefore likely that part of the inhibitory effect of Pak1DN is due to blocking Cdc42-and Rac1-dependent pathways. However, Pak1DN83,86L retained significant inhibitory activity. This activity is probably due to blocking the function of other proteins that normally interact with the Pak1 N-terminus. This result suggests that Pak1DN does not function solely by sequestering activated Rac and Cdc42, but may be a more specific inhibitor of Pak1 function, perhaps by binding to more than one protein that regulates Pak. This claim is supported further by the observation that the effects of Pak1DN are more specific than those of dominant negative Rac or Cdc42, which inhibit activation of NFAT by PMA and ionomycin as well as by the TCR (Genot et al., 1996; data not shown). Pak1DN inhibits a proximal event specific to the TCR signaling pathway To investigate further the specificity of inhibition by Pak1DN, we examined its effect on the signal transduction pathway initiated by the human muscarinic receptor (HM1R). The Jurkat-derived cell line J.HM1.2.2, which stably expresses the HM1R, was used for these studies (Goldsmith et al., 1989). The HM1R is a seven-transmembrane domain receptor, which, when expressed in Jurkat cells, can mediate activation of NFAT (Wu et al., 1995). Unlike the TCR pathway, the HM1R signal is transduced by a heterotrimeric G protein which activates PLCβ (Desai et al., 1990). Although the HM1R ultimately activates the Ras and calcium pathways required for NFAT activation, it does this through distinct proximal events. Overexpression of Pak1DN in J.HM1.2.2 inhibited NFAT activation by the TCR, but did not inhibit NFAT activation by the HM1R (Figure 5). This result suggests that Pak1DN specifically inhibits the TCR pathway at a step upstream of the point at which the TCR and HM1R pathways converge. Figure 5.Specificity of inhibition by Pak1DN. J.HM1.2.2 cells, which stably express the human muscarinic receptor, were cotransfected with 15 μg of pNFAT-Luc and 30 μg of control vector, Pak1WT or Pak1DN. Twenty-four hours later, the cells were stimulated for 6 h with medium (unstimulated) C305 (1:1000), carbachol (500 μM), or PMA (50 ng/ml) and ionomycin (1 μM), and luciferase activity was measured as in Figure 4. Results for each transfection were normalized to the activity obtained upon stimulation with PMA and ionomycin. The results shown are the average of five independent experiments. Download figure Download PowerPoint Role of the Nck adaptor protein in TCR signal transduction To obtain independent confirmation of the involvement of Pak1 in the TCR pathway, we investigated the role of Nck, an adaptor protein which has been found to associate via its second SH3 domain with the first proline-rich region of Pak1 (Bokoch et al., 1996; Galisteo et al., 1996; Lu et al., 1997). As has been reported in fibroblasts, we found that endogenously expressed Nck and Pak1 associate in Jurkat T cells although the association was not constitutive, but was induced following TCR stimulation (Figure 6A). Inducible association of Pak1 and Nck could be demonstrated by coimmunoprecipitation of Nck with anti-Pak1 (Figure 6A) or by the reverse experiment in which Pak1 is coimmunoprecipitated by anti-Nck (data not shown). Furthermore, we found that Nck becomes tyrosine phosphorylated and undergoes a mobility shift upon TCR stimulation (Figure 6B). Together with our observation of catalytic activation of Pak1 following TCR stimulation, these observations suggest that the Nck–Pak1 complex interacts functionally with the TCR-signal transduction machinery. Figure 6.Involvement of Nck in the TCR pathway. (A) Stimulation-dependent association of Nck with Pak1 in T cells. Jurkat T cells were stimulated for 1–10 min with C305, and lysates were immuno- precipitated with a rabbit polyclonal antibody directed against the C-terminal portion of Pak1. To control for specificity, 1 μg/ml of the anti-Pak1 epitope blocking peptide was included during immune complex formation where indicated. Immune complexes were analyzed by immunoblotting with anti-Pak (top) or with anti-Nck (bottom) polyclonal antisera. (B) Tyrosine phosphorylation of Nck following TCR stimulation. Jurkat T cells were stimulated for 2 min with C305 or buffer control, lysed, and Nck was immunoprecipitated with an anti-Nck mAb. Immune complexes were resolved on SDS–PAGE, transferred to PVDF, and analyzed by immunoblotting with anti-phosphotyrosine (upper panel). The blot was then stripped and reprobed with anti-Nck (lower panel). The tyrosine phosphorylated band seen in the upper panel corresponds to the slower migrating band in the anti-Nck blot. (C) Inhibition of TCR signaling by dominant negative Nck. NFAT-luciferase activity was determined in J.HM1.2.2 cells cotransfected with 15 μg of pNFAT-Luc and 30 μg of control vector or NckDN, in which a W143K mutation inactivates the second SH3 domain of Nck. Twenty-four hours later, the cells were stimulated and luciferase activity was measured as in Figure 5. Results for each transfection were normalized to the activity obtained upon stimulation with PMA and ionomycin. The results shown are the average of four independent experiments. Download figure Download PowerPoint To confirm the functional involvement of Nck in the TCR pathway, we utilized a dominant negative allele of Nck, containing a W143K mutation which inactivates the second SH3 domain of Nck and abolishes its interaction with Pak1 (Lu et al., 1997). Like Pak1DN, dominant negative Nck specifically inhibited TCR-mediated NFAT activation, but did not inhibit NFAT activation by the HM1R (Figure 6C). Taken together, our data suggest that an Nck–Pak1 signaling complex is required to mediate activation of NFAT by the TCR. The position of Pak1 in the TCR pathway relative to Ras and Rho-family G proteins It has been established previously that the catalytic activity of Pak1 is regulated by the Rho-family G proteins Rac and Cdc42 (Sells and Chernoff, 1997). However, the role of Rho-family G proteins in TCR-signal transduction is less well established than that of Ras. It has been suggested that Rac may act downstream of Ras in fibroblasts (Rodriguez-Viciana et al., 1997) as well as in the TCR pathway (Genot et al., 1996). In contrast, our experiments suggest that Pak1 does not act downstream of Ras in the TCR pathway. These considerations suggested that the relationship within the TCR pathway between Pak1 and Ras and Rho-family small G proteins should be investigated further. To test the dependence of Pak1 activation on Ras, epitope-tagged Pak1 was coexpressed with dominant negative Ras, RasN17. While RasN17 efficiently inhibited NFAT activation (Figure 7A, right), it had no effect on activation of Pak1 (Figure 7A, left). In contrast, dominant negative Cdc42 inhibited TCR-mediated Pak1 activation, as would be expected if Pak1 were regulated by Rho-family G proteins. The effect of dominant negative Rac on Pak1 activation could not be assessed owing to nonspecific effects on the level of Pak1 expression (data not shown). From this experiment we conclude that Pak1 activation is mediated by Cdc42 (perhaps together with other Rho-family GTPases), but occurs upstream or independently of Ras. Figure 7.Pak1 acts downstream of Vav and Rho-family G proteins, but is upstream of Ras and calcium flux. (A) Effect of dominant negative G proteins on TCR-mediated Pak1 activation. TAg Jurkat cells were cotransfected with 5 μg/cuvette of pEFhPak1WT and 10 μg/cuvette of pNFAT-Luc, along with 40 μg of vector, RasN17 or Cdc42N17, as indicated. Twenty-four hours later, one aliquot of the cells was stimulated for 5 min with C305 or buffer control, lysed, and anti-HA (12CA5) immune complexes were assayed for kinase activity as in Figure 1B. Results are expressed as fold-activation of Pak1 following TCR stimulation (left panel). A second aliquot was stimulated for 6 h and assayed for luciferase activity (right panel). The results are representative of at least two experiments. (B) NFAT-luciferase activity was determined in J.HM1.2.2 cells cotransfected with 20 μg of pNFAT-Luc and 15 μg of control vector, Pak1WT or Pak1DN, and stimulated with C305 alone or in combination with PMA (50 ng/ml) or ionomycin (1 μM). Results for each transfection were normalized to the activity obtained upon stimulation with PMA and ionomycin. The results shown are the average of two independent transfections. (C) Inhibition of TCR-mediated Erk2 activation by Pak1DN. Jurkat cells were cotransfected with 10 μg of myc-tagged Erk2 along with 40 μg of vector, Pak1DN or Pak1WT. Twenty-four hours later, the cells were stimulated for 5 min with anti-TCR (C305; 1:500), PMA (50 ng/ml) or buffer control, lysed, and anti-myc immune complexes were resolved by SDS–PAGE and analyzed by immunoblotting with anti-phospho-Erk (top) and anti-Erk2 (bottom). These results are representative of two experiments
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