The Ca2+-dependent Phosphatase Calcineurin Controls the Formation of the Carma1-Bcl10-Malt1 Complex during T Cell Receptor-induced NF-κB Activation
2011; Elsevier BV; Volume: 286; Issue: 9 Linguagem: Inglês
10.1074/jbc.m110.155895
ISSN1083-351X
AutoresLysann Palkowitsch, Uta Marienfeld, Cornelia Brunner, Andrea C. Eitelhuber, Daniel Krappmann, Ralf Marienfeld,
Tópico(s)Bioactive natural compounds
ResumoT cell receptor (TCR) ligation induces increased diacylglycerol and Ca2+ levels in T cells, and both secondary messengers are crucial for TCR-induced nuclear factor of activated T cells (NF-AT) and NF-κB signaling pathways. One prominent calcium-dependent enzyme involved in the regulation of NF-AT and NF-κB signaling pathways is the protein phosphatase calcineurin. However, in contrast to NF-AT, which is directly dephosphorylated by calcineurin, the molecular basis of the calcium-calcineurin dependence of the TCR-induced NF-κB activity remains largely unknown. Here, we demonstrate that calcineurin regulates TCR-induced NF-κB activity by controlling the formation of a protein complex composed of Carma1, Bcl10, and Malt1 (CBM complex). For instance, increased calcium levels induced by ionomycin or thapsigargin augmented the phorbol 12-myristate 13-acetate-induced formation of the CBM complex and activation of NF-κB, whereas removal of calcium by the calcium chelator EGTA-acetoxymethyl ester (AM) attenuated both processes. Furthermore, inhibition of the calcium-dependent phosphatase calcineurin with the immunosuppressive agent cyclosporin A (CsA) or FK506 as well as siRNA-mediated knockdown of calcineurin A strongly affected the PMA + ionomycin- or anti-CD3 + CD28-induced CBM complex assembly. Mechanistically, the positive effect of calcineurin on the CBM complex formation seems to be linked to a dephosphorylation of Bcl10. For instance, Bcl10 was found to be hyperphosphorylated in Jurkat T cells upon treatment with CsA or EGTA-AM, and calcineurin dephosphorylated Bcl10 in vivo and in vitro. Furthermore, we show here that calcineurin A interacts with the CBM complex. In summary, the evidence provided here argues for a previously unanticipated role of calcineurin in CBM complex formation as a molecular basis of the inhibitory function of CsA or FK506 on TCR-induced NF-κB activity. T cell receptor (TCR) ligation induces increased diacylglycerol and Ca2+ levels in T cells, and both secondary messengers are crucial for TCR-induced nuclear factor of activated T cells (NF-AT) and NF-κB signaling pathways. One prominent calcium-dependent enzyme involved in the regulation of NF-AT and NF-κB signaling pathways is the protein phosphatase calcineurin. However, in contrast to NF-AT, which is directly dephosphorylated by calcineurin, the molecular basis of the calcium-calcineurin dependence of the TCR-induced NF-κB activity remains largely unknown. Here, we demonstrate that calcineurin regulates TCR-induced NF-κB activity by controlling the formation of a protein complex composed of Carma1, Bcl10, and Malt1 (CBM complex). For instance, increased calcium levels induced by ionomycin or thapsigargin augmented the phorbol 12-myristate 13-acetate-induced formation of the CBM complex and activation of NF-κB, whereas removal of calcium by the calcium chelator EGTA-acetoxymethyl ester (AM) attenuated both processes. Furthermore, inhibition of the calcium-dependent phosphatase calcineurin with the immunosuppressive agent cyclosporin A (CsA) or FK506 as well as siRNA-mediated knockdown of calcineurin A strongly affected the PMA + ionomycin- or anti-CD3 + CD28-induced CBM complex assembly. Mechanistically, the positive effect of calcineurin on the CBM complex formation seems to be linked to a dephosphorylation of Bcl10. For instance, Bcl10 was found to be hyperphosphorylated in Jurkat T cells upon treatment with CsA or EGTA-AM, and calcineurin dephosphorylated Bcl10 in vivo and in vitro. Furthermore, we show here that calcineurin A interacts with the CBM complex. In summary, the evidence provided here argues for a previously unanticipated role of calcineurin in CBM complex formation as a molecular basis of the inhibitory function of CsA or FK506 on TCR-induced NF-κB activity. IntroductionUpon engagement of the T cell receptor (TCR), 2The abbreviations used are: TCR, T cell receptor; NF-κB, nuclear factor κB; NF-AT, nuclear factor of activated T cells; IKK, IκB kinase; NEMO, NF-κB essential modulator; CsA, cyclosporin A; CnA and CnB, calcineurin A and B, respectively; CaMKII, Ca2+/calmodulin-dependent protein kinase II; AM, acetoxymethyl ester; PMA, phorbol 12-myristate 13-acetate; P+I, PMA plus ionomycin. phospholipase Cγ1 (PLCγ1) is induced, which in turn hydrolyzes phosphoinositol 4,5-bisphosphate, resulting in elevated levels of diacylglycerol and inositol trisphosphate. Diacylglycerol is crucial for the activation of members of the PKC family, with PKCϴ being the key PKC family member required for the activation of AP1, NF-AT, and NF-κB signaling pathways in T cells. The other product generated by phosphoinositol 4,5-bisphosphate hydrolysis, inositol trisphosphate, is an inducer of calcium influx into the cytoplasm of T cells by a process known as store-operated calcium entry. After an initial inositol trisphosphate-induced depletion of the calcium stored in the endoplasmic reticulum, CRAC channels at the plasma membrane are opened in a STIM-dependent fashion, leading to a more sustained calcium influx (1Oh-hora M. Rao A. Curr. Opin. Immunol. 2008; 20: 250-258Crossref PubMed Scopus (293) Google Scholar). Calcium acts as a second messenger in the T cell necessary for the activation of several signaling molecules, including the protein kinases CaMKII and PKCα as well as the protein phosphatase calcineurin, which in turn are crucial for the activation of the transcription factors NF-AT, AP1, CREB1, and NF-κB. Especially calcineurin has been demonstrated to be crucial for the activation of the AP-1, NF-AT, and NF-κB signaling pathways (1Oh-hora M. Rao A. Curr. Opin. Immunol. 2008; 20: 250-258Crossref PubMed Scopus (293) Google Scholar, 2Werlen G. Jacinto E. Xia Y. Karin M. EMBO J. 1998; 17: 3101-3111Crossref PubMed Scopus (252) Google Scholar, 3Marienfeld R. Neumann M. Chuvpilo S. Escher C. Kneitz B. Avots A. Schimpl A. Serfling E. Eur. J. Immunol. 1997; 27: 1601-1609Crossref PubMed Scopus (76) Google Scholar, 4Trushin S.A. Pennington K.N. Algeciras-Schimnich A. Paya C.V. J. Biol. Chem. 1999; 274: 22923-22931Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). In contrast to the NF-AT pathway, however, which is activated due to a direct dephosphorylation of the NF-AT transcription factors by the calcium-dependent protein phosphatase calcineurin, the molecular mechanisms by which calcineurin affects the NF-κB pathway is far less understood.The transcription factor NF-κB is crucial for development, survival, homoeostasis, and function of T lymphocytes. One essential step in the antigen receptor-induced NF-κB signaling pathway is the formation of the CBM complex, which is composed of Carma1, Bcl10, and Malt1 (5Thome M. Weil R. Trends Immunol. 2007; 28: 281-288Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Whereas Bcl10 and Malt1 interact constitutively, Carma1 is recruited to this Bcl10-Malt1 complex in an inducible manner, depending on site-specific phosphorylation of Carma1. Besides PKCϴ, which seems to be the main Carma1 kinase following TCR engagement, also HPK1 and IKK2 have been reported to phosphorylate Carma1, albeit at different serine residues (6Sommer K. Guo B. Pomerantz J.L. Bandaranayake A.D. Moreno-García M.E. Ovechkina Y.L. Rawlings D.J. Immunity. 2005; 23: 561-574Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, 7Shinohara H. Maeda S. Watarai H. Kurosaki T. J. Exp. Med. 2007; 204: 3285-3293Crossref PubMed Scopus (87) Google Scholar, 8Brenner D. Brechmann M. Röhling S. Tapernoux M. Mock T. Winter D. Lehmann W.D. Kiefer F. Thome M. Krammer P.H. Arnold R. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 14508-14513Crossref PubMed Scopus (46) Google Scholar). Following CBM complex formation, Malt1 is ubiquitinated by TRAF6, and this Lys63-conjugated ubiquitin chain functions as an anchor for the recruitment of the IκB-kinase complex, which is subsequently activated by a still unknown mechanism (9Bidère N. Snow A.L. Sakai K. Zheng L. Lenardo M.J. Curr. Biol. 2006; 16: 1666-1671Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 10Oeckinghaus A. Wegener E. Welteke V. Ferch U. Arslan S.C. Ruland J. Scheidereit C. Krappmann D. EMBO J. 2007; 26: 4634-4645Crossref PubMed Scopus (158) Google Scholar). The activated IκB-kinase complex phosphorylates IκB proteins and thereby targets these proteins for proteasomal degradation. NF-κB is subsequently liberated and translocates to the nucleus, where it supports the expression of various NF-κB target genes. Finally, TCR-induced NF-κB signaling is terminated by the phosphorylation-dependent proteasomal degradation of Carma1 and Bcl10 (5Thome M. Weil R. Trends Immunol. 2007; 28: 281-288Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 11Hinz M. Scheidereit C. Sci. STKE 2007. 2007; : pe19Google Scholar, 12Moreno-García M.E. Sommer K. Shinohara H. Bandaranayake A.D. Kurosaki T. Rawlings D.J. Mol. Cell. Biol. 2010; 30: 922-934Crossref PubMed Scopus (27) Google Scholar). The calcium-dependent phosphatase calcineurin is composed of a catalytically active A subunit (CnA) of ∼61 kDa and a regulatory B subunit (CnB) of 19 kDa. In its inactive state, the active site of calcineurin is blocked by a carboxyl-terminal localized autoinhibitory domain (1Oh-hora M. Rao A. Curr. Opin. Immunol. 2008; 20: 250-258Crossref PubMed Scopus (293) Google Scholar, 13Shibasaki F. Hallin U. Uchino H. J. Biochem. 2002; 131: 1-15Crossref PubMed Scopus (183) Google Scholar). For its activation, calcineurin requires the binding of the CnB subunit as well as the calcium-calmodulin complex. The binding of the regulatory proteins causes a structural alteration of CnA and a release of the autoinhibitory domain. Immunosuppressive drugs like cyclosporin A (CsA) and tacrolimus (FK506) target calcineurin and impair the activation of key signaling pathways in lymphoid cells. For instance, pretreatment of T cells with pharmacological inhibitors specific for calcineurin, like CsA or FK506, led to a marked reduction of the TCR-induced NF-AT, JNK, and NF-κB activity (2Werlen G. Jacinto E. Xia Y. Karin M. EMBO J. 1998; 17: 3101-3111Crossref PubMed Scopus (252) Google Scholar, 3Marienfeld R. Neumann M. Chuvpilo S. Escher C. Kneitz B. Avots A. Schimpl A. Serfling E. Eur. J. Immunol. 1997; 27: 1601-1609Crossref PubMed Scopus (76) Google Scholar, 4Trushin S.A. Pennington K.N. Algeciras-Schimnich A. Paya C.V. J. Biol. Chem. 1999; 274: 22923-22931Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). However, although calcineurin dependence of the TCR-induced NF-κB signaling pathway has been known for over a decade, the molecular mechanism by which this calcium-dependent protein phosphatase regulates NF-κB has not been clarified.Here, we demonstrate that calcium influx is essential for the formation of the CBM complex in activated T cells. Furthermore, we provide evidence for a crucial role of the calcium-dependent protein phosphatase calcineurin for the assembly of the CBM complex and NF-κB activation. Blocking calcineurin activity with CsA or FK506 or suppression of the catalytic calcineurin A subunit by siRNA led to a drastic decrease in CBM complex formation. Moreover, calcineurin constitutively interacts with components of the CBM complex like Malt1, and inhibition of calcineurin augmented the basal Bcl10 phosphorylation in Jurkat T cells. Furthermore, basal Bcl10 phosphorylation was diminished by calcineurin coexpression in HEK293 cells. In conclusion, our data imply that calcineurin regulates the TCR-induced NF-κB signaling pathway by controlling the CBM complex assembly, most likely by the removal of inhibitory phosphate groups from the CBM complex component Bcl10.DISCUSSIONA multitude of signal transduction events are initiated upon engagement of the antigen receptors in T and B cells. One critical event during this process is the activation of the phospholipase Cγ1, which in turn hydrolyzes phosphoinositol 4,5-bisphosphate to diacylglycerol and inositol trisphosphate, leading to the activation of the Ras and PKC pathways on the one hand and to a calcium influx on the other. This calcium influx is known as store-operated calcium entry and depends on the calcium sensors of the STIM family and the CRAC calcium channels (1Oh-hora M. Rao A. Curr. Opin. Immunol. 2008; 20: 250-258Crossref PubMed Scopus (293) Google Scholar). Several signaling pathways, including the NF-AT, AP1, and NF-κB pathways, need both signaling branches (the diacylglycerol-induced and the calcium-dependent signals) for their full activity. One essential calcium-dependent signaling protein is the protein phosphatase calcineurin, which has been shown to be crucial for the TCR-induced activation of NF-AT as well as NF-κB (1Oh-hora M. Rao A. Curr. Opin. Immunol. 2008; 20: 250-258Crossref PubMed Scopus (293) Google Scholar, 3Marienfeld R. Neumann M. Chuvpilo S. Escher C. Kneitz B. Avots A. Schimpl A. Serfling E. Eur. J. Immunol. 1997; 27: 1601-1609Crossref PubMed Scopus (76) Google Scholar, 4Trushin S.A. Pennington K.N. Algeciras-Schimnich A. Paya C.V. J. Biol. Chem. 1999; 274: 22923-22931Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). However, in contrast to the NF-AT pathway, the molecular mechanism underlying the calcium-calcineurin dependence of the NF-κB pathway is less well understood. Therefore, we set out to determine the molecular mechanisms by which this calcium-dependent phosphatase affects the NF-κB signaling pathway. One essential step in the NF-κB signaling pathway initiated upon antigen-receptor triggering is the assembly of a multiprotein complex consisting of Carma1, Bcl10, and Malt1, also known as the CBM complex (21Schulze-Luehrmann J. Ghosh S. Immunity. 2006; 25: 701-715Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar). Here, we provide evidence that calcium influx is crucial for the proper formation of the CBM complex. For instance, we show that an additional calcium-influx, either induced by ionomycin or by thapsigargin treatment, augmented the PMA-induced Carma1-Bcl10 interaction distinctively (Fig. 1). By contrast, a pretreatment with the cell-permeable calcium chelator EGTA-AM attenuated the PMA- and P+I-induced NF-κB activity. In addition, the CBM complex formation but had no effect on the TNF-α-induced NF-κB activation (Fig. 2), thus further demonstrating the necessity of elevated cellular calcium levels for the formation of the CBM complex.An active calcineurin is composed of a catalytically active A subunit, a regulatory B subunit, and a calcium-calmodulin complex (13Shibasaki F. Hallin U. Uchino H. J. Biochem. 2002; 131: 1-15Crossref PubMed Scopus (183) Google Scholar). Inhibition of calcineurin with the pharmaceutical inhibitor CsA or FK506 impairs the IKK activation and thus the IκBα degradation specifically induced by TCR engagement but not upon TNF-α stimulation (Fig. 3, A–C) (3Marienfeld R. Neumann M. Chuvpilo S. Escher C. Kneitz B. Avots A. Schimpl A. Serfling E. Eur. J. Immunol. 1997; 27: 1601-1609Crossref PubMed Scopus (76) Google Scholar, 4Trushin S.A. Pennington K.N. Algeciras-Schimnich A. Paya C.V. J. Biol. Chem. 1999; 274: 22923-22931Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). Here, we demonstrate that the basis for the negative effect of these pharmaceutical calcineurin inhibitors is the attenuation of the CBM complex assembly. Pretreatment of Jurkat T cells as well as primary murine T cells with CsA strongly reduces the Bcl10-Carma1 interaction by P+I stimulation or by stimulation with agonistic anti-CD3 + CD28 antibodies (Fig. 3, D–F). Importantly, a similar reduction in Bcl10-Carma1 interaction was achieved by the combined transfection of siRNAs for CnAα and CnAβ but only very moderately by transfection of the individual CnA siRNAs (Fig. 5A). However, the degree of inhibition in the Carma1-Bcl10 interaction as well as NF-κB activation was always more pronounced by pretreatment with CsA than by siRNA-mediated suppression of CnA. The reason for the lower efficacy of the siRNA-mediated CnA suppression is not completely clear. However, one can speculate that the inhibition by CsA might be more efficient because CsA impairs the activity of all CnA isoforms, whereas a residual CnA expression remained in case of the siRNA-mediated knockdown experiments, which would allow a remaining calcineurin activity (Fig. 5C). Alternatively, the third isoform of CnA, CnAγ, might also contribute to the regulation of the CBM complex formation by calcineurin. Expression of CnAγ is thought to be restricted to the testis. However, data mining revealed that this calcineurin A isoform is also expressed in T and B lymphocytes at the RNA level (GeneATLAS).The formation of the CBM complex appears to be highly regulated by post-translational modifications, involving the phosphorylation of Carma1 and Bcl10. Carma1 phosphorylation upon TCR ligation, which can be mediated by PKCϴ, HPK1, IKK2, CaMKII, and casein kinase I, is required for CBM complex formation, and thus Carma1 phosphorylation represents mostly a positive signal (7Shinohara H. Maeda S. Watarai H. Kurosaki T. J. Exp. Med. 2007; 204: 3285-3293Crossref PubMed Scopus (87) Google Scholar, 8Brenner D. Brechmann M. Röhling S. Tapernoux M. Mock T. Winter D. Lehmann W.D. Kiefer F. Thome M. Krammer P.H. Arnold R. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 14508-14513Crossref PubMed Scopus (46) Google Scholar, 22Moreno-García M.E. Sommer K. Haftmann C. Sontheimer C. Andrews S.F. Rawlings D.J. J. Immunol. 2009; 183: 7362-7370Crossref PubMed Scopus (22) Google Scholar). In contrast, the phosphorylation of Bcl10 has been linked to negative functions (14Wegener E. Oeckinghaus A. Papadopoulou N. Lavitas L. Schmidt-Supprian M. Ferch U. Mak T.W. Ruland J. Heissmeyer V. Krappmann D. Mol. Cell. 2006; 23: 13-23Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 23Ishiguro K. Ando T. Goto H. Xavier R. Mol. Immunol. 2007; 44: 2095-2100Crossref PubMed Scopus (31) Google Scholar, 24Scharschmidt E. Wegener E. Heissmeyer V. Rao A. Krappmann D. Mol. Cell. Biol. 2004; 24: 3860-3873Crossref PubMed Scopus (117) Google Scholar). For instance, the phosphorylation of the five serine residues (Ser134, Ser136, Ser138, Ser141, and Ser144) in the carboxyl-terminal part of Bcl10 by IKK2 has been linked to a remodeling of the Bcl10-Malt1 heterodimer, leading to resolution of CBM complex assembly (14Wegener E. Oeckinghaus A. Papadopoulou N. Lavitas L. Schmidt-Supprian M. Ferch U. Mak T.W. Ruland J. Heissmeyer V. Krappmann D. Mol. Cell. 2006; 23: 13-23Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Phosphorylation of Bcl10 at these carboxyl-terminal serine residues does not influence the Carma1-Bcl10 interaction directly but affects CBM complex function by altering the structure of the Malt1-Bcl10 heterodimer. The disturbed Malt1-Bcl10 interaction in turn causes a reduced recruitment of Carma1 as part of a negative feedback mechanism to terminate the TCR-induced NF-κB pathway. A similar mechanism might also underlie the positive calcineurin effect shown in the present study. Although Malt1-Bcl10 interaction remained unchanged upon calcineurin inhibition by CsA or by specific siRNAs (FIGURE 3, FIGURE 5), calcineurin might dephosphorylate Bcl10, causing an alteration of the Malt1-Bcl10 heterodimer structure and thus promoting an efficient Carma1 recruitment. Interestingly, the role of basal Bcl10 phosphorylation in unstimulated T cells or during the early phase of TCR-induced signaling has not been fully elucidated. However, a basal Bcl10 phosphorylation can be observed in Jurkat T cells as well as HEK293 cells (FIGURE 6, FIGURE 7) (20Zeng H. Di L. Fu G. Chen Y. Gao X. Xu L. Lin X. Wen R. Mol. Cell. Biol. 2007; 27: 5235-5245Crossref PubMed Scopus (33) Google Scholar). The hypothesis that calcineurin might affect the CBM complex formation by removing one or more negatively acting phosphate groups from the Bcl10 protein is supported by the increased basal Bcl10 phosphorylation in CsA- or EGTA-treated Jurkat T cells (Fig. 6) (which suggests that calcineurin dephosphorylates Bcl10 in vivo). In addition, the decrease in Bcl10 phosphorylation in HEK293 and Jurkat T cells upon coexpression of calcineurin A or more pronounced upon coexpression of the constitutive active mutant ΔCam further supports our hypothesis. Moreover, we observed a protein-protein interaction of the calcineurin A subunit or a constitutive active deletion mutant of calcineurin Aα (ΔCam) with Malt1 or Carma1. In addition, calcineurin A forms a protein complex with the CBM complex in Jurkat T cells as demonstrated by the Malt1-calcineurin A interaction (Fig. 9D). The reason for the lack of Bcl10-CnA interaction with ectopically expressed proteins remains unclear. However, one explanation could be that Malt1 is required for a stable Bcl10-CnA interaction. Alternatively, the recently published Bcl10-calmodulin interaction might also contribute to the recruitment of calcineurin to Bcl10 (25Edin S. Oruganti S.R. Grundström C. Grundström T. Mol. Immunol. 2010; 47: 2057-2064Crossref PubMed Scopus (10) Google Scholar). Experiments are under way in our laboratory to clarify this question.Taken together, the evidence provided in this study clearly demonstrates that calcium influx is crucial for the formation of the CBM complex during TCR-induced NF-κB activation. Furthermore, our data strongly suggest that calcineurin is one of the calcium-dependent signaling proteins involved in the assembly of the CBM complex (Fig. 10). However, calcineurin might not be the only calcium-dependent enzyme required for this process. Blocking CaMKII with the specific inhibitor KN93, for instance, had a profound effect on the PMA- and TCR-induced NF-κB activity (26Hughes K. Edin S. Antonsson A. Grundström T. J. Biol. Chem. 2001; 276: 36008-36013Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Furthermore, PKCα has also been demonstrated to affect the antigen receptor-induced NF-κB activation in T cells. However, PKCα seems to modulate the TCR-induced NF-κB activity indirectly by affecting PKCϴ, the central regulator of CBM complex assembly (27Trushin S.A. Pennington K.N. Carmona E.M. Asin S. Savoy D.N. Billadeau D.D. Paya C.V. Mol. Cell. Biol. 2003; 23: 7068-7081Crossref PubMed Scopus (110) Google Scholar). CaMKII, on the other hand, has been identified as both a Bcl10 and a Carma1 kinase and could participate in the control of CBM complex assembly by phosphorylating Carma1 at Ser109 (23Ishiguro K. Ando T. Goto H. Xavier R. Mol. Immunol. 2007; 44: 2095-2100Crossref PubMed Scopus (31) Google Scholar, 28Ishiguro K. Green T. Rapley J. Wachtel H. Giallourakis C. Landry A. Cao Z. Lu N. Takafumi A. Goto H. Daly M.J. Xavier R.J. Mol. Cell. Biol. 2006; 26: 5497-5508Crossref PubMed Scopus (79) Google Scholar). The reduced Carma1 phosphorylation upon EGTA-AM pretreatment would support the hypothesis of an additional calcium-dependent but CsA-insensitive effect on Carma1 phosphorylation. However, although PKCα or CaMKII might contribute to the overall calcium dependence of the TCR-induced NF-κB activation, they seem not to be negatively affected by CsA (Fig. 4). From these results, especially the calineurin-mediated dephosphorylation of Bcl10 in vitro (Fig. 8), we concluded that the positive effect of calcineurin is rather direct and does not involve the activation of other calcium-dependent signaling pathways. Although we provide evidence that CBM complex formation might be affected by the calcineurin-mediated dephosphorylation of Bcl10, further work is needed to clarify the exact role of calcineurin for the TCR-induced NF-κB signaling pathway. For instance, it will be necessary to identify the serine residues in Bcl10 that are dephosphorylated by calcineurin. To this end, it seems likely that one or more of the five already identified carboxyl-terminal IKK2 phosphorylation sites are targeted by calcineurin because calcineurin coexpression reverted the IKK2-mediated Bcl10 hyperphosphorylation (Fig. 7, A–C). Furthermore, the Bcl10S5A mutant is not affected by calcineurin (Fig. 7). Another question pertains to the kinases responsible for basal Bcl10 phosphorylation. IKK2 and CaMKII have been identified as Bcl10 kinases, which are responsible for Bcl10 phosphorylation upon cell activation. However, whether the basal Bcl10 phosphorylation is also conducted by these kinases or conducted by still unknown kinases needs to be clarified in future experiments. The answers to these questions will not only provide a better understanding of the regulation of the TCR-induced NF-κB signaling pathway but might also lead to the development of more specific inhibitors for the different CnA isoforms in order to avoid the toxicity of the currently used immunosuppressors CsA and FK506. IntroductionUpon engagement of the T cell receptor (TCR), 2The abbreviations used are: TCR, T cell receptor; NF-κB, nuclear factor κB; NF-AT, nuclear factor of activated T cells; IKK, IκB kinase; NEMO, NF-κB essential modulator; CsA, cyclosporin A; CnA and CnB, calcineurin A and B, respectively; CaMKII, Ca2+/calmodulin-dependent protein kinase II; AM, acetoxymethyl ester; PMA, phorbol 12-myristate 13-acetate; P+I, PMA plus ionomycin. phospholipase Cγ1 (PLCγ1) is induced, which in turn hydrolyzes phosphoinositol 4,5-bisphosphate, resulting in elevated levels of diacylglycerol and inositol trisphosphate. Diacylglycerol is crucial for the activation of members of the PKC family, with PKCϴ being the key PKC family member required for the activation of AP1, NF-AT, and NF-κB signaling pathways in T cells. The other product generated by phosphoinositol 4,5-bisphosphate hydrolysis, inositol trisphosphate, is an inducer of calcium influx into the cytoplasm of T cells by a process known as store-operated calcium entry. After an initial inositol trisphosphate-induced depletion of the calcium stored in the endoplasmic reticulum, CRAC channels at the plasma membrane are opened in a STIM-dependent fashion, leading to a more sustained calcium influx (1Oh-hora M. Rao A. Curr. Opin. Immunol. 2008; 20: 250-258Crossref PubMed Scopus (293) Google Scholar). Calcium acts as a second messenger in the T cell necessary for the activation of several signaling molecules, including the protein kinases CaMKII and PKCα as well as the protein phosphatase calcineurin, which in turn are crucial for the activation of the transcription factors NF-AT, AP1, CREB1, and NF-κB. Especially calcineurin has been demonstrated to be crucial for the activation of the AP-1, NF-AT, and NF-κB signaling pathways (1Oh-hora M. Rao A. Curr. Opin. Immunol. 2008; 20: 250-258Crossref PubMed Scopus (293) Google Scholar, 2Werlen G. Jacinto E. Xia Y. Karin M. EMBO J. 1998; 17: 3101-3111Crossref PubMed Scopus (252) Google Scholar, 3Marienfeld R. Neumann M. Chuvpilo S. Escher C. Kneitz B. Avots A. Schimpl A. Serfling E. Eur. J. Immunol. 1997; 27: 1601-1609Crossref PubMed Scopus (76) Google Scholar, 4Trushin S.A. Pennington K.N. Algeciras-Schimnich A. Paya C.V. J. Biol. Chem. 1999; 274: 22923-22931Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). In contrast to the NF-AT pathway, however, which is activated due to a direct dephosphorylation of the NF-AT transcription factors by the calcium-dependent protein phosphatase calcineurin, the molecular mechanisms by which calcineurin affects the NF-κB pathway is far less understood.The transcription factor NF-κB is crucial for development, survival, homoeostasis, and function of T lymphocytes. One essential step in the antigen receptor-induced NF-κB signaling pathway is the formation of the CBM complex, which is composed of Carma1, Bcl10, and Malt1 (5Thome M. Weil R. Trends Immunol. 2007; 28: 281-288Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Whereas Bcl10 and Malt1 interact constitutively, Carma1 is recruited to this Bcl10-Malt1 complex in an inducible manner, depending on site-specific phosphorylation of Carma1. Besides PKCϴ, which seems to be the main Carma1 kinase following TCR engagement, also HPK1 and IKK2 have been reported to phosphorylate Carma1, albeit at different serine residues (6Sommer K. Guo B. Pomerantz J.L. Bandaranayake A.D. Moreno-García M.E. Ovechkina Y.L. Rawlings D.J. Immunity. 2005; 23: 561-574Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, 7Shinohara H. Maeda S. Watarai H. Kurosaki T. J. Exp. Med. 2007; 204: 3285-3293Crossref PubMed Scopus (87) Google Scholar, 8Brenner D. Brechmann M. Röhling S. Tapernoux M. Mock T. Winter D. Lehmann W.D. Kiefer F. Thome M. Krammer P.H. Arnold R. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 14508-14513Crossref PubMed Scopus (46) Google Scholar). Following CBM complex formation, Malt1 is ubiquitinated by TRAF6, and this Lys63-conjugated ubiquitin chain functions as an anchor for the recruitment of the IκB-kinase complex, which is subsequently activated by a still unknown mechanism (9Bidère N. Snow A.L. Sakai K. Zheng L. Lenardo M.J. Curr. Biol. 2006; 16: 1666-1671Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 10Oeckinghaus A. Wegener E. Welteke V. Ferch U. Arslan S.C. Ruland J. Scheidereit C. Krappmann D. EMBO J. 2007; 26: 4634-4645Crossref PubMed Scopus (158) Google Scholar). The activated IκB-kinase complex phosphorylates IκB proteins and thereby targets these proteins for proteasomal degradation. NF-κB is subsequently liberated and translocates to the nucleus, where it supports the expression of various NF-κB target genes. Finally, TCR-induced NF-κB signaling is terminated by the phosphorylation-dependent proteasomal degradation of Carma1 and Bcl10 (5Thome M. Weil R. Trends Immunol. 2007; 28: 281-288Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 11Hinz M. Scheidereit C. Sci. STKE 2007. 2007; : pe19Google Scholar, 12Moreno-García M.E. Sommer K. Shinohara H. Bandaranayake A.D. Kurosaki T. Rawlings D.J. Mol. Cell. Biol. 2010; 30: 922-934Crossref PubMed Scopus (27) Google Scholar). The calcium-dependent phosphatase calcineurin is composed of a catalytically active A subunit (CnA) of ∼61 kDa and a regulatory B subunit (CnB) of 19 kDa. In its inactive state, the active site of calcineurin is blocked by a carboxyl-terminal localized autoinhibitory domain (1Oh-hora M. Rao A. Curr. Opin. Immunol. 2008; 20: 250-258Crossref PubMed Scopus (293) Google Scholar, 13Shibasaki F. Hallin U. Uchino H. J. Biochem. 2002; 131: 1-15Crossref PubMed Scopus (183) Google Scholar). For its activation, calcineurin requires the binding of the CnB subunit as well as the calcium-calmodulin complex. The binding of the regulatory proteins causes a structural alteration of CnA and a release of the autoinhibitory domain. Immunosuppressive drugs like cyclosporin A (CsA) and tacrolimus (FK506) target calcineurin and impair the activation of key signaling pathways in lymphoid cells. For instance, pretreatment of T cells with pharmacological inhibitors specific for calcineurin, like CsA or FK506, led to a marked reduction of the TCR-induced NF-AT, JNK, and NF-κB activity (2Werlen G. Jacinto E. Xia Y. Karin M. EMBO J. 1998; 17: 3101-3111Crossref PubMed Scopus (252) Google Scholar, 3Marienfeld R. Neumann M. Chuvpilo S. Escher C. Kneitz B. Avots A. Schimpl A. Serfling E. Eur. J. Immunol. 1997; 27: 1601-1609Crossref PubMed Scopus (76) Google Scholar, 4Trushin S.A. Pennington K.N. Algeciras-Schimnich A. Paya C.V. J. Biol. Chem. 1999; 274: 22923-22931Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). However, although calcineurin dependence of the TCR-induced NF-κB signaling pathway has been known for over a decade, the molecular mechanism by which this calcium-dependent protein phosphatase regulates NF-κB has not been clarified.Here, we demonstrate that calcium influx is essential for the formation of the CBM complex in activated T cells. Furthermore, we provide evidence for a crucial role of the calcium-dependent protein phosphatase calcineurin for the assembly of the CBM complex and NF-κB activation. Blocking calcineurin activity with CsA or FK506 or suppression of the catalytic calcineurin A subunit by siRNA led to a drastic decrease in CBM complex formation. Moreover, calcineurin constitutively interacts with components of the CBM complex like Malt1, and inhibition of calcineurin augmented the basal Bcl10 phosphorylation in Jurkat T cells. Furthermore, basal Bcl10 phosphorylation was diminished by calcineurin coexpression in HEK293 cells. In conclusion, our data imply that calcineurin regulates the TCR-induced NF-κB signaling pathway by controlling the CBM complex assembly, most likely by the removal of inhibitory phosphate groups from the CBM complex component Bcl10.
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