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ABSTRACT: In T cells PKCh mediates the activation of critical signals downstream of TCR/CD28 stimulation. We investigated the molecular mechanisms by which PKCh regulates NFkB transactivation by examining PKCh/b single and double knockout mice and observed a redundant involvement of PKCh and PKCb in this signaling pathway. Mechanistically, we define a PKCh-CYLD protein complex and an interaction between the positive PKCh/b and the negative CYLD signaling pathways that both converge at the level of TAK1/IKK/I-kBa/NFkB and NFAT transactivation. In Jurkat leukemic T cells, CYLD is endoproteolytically processed in the initial minutes of stimulation by the paracaspase MALT1 in a PKC-dependent fashion, which is required for robust IL-2 transcription. However, in primary T cells, CYLD processing occurs with different kinetics and an altered dependence on PKC. The formation of a direct PKCh/CYLD complex appears to regulate the short-term spatial distribution of CYLD, subsequently affecting NFkB and NFAT repressional activity of CYLD prior to its MALT1-dependent inactivation. Taken together, our study establishes CYLD as a new and critical PKCh interactor in T cells and reveals that antagonistic PKCh/b-CYLD crosstalk is crucial for the adjustment of immune thresholds in primary mouse CD3 + T cells.
PLoS ONE 01/2013; · 4.09 Impact Factor
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ABSTRACT: In T cells PKCθ mediates the activation of critical signals downstream of TCR/CD28 stimulation. We investigated the molecular mechanisms by which PKCθ regulates NFκB transactivation by examining PKCθ/β single and double knockout mice and observed a redundant involvement of PKCθ and PKCβ in this signaling pathway. Mechanistically, we define a PKCθ-CYLD protein complex and an interaction between the positive PKCθ/β and the negative CYLD signaling pathways that both converge at the level of TAK1/IKK/I-κBα/NFκB and NFAT transactivation. In Jurkat leukemic T cells, CYLD is endoproteolytically processed in the initial minutes of stimulation by the paracaspase MALT1 in a PKC-dependent fashion, which is required for robust IL-2 transcription. However, in primary T cells, CYLD processing occurs with different kinetics and an altered dependence on PKC. The formation of a direct PKCθ/CYLD complex appears to regulate the short-term spatial distribution of CYLD, subsequently affecting NFκB and NFAT repressional activity of CYLD prior to its MALT1-dependent inactivation. Taken together, our study establishes CYLD as a new and critical PKCθ interactor in T cells and reveals that antagonistic PKCθ/β-CYLD crosstalk is crucial for the adjustment of immune thresholds in primary mouse CD3(+) T cells.
PLoS ONE 01/2013; 8(1):e53709. · 4.09 Impact Factor
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ABSTRACT: One of the shortcomings of vaccinia virus (VACV) as immunization vector is the down-regulation of HLA and costimulatory molecules in antigen presenting cells. To overcome this problem we investigated the use of protein kinase C (PKC) as immune stimulatory agent. Thus several classical and atypical PKCs were inserted into wild-type or attenuated VACV using recombination into the hemagglutinin gene and the expression driven by the VACV 7,5K-IE gene promoter. Recombinant constructs expressing PKC-alpha, -beta, -theta as well as wild-type, constitutive active or dominant negative PKC-zeta constructs were generated. Additional constructs expressing PKB/Akt1 and ICAM-1 were used for comparison. Immature and mature peripheral blood derived-dendritic cells (DC) as well as lymphoid cell lines capable of obtaining a DC-like phenotype upon mitogen stimulation were infected. Disappointingly, VACV-driven PKC overexpression did not significantly enhance expression of various activation markers or costimulatory molecules tested. Neither CD86 nor HLA-DR expression was upregulated and also no influence on the maturation of DC, as measured by DC-SIGN and CD83, was observed. However, VACV did not interfere with LPS induced up-regulation of CD83 and did not lead to substantial apoptosis of infected DC within the first 24 hours.
Immunological Investigations 01/2013; 42(2):164-77. · 1.47 Impact Factor
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Marlies Meisel,
Natascha Hermann-Kleiter,
Reinhard Hinterleitner,
Thomas Gruber,
Katarzyna Wachowicz,
Christa Pfeifhofer-Obermair, Friedrich Fresser,
Michael Leitges,
Cristiana Soldani,
Antonella Viola,
Sandra Kaminski,
Gottfried Baier
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ABSTRACT: Transforming growth-factor β (TGFβ) has been implicated in T helper 17 (Th17) cell biology and in triggering expression of interleukin-17A (IL-17A), which is a key Th17 cell cytokine. Deregulated TGFβ receptor (TGFβR) signaling has been implicated in Th17-cell-mediated autoimmune pathogenesis. Nevertheless, the full molecular mechanisms involved in the activation of the TGFβR pathway in driving IL-17A expression remain unknown. Here, we identified protein kinase C α (PKCα) as a signaling intermediate specific to the Th17 cell subset in the activation of TGFβRI. We have shown that PKCα physically interacts and functionally cooperates with TGFβRI to promote robust SMAD2-3 activation. Furthermore, PKCα-deficient (Prkca(-/-)) cells demonstrated a defect in SMAD-dependent IL-2 suppression, as well as decreased STAT3 DNA binding within the Il17a promoter. Consistently, Prkca(-/-) cells failed to mount appropriate IL-17A, but not IL-17F, responses in vitro and were resistant to induction of Th17-cell-dependent experimental autoimmune encephalomyelitis in vivo.
Immunity 01/2013; · 21.64 Impact Factor
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ABSTRACT: Interleukin-17A (IL-17A) is the signature cytokine produced by Th17 CD4(+) T cells and has been tightly linked to autoimmune pathogenesis. In particular, the transcription factors NFAT and RORγt are known to activate Il17a transcription, although the detailed mechanism of action remains incompletely understood. Here, we show that the nuclear orphan receptor NR2F6 can attenuate the capacity of NFAT to bind to critical regions of the Il17a gene promoter. In addition, because NR2F6 binds to defined hormone response elements (HREs) within the Il17a locus, it interferes with the ability of RORγt to access the DNA. Consistently, NFAT and RORγt binding within the Il17a locus were enhanced in Nr2f6-deficient CD4(+) Th17 cells but decreased in Nr2f6-overexpressing transgenic CD4(+) Th17 cells. Taken together, our findings uncover an example of antagonistic regulation of Il17a transcription through the direct reciprocal actions of NR2F6 versus NFAT and RORγt.
Journal of Autoimmunity 08/2012; · 7.37 Impact Factor
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ABSTRACT: Transforming growth factor β (TGFβ) plays a central role in maintaining immune homeostasis by regulating the initiation and termination of immune responses and thus preventing the development of autoimmune diseases. In this study, we describe an essential mechanism by which the actin regulatory protein Coronin 1A (Coro1A) ensures the proper response of Th17 CD4(+) T cells to TGFβ. Coro1A has been established as a key player in T cell survival, migration, activation, and Ca(2+) regulation in naive T cells. We show that mice lacking Coro1a developed less severe experimental autoimmune encephalomyelitis (EAE). Unexpectedly, upon the re-induction of EAE, Coro1a(-/-) mice exhibited enhanced EAE signs that correlated with increased numbers of IL-17 producing CD4(+) cells in the central nervous system (CNS) compared to wild-type mice. In vitro differentiated Coro1a(-/-) Th17 CD4(+) T cells consistently produced more IL-17 than wild-type cells and displayed a Th17/Th1-like phenotype in regard to the expression of the Th1 markers T-bet and IFNγ. Mechanistically, the Coro1a(-/-) Th17 cell phenotype correlated with a severe defect in TGFβR-mediated SMAD3 activation. Taken together, these data provide experimental evidence of a non-redundant role of Coro1A in the regulation of Th17 CD4(+) cell effector functions and, subsequently, in the development of autoimmunity.
Journal of Autoimmunity 06/2011; 37(3):198-208. · 7.37 Impact Factor
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ABSTRACT: Partial duplication 3q is a well defined clinical entity characterized by growth retardation, cryptorchism, microcephaly, and characteristic dysmorphisms. Most patients present with large duplications or are associated with a second chromosomal imbalance, which makes the definition of the phenotype difficult. Here, we report on a 4-year and 8-month-old girl with pre- and postnatal measurements in the high normal range, developmental delay, minor dysmorphic features, and a de novo unbalanced 3/4 translocation with trisomy 3q27 --> qter and monosomy of the subtelomeric region of 4p. Conventional karyotyping, FISH with probes from the Wolf-Hirschhorn syndrome critical region and chromosome 4p locus-specific probes, microsatellite marker-based haplotyping, and SNP microarray copy number analysis revealed a terminal 4p deletion of less than 500 kb with a breakpoint distal to the Wolf-Hirschhorn syndrome critical region, a chromosome 3q duplication of around 15.3 Mb, with origin of the rearrangement in paternal meiosis. Thus, our case clearly characterizes the phenotype of pure partial duplication 3q more exactly, and moreover, indicates that small chromosome rearrangements might lead to growth in the upper normal range or even cause overgrowth.
American Journal of Medical Genetics Part A 11/2009; 149A(11):2522-6. · 2.39 Impact Factor
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ABSTRACT: The E3 ubiquitin ligase Casitas B-lineage lymphoma (Cbl-b) is central to antigen-induced immune tolerance and regulates the CD28 dependence of T cell activation. Cbl-b undergoes ubiquitination and proteasomal degradation after adequate costimulation of T cells; however, the mechanism involved is unknown. Here, we identified protein kinase C-theta (PKC-theta) as the critical intermediary for the inactivation of Cbl-b in response to costimulation of T cells through CD28. PKC-theta associated with Cbl-b on stimulation of the T cell receptor. After costimulation of T cells through CD28, Cbl-b was ubiquitinated and degraded through a mechanism that depended on the kinase activity of PKC-theta. Consistent with this mechanism, the impaired responses of PKCtheta-deficient T cells were at least partially restored by the concomitant genetic loss of cblb. Thus, our data establish a nonredundant antagonism between PKC-theta and Cbl-b that regulates T cell activation responses.
Science Signaling 02/2009; 2(76):ra30. · 7.50 Impact Factor
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ABSTRACT: The antigen-specific interaction of a T cell with an antigen-presenting cell (APC) results in the formation of an immunologic synapse (IS) between the membranes of the 2 cells. beta(2) integrins on the T cell, namely, leukocyte function-associated antigen 1 (LFA-1) and its counter ligand, namely, immunoglobulin-like cell adhesion molecule 1 (ICAM-1) on the APC, critically stabilize this intercellular interaction. The small GTPase Rap1 controls T-cell adhesion through modulating the affinity and/or spatial organization of LFA-1; however, the upstream regulatory components triggered by the T-cell receptor (TCR) have not been resolved. In the present study, we identified a previously unknown function of a protein kinase C- theta (PKC-theta)/RapGEF2 complex in LFA-1 avidity regulation in T lymphocytes. After T-cell activation, the direct phosphorylation of RapGEF2 at Ser960 by PKC- theta regulates Rap1 activation as well as LFA-1 adhesiveness to ICAM-1. In OT-II TCR-transgenic CD4(+) T cells, clustering of LFA-1 after antigen activation was impaired in the absence of PKC- theta. These data define that, among other pathways acting on LFA-1 regulation, PKC- theta and its effector RapGEF2 are critical factors in TCR signaling to Rap1. Taken together, PKC- theta sets the threshold for T-cell activation by positively regulating both the cytokine responses and the adhesive capacities of T lymphocytes.
Blood 10/2008; 112(12):4617-27. · 9.90 Impact Factor
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Natascha Hermann-Kleiter,
Thomas Gruber,
Christina Lutz-Nicoladoni,
Nikolaus Thuille, Friedrich Fresser,
Verena Labi,
Natalia Schiefermeier,
Marei Warnecke,
Lukas Huber,
Andreas Villunger,
Gregor Eichele,
Sandra Kaminski,
Gottfried Baier
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ABSTRACT: The protein kinase C (PKC) family of serine-threonine kinases plays a central role in T lymphocyte activation. Here, we identify NR2F6, a nuclear zinc-finger orphan receptor, as a critical PKC substrate and essential regulator of CD4(+) T cell activation responses. NR2F6 potently antagonized the ability of T helper 0 (Th0) and Th17 CD4(+) T cells to induce expression of key cytokine genes such as interleukin-2 (IL-2) and IL-17. Mechanistically, NR2F6 directly interfered with the DNA binding of nuclear factor of activated T cells (NF-AT):activator protein 1 (AP-1) but not nuclear factor kappaB (NF-kappa B) and, subsequently, transcriptional activity of the NF-AT-dependent IL-17A cytokine promoter. Consistent with our model, Nr2f6-deficient mice had hyperreactive lymphocytes, developed a late-onset immunopathology, and were hypersusceptible to Th17-dependent experimental autoimmune encephalomyelitis. Our study establishes NR2F6 as a transcriptional repressor of IL-17 expression in Th17-differentiated CD4(+) T cells in vitro and in vivo.
Immunity 09/2008; 29(2):205-16. · 21.64 Impact Factor
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ABSTRACT: Using yeast two-hybrid, we isolated atypical PKCzeta as a PKCtheta-interacting kinase and demonstrated that it selectively interacted with, and was phosphorylated by, PKCtheta. Importantly, however, both atypical PKCzeta and PKCiota were functionally required in TCR/CD28-mediated activation of NF-kappaB downstream of PKCtheta in Jurkat T cells albeit, activation responses of PKCzeta-deficient CD3+ T cells were comparable with wildtype controls. This normal activation thresholds of PKCzeta-/- T cells suggested that PKCiota, the closest structural relative, might play a compensatory role in TCR/CD28-induced signalling. Consistently, both PKCzeta and PKCiota resided in the plasma membrane lipid raft microdomains of Jurkat as well as primary mouse CD3+ T cells. Thus, PKCtheta, the established constituent of the immunological synapse, physically and functionally interacted with PKCzeta and PKCiota. Together, these data demonstrate that atypical PKCzeta/iota isotypes serve as direct downstream targets of PKCtheta in the signalling pathway leading to NF-kappaB activation in T lymphocytes.
Molecular Immunology 02/2008; 45(1):117-26. · 2.90 Impact Factor
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Nikolaus Thuille,
Isabelle Heit, Friedrich Fresser,
Nina Krumböck,
Birgit Bauer,
Sabine Leuthaeusser,
Sascha Dammeier,
Caroline Graham,
Terry D Copeland,
Steve Shaw,
Gottfried Baier
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ABSTRACT: Phosphopeptide mapping identified a major autophosphorylation site, phospho (p)Thr-219, between the tandem C1 domains of the regulatory fragment in protein kinase C (PKC)theta. Confirmation of this identification was derived using (p)Thr-219 antisera that reacted with endogenous PKCtheta in primary CD3+ T cells after stimulation with phorbol ester, anti-CD3 or vanadate. The T219A mutation abrogated the capacity of PKCtheta to mediate NF-kappaB, NF-AT and interleukin-2 promoter transactivation, and reduced PKCtheta's ability in Jurkat T cells to phosphorylate endogenous cellular substrates. In particular, the T219A mutation impaired crosstalk of PKCtheta with Akt/PKBalpha in NF-kappaB activation. Yet, this novel (p)Thr-219 site did not affect catalytic activity or second-messenger lipid-binding activity in vitro. Instead, the T219A mutation prevented proper recruitment of PKCtheta in activated T cells. The PKCthetaT219A mutant defects were largely rescued by addition of a myristoylation signal to force its proper membrane localization. We conclude that autophosphorylation of PKCtheta at Thr-219 plays an important role in the correct targeting and cellular function of PKCtheta upon antigen receptor ligation.
The EMBO Journal 12/2005; 24(22):3869-80. · 9.20 Impact Factor
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ABSTRACT: LPA, the gene coding for apolipoprotein(a) [apo(a)], is the major determinant of lipoprotein(a) [Lp(a)] plasma levels, which are associated with risk for coronary heart disease (CHD) and stroke. It is not completely understood how variation in LPA relates to Lp(a) concentrations. One type of variation related to Lp(a) levels is the number of Kringle (K) IV-2 (g.61C>T; GenBank L14005.1) repeats in LPA, but sequence variation may also contribute. Human apo(a) contains from two to >40 nearly identical K IV-2 repeats of genomic size 5.5 kb, which makes it difficult to detect mutations. To elucidate the genetic variation of the apo(a) K IV-2 domain, we isolated a single "nonexpressing" apo(a) allele with 26 K IV-2 repeats, followed by PCR, cloning and sequencing of 96 clones, resulting in an average coverage of each K IV-2 repeat of approximately four-fold. The previously described K IV types 2A and 2B (K IV-2A and K IV-2B) were detected in 74% of the clones. In addition, a new type designated 2C (K IV-2C) was present. A nonsense mutation in the first exon of K IV-2 (g.61C>T) predicted to result in a truncated protein (p.R21X) was found in nine clones on a K IV-2A background. The presence of this mutation was confirmed by analysis of genomic DNA and was shown to represent the rare allele (frequency 0.02) of a SNP. Immunoblot analysis of apo(a) from plasma confirmed the presence of a truncated apo(a) isoform in the index individual and family members. Our data show that SNPs affecting Lp(a) plasma concentrations also exist in the apo(a) K IV-2 domain.
Human Mutation 12/2004; 24(6):474-80. · 5.69 Impact Factor
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ABSTRACT: Protein kinase Cθ (PKCθ) is known to induce NF-κB, an essential transcriptional element in T cell receptor/CD28-mediated interleukin-2
production but also T cell survival. Here we provide evidence that PKCθ is physically and functionally coupled to Akt1 in
this signaling pathway. First, T cell receptor/CD3 ligation was sufficient to induce activation as well as plasma membrane
recruitment of PKCθ. Second, PKCθ selectively cooperated with Akt1, known to act downstream of CD28 co-receptor signaling,
in activating a NF-κB reporter in T cells. Third, Akt1 function was shown to be required for PKCθ-mediated NF-κB transactivation.
Fourth, PKCθ co-immunoprecipitated with Akt1; however, neither Akt1 nor PKCθ served as a prominent substrate for each other
in vitro as well as in intact T cells. Finally, plasma membrane targeting of PKCθ and Akt1 exerted synergistic transactivation of
the I-κB kinase β/inhibitor of NF-κB/NF-κB signaling cascade independent of T cell activation. Taken together, these findings
suggest a direct cross-talk between PKCθ and Akt1 in Jurkat T cells.
Journal of Biological Chemistry 08/2001; 276(34):31627-31634. · 4.77 Impact Factor
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ABSTRACT: Phosphorylation of myristoylated alanine-rich protein kinase C substrate (MARCKS) in intact cells has been employed as an
indicator for activation of protein kinase C (PKC). Specific PKC isoenzymes responsible for MARCKS phosphorylation under physiological
conditions, however, remained to be identified. In our present study using stably transfected NIH 3T3 cell clones we demonstrate
that expression of constitutively active mutants of either conventional cPKC-α or novel nPKC-ϵ increased phosphorylation of
endogenous MARCKS in the absence of phorbol 12,13-dibutyrate in intact mouse fibroblasts, implicating that each of these PKC
isoforms itself is sufficient to induce enhanced MARCKS phosphorylation. Similarly, ectopic expression of a constitutively
active mutant of PKC-η significantly increased MARCKS phosphorylation compared to vector controls, identifying PKC-η as a
MARCKS kinase. The PKC-specific inhibitor GF 109203X (bisindolylmaleimide I) reduced MARCKS phosphorylation in intact cells
at a similar dose-response as enzymatic activity of recombinant isoenzymes cPKC-α, nPKC-ϵ, and nPKC-η in vitro Consistently, phorbol 12,13-dibutyrate-dependent MARCKS phosphorylation was significantly reduced in cell lines expressing
dominant negative mutants of either PKC-α K368R or (dominant negative) PKC-ϵ K436R. The fact, that the constitutively active
PKC-λ A119E mutant did not alter the MARCKS phosphorylation underscores the assumption that atypical PKC isoforms are not
involved in this process. We conclude that under physiological conditions, conventional cPKC-α and novel nPKC-ϵ, but not atypical
aPKC-λ are responsible for MARCKS phosphorylation in intact NIH 3T3 fibroblasts.
Journal of Biological Chemistry 02/1997; 272(7):4072-4078. · 4.77 Impact Factor
