The Role of the Transcription Factor NFAT in the Pathogenesis of Non-Hodgkin’s Lymphomas.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2371-2371
Author(s):  
Martin R. Mueller ◽  
Yoshiteru Sasaki ◽  
Sonia Sharma ◽  
Klaus Rajewsky ◽  
Anjana Rao
Keyword(s):  
Nuclear Export ◽  
Nuclear Translocation ◽  
Consensus Sequence ◽  
Resting Cells ◽  
Docking Site ◽  
Mutant Proteins ◽  
Nfat Activation ◽  
Hodgkin's Lymphomas ◽  
The Impact

Abstract NFAT is a family of highly phosphorylated proteins residing in the cytoplasm of resting cells. Upon dephosphorylation by the Ca2+/calmodulin-dependent serine phosphatase calcineurin, NFAT translocates to the nucleus, where it induces the transcription of a large number of genes necessary for a productive immune response. NFAT signalling has also been implicated in lymphocyte homeostasis and its deregulation has been suggested to be involved in the pathogenesis of different malignancies. A recent immunohistochemical evaluation of approximately 300 Non-Hodgkin’s Lymphoma biopsy samples showed overexpression of NFAT2 in the majority of specimens with strong nuclear translocation in certain histologic subtypes (DLBCL, Burkitt’s Lymphoma) presumably reflecting activation of the NFAT pathway as part of their pathogenesis. Other recent studies have shown that NFAT activation leads to increased expression of different cell survival factors (CD154, BLyS) in several lymphoma subtypes (DLBCL, MCL). To study the role of constitutive activation of the NFAT pathway on lymphomagenesis, we generated several hyperactivable NFAT mutants which will be used to generate transgenic mice expressing the mutant proteins from the ROSA26 locus. Here, we present the in vitro characterization of these hyperactivable NFAT proteins in cell lines and primary lymphocytes. The major docking site for calcineurin is located at the N terminus of the NFAT regulatory domain and has the consensus sequence PxIxIT (e.g. SPRIEIT in NFAT1 and NFAT2). Substitution of the SPRIEIT sequence of NFAT with HPVIVIT, a higher-affinity version obtained by peptide selection, increases the basal calcineurin sensitivity of the protein significantly. Similarly, a major kinase for NFAT is CK1, and mutation of the CK1 docking site (FSILF to ASILA in NFAT1, FDFEF to ADAEA in NFAT2) also leads to partial nuclear localization of NFAT proteins by decreasing rephosphorylation and nuclear export. In the current analysis we compared the different hyperactivable NFAT proteins with respect to dephosphorylation status and nuclear translocation using western blotting and immunocytochemistry. Whereas the wildtype NFAT protein was entirely localized in the cytoplasm and completely phosphorylated under resting conditions, we observed an increasing degree of nuclear translocation and dephosphorylation for the different mutant proteins (ASILA 20%, VIVIT 30–40%, ASILA-VIVIT 50–70%). This system will allow us to study the impact of different levels of NFAT activation on the pathogenesis of lymphomas in vivo.

scholarly journals Cytoplasmic Localization of Wis1 MAPKK by Nuclear Export Signal Is Important for Nuclear Targeting of Spc1/Sty1 MAPK in Fission Yeast

2002 ◽  
Vol 13 (8) ◽  
pp. 2651-2663 ◽  
Author(s):  
Aaron Ngocky Nguyen ◽  
Aminah D. Ikner ◽  
Mitsue Shiozaki ◽  
Sasha M. Warren ◽  
Kazuhiro Shiozaki
Keyword(s):  
Fission Yeast ◽  
Nuclear Export ◽  
Nuclear Translocation ◽  
Nuclear Export Signal ◽  
Mapk Cascade ◽  
Mitogen Activated Protein Kinase ◽  
Docking Site ◽  
Cytoplasmic Localization ◽  
Nuclear Targeting ◽  
Export Signal

Mitogen-activated protein kinase (MAPK) cascade is a ubiquitous signaling module that transmits extracellular stimuli through the cytoplasm to the nucleus; in response to activating stimuli, MAPKs translocate into the nucleus. Mammalian MEK MAPK kinases (MAPKKs) have in their N termini an MAPK-docking site and a nuclear export signal (NES) sequence, which are known to play critical roles in maintaining ERK MAPKs in the cytoplasm of unstimulated cells. Herein, we show that the Wis1 MAPKK of the stress-activated Spc1 MAPK cascade in fission yeast also has a MAPK-docking site and an NES sequence in its N-terminal domain. Unexpectedly, an inactivating mutation to the NES of chromosomal wis1 + does not affect the subcellular localization of Spc1 MAPK, whereas this NES mutation disturbs the cytoplasmic localization of Wis1. However, when Wis1 is targeted to the nucleus by fusing to a nuclear localization signal sequence, stress-induced nuclear translocation of Spc1 is abrogated, indicating that cytoplasmic Wis1 is required for nuclear transport of Spc1 upon stress. Moreover, we have observed that a fraction of Wis1 translocates into the nucleus in response to stress. These results suggest that cytoplasmic localization of Wis1 MAPKK by its NES is important for stress signaling to the nucleus.

scholarly journals A Conserved Docking Motif for CK1 Binding Controls the Nuclear Localization of NFAT1

2004 ◽  
Vol 24 (10) ◽  
pp. 4184-4195 ◽  
Author(s):  
Heidi Okamura ◽  
Carmen Garcia-Rodriguez ◽  
Holly Martinson ◽  
Jun Qin ◽  
David M. Virshup ◽  
...  
Keyword(s):  
Molecular Weight ◽  
T Cells ◽  
Transcription Factor ◽  
Circadian Rhythm ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Nuclear Export ◽  
Resting Cells ◽  
Docking Site ◽  
High Molecular Weight Complex

ABSTRACT In resting cells, the NFAT1 transcription factor is kept inactive in the cytoplasm by phosphorylation on multiple serine residues. These phosphorylated residues are primarily contained within two types of serine-rich motifs, the SRR-1 and SP motifs, which are conserved within the NFAT family. Several different kinases have been proposed to regulate NFAT, but no single candidate displays the specificity required to fully phosphorylate both types of motifs; thus, the identity of the kinase that regulates NFAT activity remains unclear. Here we show that the NFAT1 serine motifs are regulated by distinct kinases that must coordinate to control NFAT1 activation. CK1 phosphorylates only the SRR-1 motif, the primary region required for NFAT1 nuclear import. CK1 exists with NFAT1 in a high-molecular-weight complex in resting T cells but dissociates upon activation. GSK3 does not phosphorylate the SRR-1 region but can target the NFAT1 SP-2 motif, and it synergizes with CK1 to regulate NFAT1 nuclear export. We identify a conserved docking site for CK1 in NFAT proteins and show that mutation of this site disrupts NFAT1-CK1 interaction and causes constitutive nuclear localization of NFAT1. The CK1 docking motif is present in proteins of the Wnt, Hedgehog, and circadian-rhythm pathways, which also integrate the activities of CK1 and GSK3.

Expression of Hyperactivable NFAT1 from the ROSA26 Locus Leads to Detrimental Effects during Embryonic Development.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2296-2296
Author(s):  
Martin R. Mueller ◽  
Yoshiteru Sasaki ◽  
Sonia Sharma ◽  
Srimoyee Ghosh ◽  
Curtis Gelinas ◽  
...  
Keyword(s):  
T Cells ◽  
Embryonic Development ◽  
Transgenic Mice ◽  
Nuclear Export ◽  
Transgene Expression ◽  
Nuclear Translocation ◽  
Docking Site ◽  
Wild Type ◽  
T And B Cells ◽  
Rosa26 Locus

Abstract NFAT is a family of highly phosphorylated proteins residing in the cytoplasm of resting cells. Upon dephosphorylation by calcineurin, NFAT translocates to the nucleus, where it orchestrates several developmental programs, including those of the immune, cardiovascular and central nervous systems. NFAT is rephosphorylated and inactivated by different kinases (CK1, GSK-3, DYRK). The major docking site for calcineurin is located at the N terminus of the NFAT regulatory domain and has the consensus sequence PxIxIT (SPRIEIT in NFAT1). Substitution of the SPRIEIT sequence with HPVIVIT increases the basal calcineurin sensitivity of the protein significantly. Similarly, mutation of the CK1 docking site from FSILF to ASILA leads to partial nuclear localization by decreasing rephosphorylation and nuclear export. To assess the impact of hyperactivable NFAT mutants on activation kinetics and signal responsiveness, we retrovirally transduced T cells from NFAT1−/− mice with contructs expressing wild type NFAT1, NFAT1-ASILA, NFAT1-HPVIVIT or NFAT1-ASILA-HPVIVIT. Analysis by western blotting and immunocytochemistry revealed, that the wild type protein was entirely localized in the cytoplasm and completely phosphorylated under resting conditions, whereas the hyperactivable mutants exhibited an increasing degree of nuclear translocation and dephosphorylation (ASILA 20%, HPVIVIT 30–40%, ASILA-HPVIVIT 50–70%). Upon stimulation with PMA and ionomycin, the hyperactivable mutants exhibited an incremental acceleration of nuclear translocation and delay of nuclear export as compared to the wild type protein. Furthermore, T cells expressing hyperactivable NFAT proteins exhibited significantly higher expression rates of different cytokines (IL-2, TNF-α, IFN-γ) upon stimulation with low doses of ionomycin documenting their hyperresponsiveness and biological activity. To provide a new tool for the analysis of the calcineurin-NFAT pathway in vivo and to assess the effect of modulating the affinity of signaling molecules for their upstream regulators, which are consistently kept at low to moderate affinity during evolution, we subsequently generated transgenic mice conditionally expressing different hyperactivable NFAT1 mutants from the ROSA26 locus (NFAT1-HPVIVIT, NFAT1-ASILA-HPVIVIT). Transgene expression in the T cell lineage was achieved by breeding the ROSA26 transgenic mice to CD4-Cre mice, which express the Cre recombinase under the control of the CD4 promoter. CD4 and CD8 T cells from these mice showed significant hyperactivability as assessed by accelerated nuclear translocation and delayed nuclear export of NFAT and substantially increased cytokine expression upon stimulation. Expression of the hyperactivable NFAT proteins early in the germline was achieved by breeding the ROSA26 mice to Cre-Del mice, which express the Cre recombinase under the control of the ACE promoter. While breeding of ROSA26-YFP control mice to Cre-Del mice resulted in transgene expression in all T and B cells, expression of hyperactivable NFAT proteins early in the germline resulted in incremental mosaicism in T and B cells (70% transgene expression in NFAT1-HPVIVIT and 20–30% transgene expression in NFAT1-ASILA-HPVIVIT). This data demonstrate that hyperactivable NFAT1 proteins result in a selective disadvantage for the expressing cells in embryonic development and provide a potential explanation why evolution chose to keep the respective docking sites at moderate affinity.

Role of the MAPK and PI3-Kinase/Akt Pathways in the Pre-B Cell Receptor (pre-BCR)-Induced NF-κb Activation and Rag1 and Rag2 Down Regulation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2667-2667
Author(s):  
Kaiss Lassoued ◽  
Vincent Fuentes ◽  
Hussein Gamlouch ◽  
Eliane Bissac ◽  
Jean-Pierre Marolleau ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Signaling Pathways ◽  
Nuclear Translocation ◽  
Conflicts Of Interest ◽  
Consensus Sequence ◽  
Pi3 Kinase ◽  
Strong Increase ◽  
Down Regulation

Abstract Abstract 2667 Poster Board II-643 The pre-BCR acts as a critical checkpoint in pre-B cell development and might be also involved in leukemogenesis. Using the 697 and Nalm6 human pre-B cell lines, we have previously shown that pre-BCR stimulation resulted in cell cycle progression associated with activation of number of adaptors and signaling pathways including the PI3-Kinase/Akt, Ras/MAPK, AP1 and the canonical NFkB pathway. We have also demonstrated that Src kinases together with Syk played a crucial role in controlling the pre-BCR-associated functions, acting upstream the above-mentioned signaling pathways. Pre-BCR crosslinking also induced down regulation of Rag1 and Rag2 transcription. In this study we aimed to evaluate the role of MAPK and Akt in the pre-BCR-induced NF-kB activation and Rag1/2 down modulation. For this purpose the 697 pre-B cells and normal bone marrow primary pre-B cells were treated with the U0126 and LY294002, and with MEK1/2 and Akt inhibitors, respectively. A dominant negative form of Akt fused to the HIV1 Tat peptide was also used to inhibit the PI3-Kinase/Akt pathway. We bring evidence that LY294002 could alter the pre-BCR-induced NF-kB activation by inhibiting : i) p105 degradation, ii) p50 NF-kB1 nuclear translocation and, iii) the binding of p50 to an oligonucleotide containing a specific consensus sequence. On the contrary, U0126 significantly enhanced p105 degradation, indicating that MAPK and Akt exerted antagonistic effects on the pre-BCR-induced NF-kB activation. Strikingly the baseline levels of Rag1 and Rag2 transcripts were increased in the LY294002 but not the U0126-treated pre-B cells. Futhermore, both inhibitors were shown to induce a strong increase in the expression of Rag1 and Rag2 transcripts upon pre-BCR crosslinking, suggesting that this receptor exerts dual effects on Rag1/2 expression with a predominant negative regulatory component mediated by both PI3-K and MAPK. No changes in the levels of Pax5, E2A, EBF, IFR4, IRF8, FOXO1, FOXO3, Myb, MAZ, LEF1 and SP1 (transcription factors implied in the regulation of Rag1 and Rag2 transcription) were observed in the pre-BCR stimulated or unstimulated-697 cells, treated or not with the MAPK and Akt inhibitors. Our results suggest that the pre-BCR signaling is a complex and tightly self-controlled process, which deregulation might alter cell growth and survival pathways via NF-kB as well as genomic stability trough Rag1/2 expression. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The Impact of m6A RNA Modification in Therapy Resistance of Cancer: Implication in Chemotherapy, Radiotherapy, and Immunotherapy

2021 ◽  
Vol 10 ◽  
Author(s):  
Omprakash Shriwas ◽  
Pallavi Mohapatra ◽  
Sibasish Mohanty ◽  
Rupesh Dash
Keyword(s):  
Nuclear Export ◽  
Potential Role ◽  
Therapy Resistance ◽  
Rna Modification ◽  
Cancer Cell Proliferation ◽  
Rna Translation ◽  
Regulation Mechanisms ◽  
M6a Rna Methylation ◽  
The Impact

m6A RNA methylation, which serves as a critical regulator of transcript expression, has gathered tremendous scientific interest in recent years. From RNA processing to nuclear export, RNA translation to decay, m6A modification has been studied to affect various aspects of RNA metabolism, and it is now considered as one of the most abundant epitranscriptomic modification. RNA methyltransferases (writer), m6A-binding proteins (readers), and demethylases (erasers) proteins are frequently upregulated in several neoplasms, thereby regulating oncoprotein expression, augmenting tumor initiation, enhancing cancer cell proliferation, progression, and metastasis. Though the potential role of m6A methylation in growth and proliferation of cancer cells has been well documented, its potential role in development of therapy resistance in cancer is not clear. In this review, we focus on m6A-associated regulation, mechanisms, and functions in acquired chemoresistance, radioresistance, and resistance to immunotherapy in cancer.

Intergroup Threat and Outgroup Attitudes

2013 ◽  
Vol 44 (5) ◽  
pp. 311-319 ◽  
Author(s):  
Marco Brambilla ◽  
David A. Butz
Keyword(s):  
Gay Men ◽  
Social Group ◽  
Welfare Recipients ◽  
Intergroup Attitudes ◽  
Social Policies ◽  
Intergroup Threat ◽  
Economic Threat ◽  
The Impact ◽  
General Climate

Two studies examined the impact of macrolevel symbolic threat on intergroup attitudes. In Study 1 (N = 71), participants exposed to a macrosymbolic threat (vs. nonsymbolic threat and neutral topic) reported less support toward social policies concerning gay men, an outgroup whose stereotypes implies a threat to values, but not toward welfare recipients, a social group whose stereotypes do not imply a threat to values. Study 2 (N = 78) showed that, whereas macrolevel symbolic threat led to less favorable attitudes toward gay men, macroeconomic threat led to less favorable attitudes toward Asians, an outgroup whose stereotypes imply an economic threat. These findings are discussed in terms of their implications for understanding the role of a general climate of threat in shaping intergroup attitudes.

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