Isolation of the Cyclosporin-Sensitive T Cell Transcription Factor NFATp

McCaffrey, Patricia G.; Luo, Chun; Kerppola, Tom K.; Jain, Jugnu; Badalian, Tina M.; Ho, Ayy; Burgeon, Emmanuel; Lane, William S.; Lambert, John N.; Curran, Tom; Verdine, Gregory L.; Rao, Anjana; Hogan, Patrick G.

doi:10.1126/science.8235597

Cited by 385 publications

(287 citation statements)

References 25 publications

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“…Activation of these genes is mediated in part by nuclear factor of activated T-cells (NFAT), a DNA-binding protein that binds to specific sites on the regulatory regions of these genes (reviewed in [1,2]). Molecular cloning studies have demonstrated that at least four different NFAT genes exist, including NFAT p , NFAT c , NFAT3 and NFAT x , all of which contain a Rel homology region that is important for DNA binding [3][4][5][6]. In addition, a serine\proline repeat region of unknown function is present in each of the family members, as well as a putative nuclear localization sequence.…”

Section: Introductionmentioning

confidence: 99%

Dynamic equilibrium between calcineurin and kinase activities regulates the phosphorylation state and localization of the nuclear factor of activated T-cells

Scott¹,

Ruff²,

Leach³

1997

Biochemical Journal

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The nuclear factor of activated T-cells (NFAT p ) is a phosphorylated transcription factor that resides in the cytoplasm of unactivated T-cells. T-cell activation results in the activation of the phosphatase calcineurin (CaN), which leads to the dephosphorylation and subsequent nuclear localization of NFAT p . We have investigated the role of kinases in the phosphorylation state and subcellular localization of NFAT p . The phosphorylation state and nuclear\cytoplasmic location of NFAT p were determined in unstimulated murine HT-2 cells treated with a panel of kinase inhibitors. Two of the seven kinase inhibitors, staurosporine (St) and bisindolylmaleimide I (BI), resulted in the dephosphorylation and nuclear localization of NFAT p . These Stinduced effects were inhibited by pretreatment with FK506,

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Section: Introductionmentioning

confidence: 99%

Dynamic equilibrium between calcineurin and kinase activities regulates the phosphorylation state and localization of the nuclear factor of activated T-cells

Scott¹,

Ruff²,

Leach³

1997

Biochemical Journal

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“…The bZIP domains were modified to contain unique cysteine residues through replacement of the native cysteines by serines and addition of cysteine residues on the amino terminal sides of the bZIP domains. The extended rel-homology region of NFAT1 (residues 396-692) and full-length Fos and Jun were expressed and purified as described (16). Jun was labeled by incubation with Texas red C2 maleimide (Molecular Probes) and purified by either nickel chelate affinity or size exclusion chromatography (4,5).…”

Section: Methodsmentioning

confidence: 99%

“…A total of 3 M of an oligonucleotide competitor containing the composite AP-1-NFAT binding site was added. After 1-min incubation at 30°C, 50 g of nuclear extract protein from Namalwa cells (16) was added. This extract contained low endogenous Fos-Jun and NFAT1 activities.…”

Section: Methodsmentioning

confidence: 99%

Dynamics of Fos-Jun-NFAT1 complexes

Ramirez-Carrozzi

Kerppola

2001

Proc. Natl. Acad. Sci. U.S.A.

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Transcription initiation in eukaryotes is controlled by nucleoprotein complexes formed through cooperative interactions among multiple transcription regulatory proteins. These complexes may be assembled via stochastic collisions or defined pathways. We investigated the dynamics of Fos-Jun-NFAT1 complexes by using a multicolor fluorescence resonance energy transfer assay. Fos-Jun heterodimers can bind to AP-1 sites in two opposite orientations, only one of which is populated in mature Fos-Jun-NFAT1 complexes. We studied the reversal of Fos-Jun binding orientation in response to NFAT1 by measuring the efficiencies of energy transfer from donor fluorophores linked to opposite ends of an oligonucleotide to an acceptor fluorophore linked to one subunit of the heterodimer. The reorientation of Fos-Jun by NFAT1 was not inhibited by competitor oligonucleotides or heterodimers. The rate of Fos-Jun reorientation was faster than the rate of heterodimer dissociation at some binding sites. The facilitated reorientation of Fos-Jun heterodimers therefore can enhance the efficiency of Fos-Jun-NFAT1 complex formation. We also examined the influence of the preferred orientation of Fos-Jun binding on the stability and transcriptional activity of Fos-Jun-NFAT1 complexes. Complexes formed at sites where Fos-Jun favored the same binding orientation in the presence and absence of NFAT1 exhibited an 8-fold slower dissociation rate than complexes formed at sites where Fos-Jun favored the opposite binding orientation. Fos-Jun-NFAT1 complexes also exhibited greater transcription activation at promoter elements that favored the same orientation of Fos-Jun binding in the presence and absence of NFAT1. Thus, the orientation of heterodimer binding can influence both the dynamics and promoter selectivity of multiprotein transcription regulatory complexes.M any transcription regulatory proteins form heterodimers that bind to palindromic DNA sequences. Such heterodimers can potentially bind their recognition sites in either of two opposite orientations (1-5). Heterodimers that bind in opposite orientations present different protein surfaces for interactions with adjacent DNA binding proteins. Thus, the orientation of heterodimer binding may influence interactions with other transcription factors. Opposite orientations of heterodimer binding can result in different transcriptional activities (6-9). The mechanisms whereby the orientation of heterodimer binding controls transcriptional activity have not been defined.Fos and Jun family basic region-leucine zipper (bZIP) proteins regulate the expression of different genes in different cell types through cooperative interactions with other transcription factor families (10). Complexes formed by Fos and Jun with members of the NFAT family at composite regulatory elements induce cytokine gene expression in T cells activated by antigen presentation (11,12). The x-ray crystal structures of DNA-bound complexes formed by the bZIP domains of Fos and Jun alone as well as by the DNA binding domains of Fos, Jun...

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“…Although these agents bind primarily to different molecular targets , respectively], they exert similar pharmacological effects on signal-transduction pathways in T lymphocytes. It is generally accepted that by blocking dephosphorylation and subsequent translocation of the nuclear factor of activated T-cells (NFAT), CNIs inhibit the production of interleukin-2 (IL-2) (Shaw et al 1988;McCaffrey et al 1993). Extensive clinical experience has demonstrated that although both drugs are indispensable for immunosuppression after organ transplantation, due to a narrow window for therapeutic dosage it is difficult to find the dose appropriate to each patient.…”

Section: Introductionmentioning

confidence: 99%

A model of prediction system for adverse cardiovascular reactions by calcineurin inhibitors among patients with renal transplants using gene-based single-nucleotide polymorphisms

et al. 2005

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The application of pharmacogenomic information to diagnostic assays is expected to improve the prediction of drug efficacy and toxicity, leading to appropriate therapeutic regimens for individual patients. Cardiovascular events are common and severe adverse drug reactions (ADRs) among transplant patients treated with calcineurin inhibitors (CNIs). We conducted case-control association studies using 50,947 gene-based single-nucleotide polymorphisms (SNPs) to identify genetic variations that might be associated with cardiovascular risk factors in 72 renal transplant recipients with CNI therapy. The overall incidence of cardiovascular events was 13.9% (10/72) among patients receiving cyclosporine or tacrolimus; arrhythmias in six patients (8.3%), ischemic heart diseases in two patients (2.8%), and heart failure in two patients (2.8%). On the basis of results of the genome-wide association studies, we attempted to establish a scoring system to predict individual risks for cardiovascular toxicity of cyclosporine and tacrolimus. Estimation of the predictive performance was carried out by the use of internal leave-one-out cross-validation test. When we combined arrhythmia, ischemic heart disease and heart failure cases as subjects with a cardiotoxicity phenotype, nine of ten ADR patients and 50 of 62 non-ADR patients were correctly classified into the respective categories using the top eight SNPs. In addition, the proportion of individuals in the control population (n=246) with scores over the cut-off (11.0%) was close to the cardiovascular ADR frequency (8.3%) among renal transplant patients in the previous clinical study. Our results open the possibility that prediction of CNI-induced cardiovascular complications can lead to better prognosis and quality of life among kidney-transplant patients, and to improved immunosuppressive regimens.

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Isolation of the Cyclosporin-Sensitive T Cell Transcription Factor NFATp

Cited by 385 publications

References 25 publications

Dynamic equilibrium between calcineurin and kinase activities regulates the phosphorylation state and localization of the nuclear factor of activated T-cells

Dynamic equilibrium between calcineurin and kinase activities regulates the phosphorylation state and localization of the nuclear factor of activated T-cells

Dynamics of Fos-Jun-NFAT1 complexes

A model of prediction system for adverse cardiovascular reactions by calcineurin inhibitors among patients with renal transplants using gene-based single-nucleotide polymorphisms

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