Detection of the FK506-FKBP-Calcineurin Complex by a Simple Binding Assay

doi:10.1006/bbrc.1993.1570

Biochemical and Biophysical Research Communications

Volume 192, Issue 3, 14 May 1993, Pages 1388-1394

https://doi.org/10.1006/bbrc.1993.1570 Get rights and content

Abstract

[³H]FK506-FK506-binding protein (FKBP) complex does not bind polyethylenimine-treated glass fiber filters, on the other hand, it binds the filter when it makes a complex with calcineurin. This enabled us to develop a simple binding assay to detect the FK506-FKBP-calcineurin complex in the mixture and to estimate the amount of FKBP or calcineurin which can form the complex in the soluble extracts from rat tissues. The molar concentration of FKBP of every rat tissue is much higher than that of calcineurin, and FKBP/calcineurin molar ratio varies from 13 to 33 in various brain regions, and from 42 to 343 in peripheral tissues. These results suggest that most of the FKBP molecules have physiological roles which are not related to calcineurin, in spite of their potential ability to form the complex with FK506 and calcincurin.

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Cited by (33)

The role of the regulatory subunit of fission yeast calcineurin for in vivo activity and its relevance to FK506 sensitivity
2005, Journal of Biological Chemistry
Citation Excerpt :
However, in our previous in vitro binding study using a rapid filtration assay, we showed that the FKBP12-FK506 complex binds the calcineurin-calmodulin-Ca2+ complex but does not bind to the calcineurin-calmodulin-Mg2+ complex (37), suggesting the correlation between calcineurin activity and its binding to the FKBP12-FK506 complex. In addition, we showed that although the calcineurin-Ca2+ complex binds the FKBP12-FK506 complex, its binding affinity was ∼8 times lower than that of the calcineurin-calmodulin-Ca2+ complex (37). Together, these results suggest that the in vivo pharmacological target of the FKBP12-FK506 complex is the active calcineurin-calmodulin-Ca2+ complex.
Calcineurin, a protein phosphatase required for Ca²⁺signaling in many cell types, is a heterodimer composed of catalytic and regulatory subunits. The fission yeast genome encodes a single set of catalytic (Ppb1) and regulatory (Cnb1) subunits, providing an ideal model system to study the functions of these subunits in vivo. Here, we cloned the cnb1⁺ gene and showed that the cnb1 knock-out (Δcnb1) exhibits identical phenotypes with Δppb1 and that overexpression of Ppb1 failed to suppress the phenotypes of Δcnb1. Interestingly, overexpression of the C-terminal-deleted Ppb1 (Ppb1ΔC), the constitutively active form of Ppb1, also failed to suppress the phenotypes of Δcnb1. FK506 caused MgCl₂ sensitivity to the wild-type cells in an FKBP12-dependentmanner. Co-overexpression of Ppb1 and Cnb1 suppressed the FK506-induced MgCl₂ sensitivity, but the suppression was only partial, suggesting that an excess amount of the Ppb1-Cnb1 complex cannot compete out the FKBP12-FK506 complex. Although overexpression of Ppb1ΔC alone had little effect on cell growth, co-overexpression of Ppb1ΔC and Cnb1 caused a distinct growth defect. FK506 suppressed the growth defect when Cnb1 was co-expressed using the attenuated nmt1 promoter, but it failed to suppress the defect when Cnb1 was co-expressed using the wild-type nmt1 promoter. Knock-out of the prz1⁺ gene, encoding a downstream target transcription factor of calcineurin, suppressed the growth defect irrespective of the promoter potency. These results suggest that Cnb1 is essential for the activation of calcineurin and that the activated calcineurin is the pharmacological target of the FKBP12-FK506 complex in vivo.
Calcineurin: From structure to function
2001, Current Topics in Cellular Regulation
The use of the calcineurin inhibitors—namely, FK506 and CsA, together with yeast genetics and the overexpression of calcineurin by transgenic mice, has established the critical roles of calcineurin in the regulation of many cellular processes that are induced by changes in the concentration of intracellular Ca²⁺ in response to external signals. None of the physiological roles of calcineurin is better documented than the regulation of gene expression mediated by the broadly distributed NFAT family of transcription factors in mammalian cells. Equally well documented is the role of calcineurin in the regulation of expression of many genes that are under the control of the TCNI/CRZl transcription factor in yeast. Calcineurim was originally identified as a major calmodulin-binding protein in the brain and later shown to be the only Ca²⁺/calmodulin-regulated serine/threonine protein phosphatase. Since then, this enzyme has been shown to be expressed in every tissue and to be highly conserved phylogenetically.
Potent immunosuppressive C32-o-arylethyl ether derivatives of ascomycin with reduced toxicity
1999, Bioorganic and Medicinal Chemistry Letters
Immunosuppressant FK506 inhibits inducible nitric oxide synthase gene expression at a step of NF-κB activation in rat hepatocytes
1999, Journal of Hepatology
Background/Aims: Recent evidence indicates that an increase in nitric oxide production after liver transplantation is associated with acute allograft rejection. Nitric oxide mediates cellular injury under various pathological conditions in the liver. Studies were performed to determine whether the immunosuppressants FK506 and cyclosporin A directly influence gene expression of inducible nitric oxide synthase by interleukin 1β in hepatocytes.
Methods: Primary cultures of rat hepatocytes were treated with interleukin 1β in the presence and absence of FK506 or cyclosporin A. Release of nitrite (nitric oxide metabolite) into culture medium, levels of inducible nitric oxide synthase protein and mRNA, and activation of nuclear factor-κB were compared with the two drugs.
Results: Interleukin 1β increased levels of inducible nitric oxide synthase protein and inducible nitric oxide synthase mRNA, as well as nitric oxide production, in the cultured hepatocytes. Nuclear factor-κB, an important transcription factor in inducible nitric oxide synthase gene expression in response to inflammation, also appeared in the nuclear fraction of hepatocytes after addition of interleukin 1β. FK506 markedly inhibited the nitric oxide formation, inducible nitric oxide synthase protein synthesis and inducible nitric oxide synthase mRNA expression induced by interleukin 1β, but cyclosporin A had no effects. Furthermore, FK506 inhibited nuclear factor-κB activation and decreased mRNA levels of the p50/p65 subunits of nuclear factor-κB.
Conclusions: These results demonstrate that FK506, but not cyclosporin A, inhibits the induction of inducible nitric oxide synthase expression during nuclear factor-κB activation. FK506 may influence liver function during diseases by modulating the nitric oxide pathway, in addition to its immunosuppressive effect.
Inhibition of iNOS induction by FK506, but not by cyclosporine, in rat hepatocytes
1999, Transplantation Proceedings
The effect of cyclosporine A and amlodipine on the activity of γ-glutamyl transpeptidase in mouse cerebral cortex
1997, Pharmacological Research
The effect of cyclosporine A (CsA) and dihydropyridine calcium channel blocker (CCB) amlodipine on γ-glutamyl transpeptidase (GGT; EC 2.3.2.2) activity in mouse cerebral cortex was investigated. Mice received amlodipine (0.07 mg kg⁻¹), CsA (10, 20 and 40 mg kg⁻¹) and CsA in the above doses combined with amlodipine (0.07 mg kg⁻¹) once daily, intraperitoneally for 5 days. The control group received saline. Amlodipine decreased GGT activity when compared to the control group. GGT activity in the cerebral cortex was decreased after treatment with CsA (20 and 40 mg kg⁻¹), but was not changed after treatment with CsA in the 10 mg kg⁻¹dose as compared to the control group. CsA in doses of 20 and 40 mg kg⁻¹, combined with amlodipine, increased GGT activity as compared to the control group groups received the same doses of CsA without amlodipine and received only amlodipine. However, CsA in the 10 mg kg⁻¹dose combined with amlodipine decreased GGT activity when compared to the control group, but did not show any statistical difference when compared to the groups treated only with amlodipine or CsA in the same doses. These results suggest that CsA and amlodipine may modulate GGT activity in mouse cerebral cortex.

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Regular ArticleDetection of the FK506-FKBP-Calcineurin Complex by a Simple Binding Assay

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Regular Article
Detection of the FK506-FKBP-Calcineurin Complex by a Simple Binding Assay