Group:MUZIC:Calcineurin

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Contents

Introduction

Calcineurin, a serine-threonine phosphatase also known as protein phosphatase 2B (PP2B), is a heterodimeric protein composed of a catalytic subunit, with phosphatase activity, and a regulatory subunit, Ca2+ regulated. Calcineurin is located in the cytoplasm, where sustained low-amplitude Ca2+ waves trigger its phosphatase activity [1]. Calcineurin’s substrates are different members of the nuclear factor activated T-cells (NFAT) transcription factor family, which are dephosphorylated, and consequently, translocate to the nucleus for activating expression of different gene networks.[2]. The presence of Calcineurin in different cell types and organisms, ranging from T-lymphocytes to skeletal muscle, and from yeast to humans, highlights its important role within the cell.

Sequence Annotation

The heterodimeric Calcineurin is composed of two subunits: the serine/threonine-protein phosphatase 2B catalytic subunit alpha isoform chain A (CnA), and the Calcineurin subunit B type 1 (CnB) [3]. Each of the subunits has alternative names in the literature, as it is shown in their uniprotKB entries Q08209 and P63098. CnA comprises a globular catalytic domain (residues 1 to 301), a CnB binding region (residues 247 to 253 and 296 to 301), a calmodulin-binding region (residues 392 to 414), and an autoinhibitory peptide (residues 465 to 487). CnB is composed of four EF hands spanning from residue 18 to 163 [4].

Structure

The structure was solved by macromolecular crystallography at a resolution of 2.1 Å. 1AUI

Drag the structure with the mouse to rotate

CnA contains a followed by an alpha-helical region, forming the . It also has a calmodulin-binding region, which is critical for Calcineurin's catalytic activity. In the C-terminal region of CnA there are 18 residues considered an , because it blocks the substrate binding cleft on the catalytic domain. The CnB is a 168 polipeptide chain, belonging to the EF-hand calcium binding protein family. This subunit is composed of two lobes with two calcium ions bound by in each lobe [5].

Function and Interactions

Calcineurin is specifically activated by low-amplitude Ca2+ waves [6], promoting association of Calmodulin with CnA and a conformational change on CnB, which result in activation of the phosphatase catalytic domain. Different isoforms of the NFAT transcription factors are Calcineurin substrates, which after dephosphorylation translocate from the cytoplasm to the nucleus, finding their different target genes [7].

The Calcineurin/NFAT pathway controls the response to foreign antigens in T-lymphocytes, where the activated genes determine T-cell response to the external stimulus. Such response can be inhibited by cyclosporin A (CsA) and FK-506, Calcineurin inhibitors, used to immunosuppress organ rejection in human transplants [8].

The Calcineurin/NFAT pathway determines the fiber type composition of skeletal muscle in response to motor neuron activity. Dephosphorylation of isoform NFTAc1 activates expression of the slow myosin heavy chain (MyHC) and other slow fiber type specific genes [9]. This was experimentally confirmed in regenerating and adult mouse muscles by using VIVIT, a blocker of the interaction between Calcineurin and NFATc1, which inhibited the activation of MyHC-slow promoter. Additionally, it was possible to rescue the slow fiber phenotype by a constitutively active form of NFATc, demonstrating that the slow gene activation program is controlled by the Calcineurin/NFAT pathway [10]. Different experiments suggest that Calcineurin also controls the slow fiber type composition via MEF-2, another family of transcription factors which appears during early stages of myocytes differentiation and activate the expression of proteins associated with oxidative metabolism. The treatment with CsA blocks the response of MEF-2 to exercise stimulus, the over-expression of Calcineurin increases dramatically the activity of MEF-2, and the activation of both Calcineurin and MEF-2 pathways promotes expression of myoglobin, myosin heavy chain, and slow troponin I [11]. These observations suggest that the slow fiber phenotype could be result of the cross-talking between Calcineurin and different signaling pathways.

Calcineurin interacts with the protein family FATZ [[1]](also known as Calsarcins or Myozenins), composed by three isoforms. The isoform FATZ-1 (Calsarcin-2 and Myozenin-1) is a negative regulator of the Calcineurin/NFAT pathway in fast fibers. In absence of FATZ-1 the activity of the Calcineurin/NFAT signaling pathway increases, consequently, there are a major number of oxidative fibers, and less fatigue of FATZ-1 knockdown compared to FATZ-1 wild type mice subjected to long endurance exercise. The interaction between FATZ-1 and Calcineurin is another check point in the signaling pathways controlling skeletal muscle fiber type composition and response to exercise performance [12].

Besides controlling fiber type composition, the Calcineurin/NFAT pathway controls the hypertrophic response to pressure overload in both, cardiac and skeletal muscle. The dephosphorylation of NFATc4, and cooperation with GATA4 and MEF-2, activates the hypertrophic gene program in mouse cardiomyocytes [13]. Additionally, cardiomyocytes exposed to hypertrophic agonist and treated with inhibitors do not develop the hypertrophic phenotype. Calcineurin activity is also modulated by FATZ-2 (calsarcin-1/Myozenin-2), a negative regulator which leads to the hypertrophic phenotype in FATZ-2 deficient mouse cardiomyocytes, subjected to hypertrophic agonists [14]. Furthermore, it is involved in skeletal muscle hypertrophy driven by insulin growth factor (IGFs). Experimental data showed that treatment of muscle cultured cells with IGFs up-regulates the transcriptional levels of CnA, promotes association between NFATc1 and GATA-2, which isoforms in heart control the hypertrophic response, and displays phenotypic characteristics of cellular hypertrophy. In support to these findings it was seen that treatment with CsA, prevents activation of the hypertrophic program [15]. Other experiments showed that constitutive expression of activated Calcineurin is not enough to induce hypertrophy, suggesting that other signaling pathways may be responsible for muscle hypertrophy in response to IGFs [16].

Other Z-disc proteins interact with Calcineurin and regulated its activity. The interaction with MLP [[2]] suggests that Calcineurin is anchored to the Z-disc, and changes its mobility between the Z-disc and the cytoplasm in response to mechanical stress[17]. PICOT displaces Calcineurin from the Z-disc, preventing its interaction with other partners [18]. Lmcd1/Dyxin is a positive modulator, which up-regulation in experimental models causes hypertrophy accompanied by strong activation of Calcineurin [19].

Pathology

It is well established in animal models that over activation of the Calcineurin/NFAT pathway leads to cardiac hypertrophy [20], [21]. However, the treatment of organ transplant patients with Calcineurin inhibitors can lead to a human hypertrophic cardiomyopathy phenotype, being reversible upon discontinuation [22]. Furthermore, there is controversy about whether the Calcineurin enzymatic activity is increased or not in biopsies from hypertrophic and failing human hearts [23]. Despite being a crucial signaling pathway controlling the hypertrophic response in cardiomyocytes, the role of Calcineurin in human hypertrophic cardiomyopathy remains unclear.


References

  1. ↑ Dolmetsch RE, Lewis RS, Goodnow CC, Healy JI. Differential activation of transcription factors induced by Ca2+ response amplitude and duration. Nature. 1997 Apr 24;386(6627):855-8. PMID:9126747 doi:10.1038/386855a0
  2. ↑ Rao A, Luo C, Hogan PG. Transcription factors of the NFAT family: regulation and function. Annu Rev Immunol. 1997;15:707-47. PMID:9143705 doi:10.1146/annurev.immunol.15.1.707
  3. ↑ Kissinger CR, Parge HE, Knighton DR, Lewis CT, Pelletier LA, Tempczyk A, Kalish VJ, Tucker KD, Showalter RE, Moomaw EW, et al.. Crystal structures of human calcineurin and the human FKBP12-FK506-calcineurin complex. Nature. 1995 Dec 7;378(6557):641-4. PMID:8524402 doi:http://dx.doi.org/10.1038/378641a0
  4. ↑ Guerini D. Calcineurin: not just a simple protein phosphatase. Biochem Biophys Res Commun. 1997 Jun 18;235(2):271-5. PMID:9199180 doi:10.1006/bbrc.1997.6802
  5. ↑ Kissinger CR, Parge HE, Knighton DR, Lewis CT, Pelletier LA, Tempczyk A, Kalish VJ, Tucker KD, Showalter RE, Moomaw EW, et al.. Crystal structures of human calcineurin and the human FKBP12-FK506-calcineurin complex. Nature. 1995 Dec 7;378(6557):641-4. PMID:8524402 doi:http://dx.doi.org/10.1038/378641a0
  6. ↑ Dolmetsch RE, Lewis RS, Goodnow CC, Healy JI. Differential activation of transcription factors induced by Ca2+ response amplitude and duration. Nature. 1997 Apr 24;386(6627):855-8. PMID:9126747 doi:10.1038/386855a0
  7. ↑ Guerini D. Calcineurin: not just a simple protein phosphatase. Biochem Biophys Res Commun. 1997 Jun 18;235(2):271-5. PMID:9199180 doi:10.1006/bbrc.1997.6802
  8. ↑ Crabtree GR. Generic signals and specific outcomes: signaling through Ca2+, calcineurin, and NF-AT. Cell. 1999 Mar 5;96(5):611-4. PMID:10089876
  9. ↑ Schiaffino S. Fibre types in skeletal muscle: a personal account. Acta Physiol (Oxf). 2010 Aug;199(4):451-63. doi:, 10.1111/j.1748-1716.2010.02130.x. Epub 2010 Mar 26. PMID:20353491 doi:10.1111/j.1748-1716.2010.02130.x
  10. ↑ Dieminger L, Schultz DR, Arnold PI. Activation of the classical complement pathway in human serum by a small oligosaccharide. J Immunol. 1979 Nov;123(5):2201-11. PMID:489979
  11. ↑ Bassel-Duby R, Olson EN. Signaling pathways in skeletal muscle remodeling. Annu Rev Biochem. 2006;75:19-37. PMID:16756483 doi:10.1146/annurev.biochem.75.103004.142622
  12. ↑ Conlon KC, Bading JR, DiResta GR, Corbally MT, Gelbard AS, Brennan MF. Validation of transport measurements in skeletal muscle with N-13 amino acids using a rabbit isolated hindlimb model. Life Sci. 1989;44(13):847-59. PMID:2564612
  13. ↑ Molkentin JD, Lu JR, Antos CL, Markham B, Richardson J, Robbins J, Grant SR, Olson EN. A calcineurin-dependent transcriptional pathway for cardiac hypertrophy. Cell. 1998 Apr 17;93(2):215-28. PMID:9568714
  14. ↑ Frey N, Barrientos T, Shelton JM, Frank D, Rutten H, Gehring D, Kuhn C, Lutz M, Rothermel B, Bassel-Duby R, Richardson JA, Katus HA, Hill JA, Olson EN. Mice lacking calsarcin-1 are sensitized to calcineurin signaling and show accelerated cardiomyopathy in response to pathological biomechanical stress. Nat Med. 2004 Dec;10(12):1336-43. Epub 2004 Nov 14. PMID:15543153 doi:nm1132
  15. ↑ PMID: 10448862; 10448861
  16. ↑ Naya FJ, Mercer B, Shelton J, Richardson JA, Williams RS, Olson EN. Stimulation of slow skeletal muscle fiber gene expression by calcineurin in vivo. J Biol Chem. 2000 Feb 18;275(7):4545-8. PMID:10671477
  17. ↑ Heineke J, Ruetten H, Willenbockel C, Gross SC, Naguib M, Schaefer A, Kempf T, Hilfiker-Kleiner D, Caroni P, Kraft T, Kaiser RA, Molkentin JD, Drexler H, Wollert KC. Attenuation of cardiac remodeling after myocardial infarction by muscle LIM protein-calcineurin signaling at the sarcomeric Z-disc. Proc Natl Acad Sci U S A. 2005 Feb 1;102(5):1655-60. Epub 2005 Jan 21. PMID:15665106 doi:10.1073/pnas.0405488102
  18. ↑ Jeong D, Kim JM, Cha H, Oh JG, Park J, Yun SH, Ju ES, Jeon ES, Hajjar RJ, Park WJ. PICOT attenuates cardiac hypertrophy by disrupting calcineurin-NFAT signaling. Circ Res. 2008 Mar 28;102(6):711-9. Epub 2008 Feb 7. PMID:18258855 doi:10.1161/CIRCRESAHA.107.165985
  19. ↑ Frank D, Frauen R, Hanselmann C, Kuhn C, Will R, Gantenberg J, Fuzesi L, Katus HA, Frey N. Lmcd1/Dyxin, a novel Z-disc associated LIM protein, mediates cardiac hypertrophy in vitro and in vivo. J Mol Cell Cardiol. 2010 Oct;49(4):673-82. Epub 2010 Jun 30. PMID:20600098 doi:10.1016/j.yjmcc.2010.06.009
  20. ↑ Molkentin JD, Lu JR, Antos CL, Markham B, Richardson J, Robbins J, Grant SR, Olson EN. A calcineurin-dependent transcriptional pathway for cardiac hypertrophy. Cell. 1998 Apr 17;93(2):215-28. PMID:9568714
  21. ↑ Frey N, Barrientos T, Shelton JM, Frank D, Rutten H, Gehring D, Kuhn C, Lutz M, Rothermel B, Bassel-Duby R, Richardson JA, Katus HA, Hill JA, Olson EN. Mice lacking calsarcin-1 are sensitized to calcineurin signaling and show accelerated cardiomyopathy in response to pathological biomechanical stress. Nat Med. 2004 Dec;10(12):1336-43. Epub 2004 Nov 14. PMID:15543153 doi:nm1132
  22. ↑ Turska-Kmiec A, Jankowska I, Pawlowska J, Kalicinski P, Kawalec W, Tomyn M, Markiewicz M, Teisseyre J, Czubkowski P, Rekawek J, Socha J. Reversal of tacrolimus-related hypertrophic cardiomyopathy after conversion to rapamycin in a pediatric liver transplant recipient. Pediatr Transplant. 2007 May;11(3):319-23. PMID:17430490 doi:10.1111/j.1399-3046.2006.00633.x
  23. ↑ Tsao L, Neville C, Musaro A, McCullagh KJ, Rosenthal N. Revisiting calcineurin and human heart failure. Nat Med. 2000 Jan;6(1):2-3. PMID:10613792 doi:10.1038/71478

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