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|Mutation Type||critical splice donor site (2 bp from exon)|
|Coordinate||20,530,942 bp (GRCm38)|
|Base Change||A ⇒ G (forward strand)|
|Gene Name||protein phosphatase 3, catalytic subunit, beta isoform|
|Synonym(s)||Calnb, PP2BA beta, Cnab, CnAbeta, 1110063J16Rik|
|Chromosomal Location||20,499,364-20,546,573 bp (-)|
|MGI Phenotype||Homozygous null mice have small hearts and thymi, and reduced body weight. Cardiac function is normal, but mice lack a cardiac hypertrophic response to pressure overload, angiotensin II, or isopreteronol. Thymi are hypoplastic, with abnormal T cell development and reduced numbers of T cells.|
|Amino Acid Change|
|Institutional Source||Beutler Lab|
|Gene Model||predicted gene model for protein(s): [ENSMUSP00000022355] [ENSMUSP00000125722] [ENSMUSP00000125630] [ENSMUSP00000125582]|
|Predicted Effect||probably null|
|Predicted Effect||probably null|
|Predicted Effect||probably null|
|Predicted Effect||probably null|
|Phenotypic Category||Autosomal Semidominant|
|Alleles Listed at MGI|
|Mode of Inheritance||Autosomal Semidominant|
|Last Updated||2018-04-04 7:44 PM by Emre Turer|
|Record Created||2015-11-03 8:56 AM by Bruce Beutler|
The Copacabana phenotype was identified among G3 mice of the pedigree R3726, some of which showed an increase in the B:T cell ratio (Figure 1) due to a reduced frequency of T cells (Figure 2) including CD4+ T cells (Figure 3) and CD8+ T cells (Figure 4), and an increased frequency of central memory CD8+ T cells (Figure 5), all in the peripheral blood. Some mice also showed increased expression of CD44 on CD8+ T cells (Figure 6) and an increased frequency of NK cells (Figure 7) in the peripheral blood.
|Nature of Mutation|
Whole exome HiSeq sequencing of the G1 grandsire identified 53 mutations. All of the above anomalies were linked by continuous variable mapping to a mutation in Ppp3cb: a T to C transition at base pair 20,530,942 (v38) on chromosome 14, or base pair 15,632 in the GenBank genomic region NC_000080 encoding Ppp3cb. The mutation is within the donor splice site of intron 3, two base pairs from exon 3 (out of 14 total exons). The strongest association was found with an additive model of linkage to the normalized frequency of peripheral blood CD8+ T cells, wherein 12 variant homozygotes and 36 heterozygotes departed phenotypically from 17 homozygous reference mice with a P value of 2.435 x 10-9 (Figure 8). A substantial semidominant effect was observed in most of the assays. The effect of the mutation at the cDNA and protein level have not examined, but the mutation is predicted to result in use of a cryptic splice site in intron 3, resulting in a 67-base pair insertion of intron 3. The insertion would result in a frame-shifted protein product beginning after amino acid 137 of the protein, which is normally 525 amino acids in length, and terminating after the inclusion of seven aberrant amino acids.
Genomic numbering corresponds to NC_000080. The donor splice site of intron 3, which is destroyed by the Copacabana mutation, is indicated in blue lettering and the mutated nucleotide is indicated in red.
Calcineurin (alternatively, PP2B) is a calcium- and calmodulin (CaM)-dependent serine/threonine protein phosphatase. Calcineurin has a catalytic subunit and a regulatory calcium-binding subunit, termed calcineurin A (CnA) and calcineurin B (CnB), respectively. Three genes (CnAα, CnAβ, and CnAγ) encode calcineurin catalytic subunits, while two genes (CnB1 and CnB2) encode calcineurin regulatory subunits in mammals. Ppp3cb encodes calcineurin Aβ (CnAβ), a calcineurin catalytic subunit isoform.
All of the CnA proteins (CnAα, CnAβ, and CnAγ) have a catalytic domain (amino acids 2-310 in CnAβ) that is highly homologous to other serine/threonine protein phosphatases (Figure 9). The CnAβ catalytic subunit has a poly-proline motif (amino acids 11-20 in CnAβ) within the catalytic domain (1). The CnA proteins have three C-terminal regulatory domains that include a CnB binding domain (amino acids 256-262 and 305-310 in CnAβ), a CaM-binding domain (amino acids 401-423 in CnAβ), and an autoinhibitory domain (amino acids 474-496 in CnAβ) (1;2). The CaM- and the CnB-binding domains are required for calcineurin association with tau, a neuronal protein that is associated with Alzheimer’s disease (3). CaM binding impairs the association between calcineurin and tau. The autoinhibitory domain binds in the active site cleft in the absence of Ca2+/CaM to inhibit the enzyme (4). Upon Ca2+/CaM binding, inhibition is removed due to a conformation change that exposes the active site. The CnA proteins differ at the N- and C-termini; the sequence differences are proposed to mediate substrate recognition and/or localization.
CnAβ can be phosphorylated by protein kinase C, casein kinase I, and casein kinase II (5-7). Ppp3cb can be alternatively spliced upon insulin-like growth factor 1 induction to generate a CnAβ1 isoform (8). The CnAβ1 isoform does not have an autoinhibitory domain, and contains a unique C-terminal domain to CnAβ (9). The CnAβ1 isoform improves cardiac function after myocardial infarction by reducing inflammation and scar formation (9). In skeletal muscle, the CnAβ1 isoform is essential for myoblast proliferation, stimulates regeneration, and accelerates the resolution of inflammation (8).
The Copacabana mutation is predicted to result in aberrant splicing leading to a frame-shift and coding of a premature stop codon after amino acid 144 within the catalytic domain of the encoded protein.
CnAβ, CnAα, and CnB1 are ubiquitously expressed, while CnAγ and CnB2 are expressed in the testis and portions of the brain (10;11). In the rat brain and heart, CnAα is more abundantly expressed than CnAβ. CnAβ is more abundantly expressed than CnAα in the spleen, thymus, and lymphocytes (12). CnAβ expression in the heart is increased by stress, agonist stimulation, or growth factor stimulation (13-15). CnAβ is also activated by high glucose (16).
Calcineurin has functions in T cell activation, activation-induced cell death (AICD), T cell tolerance, ion channel regulation, cardiac myocyte hypertrophy, sperm motility, synaptic endocytosis, and Alzheimer’s disease (17-20). In lymphocytes, antigen engagement of lymphocyte receptors promotes the activation of phospholipase C-γ (PLC-γ) (Figure 10). Activated PLC-γ hydrolyzes phosphatidylinositol-4,5-bisphosphate into inositol-1,4,5-trisphosphate (IP3) and diacylglycerol. IP3 then binds to receptors on the endoplasmic reticulum and drives Ca2+ release from the endoplasmic reticulum into the cytoplasm, which triggers opening of Ca2+ release-activated Ca2+ channels. Calcineurin is activated by binding of CaM in response to sustained increased levels of intracellular calcium (11;21). Upon calcineurin activation, it dephosphorylates members of the nuclear factor of activated T cells (NFAT) family, promoting their translocation from the cytosol to the nucleus and subsequent induction of the transcription of NFAT target genes such as IL-2 growth factor, IFN-γ, and IL-4. The immunosuppressive drugs cyclosporine A and FK506 inhibit calcineurin, subsequently preventing NFAT nuclear translocation and the induction of cytokine gene expression (10;11;21). In addition to cytokine production, calcineurin-NFAT signaling mediates T cell maturation, synaptic transmission in neurons, vascular patterning in embryonic development, hypertrophic growth of the heart, and regulation of oxidative/slow fiber content in glycolytic/fast muscles (i.e., gastrocnemius, tibialis anterior, biceps, and triceps) (22;23). Calcineurin has a several functions including a role in apoptosis of T and B cells (24-26) and neuronal cells (27). In lymphocytes, calcineurin and NFAT function in apoptosis by mediating the induction of Fas (see the record for cherry) and FasL (see the record for riogrande), which transduce an apoptotic signal upon T cell activation (28;29). The specific functions of CnAβ are described in more detail, below.
CnAβ has many anti-inflammatory functions including limiting spontaneous pro-inflammatory Th1 and Th17-cell generation, the control of Treg-cell generation from the thymus, and the generation of inducible Treg cells (20). CnAβ is required for the spontaneous survival of naïve T cells (30). Naïve T cells from CnAβ-deficient (Ppp3cb-/-) mice exhibited increased spontaneous apoptosis that was blocked by IL-7 and IL-15. Ppp3cb-/- mice exhibited a reduction in CD3+ T cells in the peripheral blood compared to that in wild-type mice (31). In addition, the frequency of both thymic and splenic CD4+ and CD8+ cells in the Ppp3cb-/- mice was reduced compared to that in wild-type mice. The numbers of single positive T cells increased in the Ppp3cb-/- mice with age to levels comparable to wild-type mice (20). Thymus cellularity was also reduced in the Ppp3cb-/- mice. Splenic T cells from Ppp3cb-/- mice exhibited reduced proliferation induced by CD3 cross-linking or PMA/ionomycin. CnAα is able to partially compensate for the function of CnAβ in T cell activation, but both are required for efficient cell activation. After calcineurin-induced activation, NFAT forms a complex with FOXP3 to induce regulatory T cell (Treg) cell generation through the induction of Il2ra (CD25) and Ctla4 (CD152) (32). Loss of CnAβ expression results in deficient Treg cell generation with a concomitant expansion of mature T cells with an activated phenotype (20).
Calcineurin and NFAT function in cardiac morphogenesis and the induction of cardiac hypertrophy (33;34). Calcineurin overexpression in a transgenic mouse model resulted in cardiac hypertrophy and heart failure (34). In the heart, microRNA-499 (miR-499) targets both the CnAα and CnAβ catalytic subunits to inhibit anoxia-induced cardiomyocyte apoptosis (35). Ppp3cb-/- mice exhibited a greater loss of viable myocardium, an increase in cell death, and loss of cardiac function after acute ischemia-reperfusion injury to the heart (36).
CnAβ is essential for lipid homeostasis. Ppp3cb-/- mice exhibit hyperlipidemia and develop age-dependent insulin resistance (37). The hyperlipidemia exhibited by the Ppp3cb-/- mice is due to increased β-adrenergic receptor signaling-associated lipolysis in adipose tissues. Ppp3cb-/- mice treated with STZ to induce type 1 diabetes exhibited increased sensitivity and higher glucose levels at one and two weeks than STZ-treated wild-type mice. At 3 weeks, the levels of hyperglycemia were comparable between the Ppp3cb-/- and wild-type mice. The two groups exhibited comparable renal function, glomerular filtration rate, urine excretion, and concentration. However, the Ppp3cb-/- mice exhibited more loss of albumin and total protein excretion in the urine than wild-type mice. Wild-type mice exhibited increased hypertrophy in the whole kidney as early as one week after diabetes induction. However, the Ppp3cb-/- mice did not display whole kidney hypertrophy. After 6 weeks of diabetes, wild-type and Ppp3cb-/- mice exhibited both whole kidney and glomeruli hypertrophy, with the Ppp3cb-/- mice exhibiting a lesser degree of hypertrophy than wild-type mice (38).
The frequency of thymic and splenic CD4+ and CD8+ cells was reduced in Ppp3cb-/- mice compared to that in wild-type mice. CnAβ is required for the spontaneous survival of naïve T cells (30). In addition, calcineurin has a role in apoptosis of T and B cells (24-26). In lymphocytes, calcineurin and NFAT function in apoptosis by mediating the induction of Fas and FasL, which transduce an apoptotic signal upon T cell activation (28;29). The reduced frequency of single positive T cells in the Copacabana mice indicates that CnAβCopacabana exhibits loss of function.
Copacabana(F):5'- CACAAGGAAAACTAATTGGATGGTC -3'
Copacabana(R):5'- TGATGACTCTTCATAGGCCAC -3'
Copacabana_seq(F):5'- ACTAATTGGATGGTCTGAAAAAGAAG -3'
Copacabana_seq(R):5'- GGCCACTCCAGATATTTCTTGATAG -3'
1. Guerini, D., and Klee, C. B. (1989) Cloning of Human Calcineurin A: Evidence for Two Isozymes and Identification of a Polyproline Structural Domain. Proc Natl Acad Sci U S A. 86, 9183-9187.
2. Hubbard, M. J., and Klee, C. B. (1989) Functional Domain Structure of Calcineurin A: Mapping by Limited Proteolysis. Biochemistry. 28, 1868-1874.
3. Yu, D. Y., Tong, L., Song, G. J., Lin, W. L., Zhang, L. Q., Bai, W., Gong, H., Yin, Y. X., and Wei, Q. (2008) Tau Binds both Subunits of Calcineurin, and Binding is Impaired by Calmodulin. Biochim Biophys Acta. 1783, 2255-2261.
4. Kissinger, C. R., Parge, H. E., Knighton, D. R., Lewis, C. T., Pelletier, L. A., Tempczyk, A., Kalish, V. J., Tucker, K. D., Showalter, R. E., and Moomaw, E. W. (1995) Crystal Structures of Human Calcineurin and the Human FKBP12-FK506-Calcineurin Complex. Nature. 378, 641-644.
5. Hashimoto, Y., and Soderling, T. R. (1989) Regulation of Calcineurin by Phosphorylation. Identification of the Regulatory Site Phosphorylated by Ca2+/calmodulin-Dependent Protein Kinase II and Protein Kinase C. J Biol Chem. 264, 16524-16529.
6. Martensen, T. M., Martin, B. M., and Kincaid, R. L. (1989) Identification of the Site on Calcineurin Phosphorylated by Ca2+/CaM-Dependent Kinase II: Modification of the CaM-Binding Domain. Biochemistry. 28, 9243-9247.
7. Singh, T. J., and Wang, J. H. (1987) Phosphorylation of Calcineurin by Glycogen Synthase (Casein) Kinase-1. Biochem Cell Biol. 65, 917-921.
8. Lara-Pezzi, E., Winn, N., Paul, A., McCullagh, K., Slominsky, E., Santini, M. P., Mourkioti, F., Sarathchandra, P., Fukushima, S., Suzuki, K., and Rosenthal, N. (2007) A Naturally Occurring Calcineurin Variant Inhibits FoxO Activity and Enhances Skeletal Muscle Regeneration. J Cell Biol. 179, 1205-1218.
9. Felkin, L. E., Narita, T., Germack, R., Shintani, Y., Takahashi, K., Sarathchandra, P., Lopez-Olaneta, M. M., Gomez-Salinero, J. M., Suzuki, K., Barton, P. J., Rosenthal, N., and Lara-Pezzi, E. (2011) Calcineurin Splicing Variant Calcineurin Abeta1 Improves Cardiac Function After Myocardial Infarction without Inducing Hypertrophy. Circulation. 123, 2838-2847.
11. Crabtree, G. R. (1999) Generic Signals and Specific Outcomes: Signaling through Ca2+, Calcineurin, and NF-AT. Cell. 96, 611-614.
12. Jiang, H., Xiong, F., Kong, S., Ogawa, T., Kobayashi, M., and Liu, J. O. (1997) Distinct Tissue and Cellular Distribution of Two Major Isoforms of Calcineurin. Mol Immunol. 34, 663-669.
13. Haq, S., Choukroun, G., Lim, H., Tymitz, K. M., del Monte, F., Gwathmey, J., Grazette, L., Michael, A., Hajjar, R., Force, T., and Molkentin, J. D. (2001) Differential Activation of Signal Transduction Pathways in Human Hearts with Hypertrophy Versus Advanced Heart Failure. Circulation. 103, 670-677.
14. Taigen, T., De Windt, L. J., Lim, H. W., and Molkentin, J. D. (2000) Targeted Inhibition of Calcineurin Prevents Agonist-Induced Cardiomyocyte Hypertrophy. Proc Natl Acad Sci U S A. 97, 1196-1201.
15. Oka, T., Dai, Y. S., and Molkentin, J. D. (2005) Regulation of Calcineurin through Transcriptional Induction of the Calcineurin A Beta Promoter in Vitro and in Vivo. Mol Cell Biol. 25, 6649-6659.
16. Williams, C. R., and Gooch, J. L. (2014) Calcineurin Abeta Regulates NADPH Oxidase (Nox) Expression and Activity Via Nuclear Factor of Activated T Cells (NFAT) in Response to High Glucose. J Biol Chem. 289, 4896-4905.
17. Kincaid, R. L., Takayama, H., Billingsley, M. L., and Sitkovsky, M. V. (1987) Differential Expression of Calmodulin-Binding Proteins in B, T Lymphocytes and Thymocytes. Nature. 330, 176-178.
18. Tash, J. S., Krinks, M., Patel, J., Means, R. L., Klee, C. B., and Means, A. R. (1988) Identification, Characterization, and Functional Correlation of Calmodulin-Dependent Protein Phosphatase in Sperm. J Cell Biol. 106, 1625-1633.
19. Sun, T., Wu, X. S., Xu, J., McNeil, B. D., Pang, Z. P., Yang, W., Bai, L., Qadri, S., Molkentin, J. D., Yue, D. T., and Wu, L. G. (2010) The Role of calcium/calmodulin-Activated Calcineurin in Rapid and Slow Endocytosis at Central Synapses. J Neurosci. 30, 11838-11847.
20. Doetschman, T., Sholl, A., Chen, H., Gard, C., Hildeman, D. A., and Bommireddy, R. (2011) Divergent Effects of Calcineurin Abeta on Regulatory and Conventional T-Cell Homeostasis. Clin Immunol. 138, 321-330.
21. Klee, C. B., Ren, H., and Wang, X. (1998) Regulation of the Calmodulin-Stimulated Protein Phosphatase, Calcineurin. J Biol Chem. 273, 13367-13370.
22. Parsons, S. A., Wilkins, B. J., Bueno, O. F., and Molkentin, J. D. (2003) Altered Skeletal Muscle Phenotypes in Calcineurin Aalpha and Abeta Gene-Targeted Mice. Mol Cell Biol. 23, 4331-4343.
23. Crabtree, G. R., and Olson, E. N. (2002) NFAT Signaling: Choreographing the Social Lives of Cells. Cell. 109 Suppl, S67-79.
24. Bonnefoy-Berard, N., Genestier, L., Flacher, M., and Revillard, J. P. (1994) The Phosphoprotein Phosphatase Calcineurin Controls Calcium-Dependent Apoptosis in B Cell Lines. Eur J Immunol. 24, 325-329.
25. Fruman, D. A., Mather, P. E., Burakoff, S. J., and Bierer, B. E. (1992) Correlation of Calcineurin Phosphatase Activity and Programmed Cell Death in Murine T Cell Hybridomas. Eur J Immunol. 22, 2513-2517.
26. Zhao, Y., Tozawa, Y., Iseki, R., Mukai, M., and Iwata, M. (1995) Calcineurin Activation Protects T Cells from Glucocorticoid-Induced Apoptosis. J Immunol. 154, 6346-6354.
27. Asai, A., Qiu, J., Narita, Y., Chi, S., Saito, N., Shinoura, N., Hamada, H., Kuchino, Y., and Kirino, T. (1999) High Level Calcineurin Activity Predisposes Neuronal Cells to Apoptosis. J Biol Chem. 274, 34450-34458.
28. Toth, R., Szegezdi, E., Molnar, G., Lord, J. M., Fesus, L., and Szondy, Z. (1999) Regulation of Cell Surface Expression of Fas (CD95) Ligand and Susceptibility to Fas (CD95)-Mediated Apoptosis in Activation-Induced T Cell Death Involves Calcineurin and Protein Kinase C, Respectively. Eur J Immunol. 29, 383-393.
29. Latinis, K. M., Norian, L. A., Eliason, S. L., and Koretzky, G. A. (1997) Two NFAT Transcription Factor Binding Sites Participate in the Regulation of CD95 (Fas) Ligand Expression in Activated Human T Cells. J Biol Chem. 272, 31427-31434.
30. Manicassamy, S., Gupta, S., Huang, Z., Molkentin, J. D., Shang, W., and Sun, Z. (2008) Requirement of Calcineurin a Beta for the Survival of Naive T Cells. J Immunol. 180, 106-112.
31. Bueno, O. F., Brandt, E. B., Rothenberg, M. E., and Molkentin, J. D. (2002) Defective T Cell Development and Function in Calcineurin A Beta -Deficient Mice. Proc Natl Acad Sci U S A. 99, 9398-9403.
32. Wu, Y., Borde, M., Heissmeyer, V., Feuerer, M., Lapan, A. D., Stroud, J. C., Bates, D. L., Guo, L., Han, A., Ziegler, S. F., Mathis, D., Benoist, C., Chen, L., and Rao, A. (2006) FOXP3 Controls Regulatory T Cell Function through Cooperation with NFAT. Cell. 126, 375-387.
33. de la Pompa, J. L., Timmerman, L. A., Takimoto, H., Yoshida, H., Elia, A. J., Samper, E., Potter, J., Wakeham, A., Marengere, L., Langille, B. L., Crabtree, G. R., and Mak, T. W. (1998) Role of the NF-ATc Transcription Factor in Morphogenesis of Cardiac Valves and Septum. Nature. 392, 182-186.
34. Molkentin, J. D., Lu, J. R., Antos, C. L., Markham, B., Richardson, J., Robbins, J., Grant, S. R., and Olson, E. N. (1998) A Calcineurin-Dependent Transcriptional Pathway for Cardiac Hypertrophy. Cell. 93, 215-228.
35. Wang, J. X., Jiao, J. Q., Li, Q., Long, B., Wang, K., Liu, J. P., Li, Y. R., and Li, P. F. (2011) MiR-499 Regulates Mitochondrial Dynamics by Targeting Calcineurin and Dynamin-Related Protein-1. Nat Med. 17, 71-78.
36. Bueno, O. F., Lips, D. J., Kaiser, R. A., Wilkins, B. J., Dai, Y. S., Glascock, B. J., Klevitsky, R., Hewett, T. E., Kimball, T. R., Aronow, B. J., Doevendans, P. A., and Molkentin, J. D. (2004) Calcineurin Abeta Gene Targeting Predisposes the Myocardium to Acute Ischemia-Induced Apoptosis and Dysfunction. Circ Res. 94, 91-99.
37. Suk, H. Y., Zhou, C., Yang, T. T., Zhu, H., Yu, R. Y., Olabisi, O., Yang, X., Brancho, D., Kim, J. Y., Scherer, P. E., Frank, P. G., Lisanti, M. P., Calvert, J. W., Lefer, D. J., Molkentin, J. D., Ghigo, A., Hirsch, E., Jin, J., and Chow, C. W. (2013) Ablation of Calcineurin Abeta Reveals Hyperlipidemia and Signaling Cross-Talks with Phosphodiesterases. J Biol Chem. 288, 3477-3488.
|Science Writers||Anne Murray|
|Authors||Ming Zeng, Xue Zhong, and Bruce Beutler|
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