Lab Interest: Calcineurin/NFAT Signaling in Development and Disease
How do cells percieve and respond to their environment? This question inspired the origin of our lab and lead to the elucidation of the NFAT signaling pathway that plays essential roles in vertebrate organogenesis and development. In this pathway. Calcium trigers the activation of calcineuirn and the dephosphorylation and nuclear entry of the different cytoplasmic subunits (NFATc proteins). In the nucleus these assemble with NFATn proteins on DNA target sites and activate or inactivate downstream genes. Calcium entry and NFAT activation can be triggered by tyrosine kinase receptors, non-receptor tyrosine kinases, L-type Calcium channels, NMDA receptors, gap junctions and non-canonical Wnt signaling. More recently we have found that, NFAT signaling can be activated by preventing export of NFATc proteins by activation of PI3 kinase and subsequent inhibition of GSK3 in the nucleus. Present studies indicate that there are likely to be many as yet undefined modulators of this pathway.
Mathematical Modeling of Calcineurin/NFAT signaling
We are mathematically modeling and fully perimeterizing the calcineurin-NFAT pathway because of its interesting role in human diseases and immune responses. Because calcium concentrations can be measured on a second-by-second basis, calcium activated pathways may prove to be the best for mathematical modeling.
Our recent studies (Arron et al Nature 2006) indicate that many of the characteristics of Down Syndrome arise from a compromise of NFAT signaling. Trisomy 21 produces the triplication of the DSCR1 and Dyrk1a genes. DSCR1 prevents NFATc nuclear import by inhibiting calcineurin, while Dyrk1a exports NFATc proteins from the nucleus. The coupling of these mechanisms with postive autofeedback loops within the pathway give rise to extrarodinary sensitivity to small variations in these kinases and phohpatase inhibitors. Current evidence indicates that similar circuit sensitivity underlies immunosuppression with the calcineurin inhibitors FK506 and Cyclosporin A.
NFAT Signaling and the Invention of Vertebrates
While nearly all signaling pathways are ancient, the NFATc family of genes originated at the origin of vertebrates by the recombination of a Calcium sensiing domain with a DNA binding domain. We believe that this event may have directed calcium signals to a new morphogenic pathway and given the raw material needed for the origin of many vertebrate-specific tissues. A prediction of this hypothesis is that calcineurin/NFAT signaling should be essential for the formation of vertebrate specific structures; indeed this appears to be the case. Using conditional alleles of the NFATc and calcineurin genes we find that they are required for the formation a recombinational immune system, organizing and patterning vessels, heart valves, skin, lungs, neural crest diversification, myelination, bone and skeleton development. The NFATc gene family appears in the genomes of vertebrates coincident with receptors such as the T cell receptor, the B cell receptor, Neurotrophins, VEGF, Tie1, Tie 2 and other ligand-receptors that are found only in vertebrates. The use of conditional alleles of the genes in this pathway has given us a systematic approach to understanding vertebratte organogenesis.
Websites and Articles about Calcineurin/NFAT Signaling
NFAT Nomenclature- Recently the Genome Nomenclature Committee at HUGO called for a vote on the nomenclature and the following designation was approved for the calcineurin-dependent subunits of NFAT complexes:
NFATc1- also called NFATc-
Human protein from cDNA, Murine protein from cDNA, Human gene, Murine gene
NFATc2- also called NFATp or NFAT1-
Human protein, Murine protein, Human gene, Murine gene,
NFATc3- also called NFATx or (4)-
Human protein, Murine Protein Human gene, Murine gene
NFATc4- also called NFAT3 -
Human protein, Murine protein Human gene, Murine gene
First Description of NFAT transcription complexes: Science. 1988;241,:202-5; :Mol Cell Biol. 1988:1715-24; Mol Cell Biol. 1986 9:3042-9
Signal Integration by NFAT transcription complexes Science. 1989;243:355-61
Cyclosporin Sensitivity Science. 1989;246:1617-20
Nuclear Translocation of cytoplasmic subunits (NFATc) Nature. 1991;352:803-7
Calcineurin requirement for NFATc translocation Nature. 1992 Jun 25;357(6380):695-7.
Cloning of Cytoplasmic subunits
Nature. 1994;369:497-502- Cloning of C1 and C2 (2 or c)
Science. 1993;262:750-4 Cloining of C2 (1 or p)
J Biol Chem. 1995;270:19898-907- Cloning of C3 (4)
Immunity. 1995 May;2(5):461-72- Cloning of C4 (3)
Identification of the SP-repeat motif
Mechanism of Nuclear Import
Mechanism of Nuclear Export
Structure of the DNA binding domains
NFATc1: Wolfe SA et al Nature1997 Jan 9;385(6612):172-6
NFATc1: Zhou P et al Cell. 1998 Mar 6;92(5):687-96
NFATc2 Chen et al Nature. 1998 Mar 5;392(6671):42-8
NFAT Signaling in the Development and Function of the Nervous System
Graef, Wang et al Cell. 2003 May 30;113(5):657-702003
Zeng H et al Cell. 2001 Nov 30;107(5):617-29.
Mansuy IM, et al Cell. 1998 Jan 9;92(1):39-49.
Graef et al . Nature 1999 Oct 14;401(6754):703-8
NFAT Signaling in the Development and Function of the Cardiovascular System
Molkentin JD et al Cell. 1998 Apr 17;93(2):215-28
Sussman et al Science. 1998 Sep 11;281(5383):1690-3
Graef et al Cell. 2001 Jun 29;105(7):863-75
NFAT Signaling in the Development and Function of the Immue System
Macian F, et al Cell. 2002 Jun 14;109(6):719-31
Rengarajan J, Tang B, and LH Glimcher Nat Immunol. 2002 Jan;3(1):48-54
Peng SL et al Immunity. 2001 Jan;14(1):13-20
Xanthoudakis S, et al Science. 1996 May 10;272(5263):892-5.
Caution- NFAT complexes are not present in conventional Dignham nuclear extracts- See our Protocols for the preparation of extracts.
NFATc1 monoclonal-7A6 Nature. 1994;369:497-502- J. Immunology 1997 Our 7A6 monoclonal antibody western blots, is good for IP and quantiatively supershifts NFATc1-based complexes. It is sold by BD Pharmagene, Santa Cruz, ABI, Serotec and other companies. It is also available through the Developmental Studies Hybridoma Bank.
NFATc2 monoclonal-Nature. 1994;369:497-502- J. Immunology 1997 We isolated three useful NFATc2 antibodies: G1D10 and G1G9, which give quantiative supershifts of both murine and human NFATc2-based complexes, but do not Western blot. In contrast clone 4G6-G5 Western blots well, but does not supershift and is not very useful for immunofluorescence.
and 4G5D, they are good for western blots, are good for IP and supershift experiments. Theu are sold by BD Pharmagene, Santa Cruz, ABI, Serotec and other companies.
NFATc3 monoclonal- Not available
NFATc3 polyclonal. J Biol Chem. 1995;270:19898-907 Sold by BD Pharmagene, Santa Cruz, ABI and other companies.
NFATc4 polyclonal- Limited quantities available. Requires purification to be effective.
WebSites on NFAT Signaling
Schreiber Lab Website
Greg Verdine's Web Site
Anjana Rao's Web Site
Chen Lab Web Site
Reviews on NFAT Signaling from our lab and others
Crabtree GR, Olson EN. Cell. 2002 Apr;109 Suppl:S67-79
Olson EN, Williams RS.Cell. 2000 Jun 23;101(7):689-92
Graef and Crabtree Curr Opin Genet Dev. 2001 Oct;11(5):505-12
Crabtree GR.Cell. 1999 Mar 5;96(5):611-4
Rao A, Luo C, Hogan PG. Annu Rev Immunol. 1997;15:707-47
Crabtree and Clipstone Annu Rev Biochem. 1994;63:1045-83.
Crabtree GR Science. 1989 Jan 20;243(4889):355-61.