Expression of Hyperactivable NFAT1 from the ROSA26 Locus Leads to Detrimental Effects during Embryonic Development.
Authors: Mueller Martin R. , Sasaki Yoshiteru , Sharma Sonia , Ghosh Srimoyee , Gelinas Curtis , Lamperti Edward L. , Rajewsky Klaus , Rao Anjana 
Published: April 2020

Abstract
NFAT is a family of highly phosphorylated proteins residing in the cytoplasm of resting cells. Upon dephosphorylation by calcineurin, NFAT translocates to the nucleus, where it orchestrates several developmental programs, including those of the immune, cardiovascular and central nervous systems. NFAT is rephosphorylated and inactivated by different kinases (CK1, GSK-3, DYRK). The major docking site for calcineurin is located at the N terminus of the NFAT regulatory domain and has the consensus sequence PxIxIT (SPRIEIT in NFAT1). Substitution of the SPRIEIT sequence with HPVIVIT increases the basal calcineurin sensitivity of the protein significantly. Similarly, mutation of the CK1 docking site from FSILF to ASILA leads to partial nuclear localization by decreasing rephosphorylation and nuclear export. To assess the impact of hyperactivable NFAT mutants on activation kinetics and signal responsiveness, we retrovirally transduced T cells from NFAT1−/− mice with contructs expressing wild type NFAT1, NFAT1-ASILA, NFAT1-HPVIVIT or NFAT1-ASILA-HPVIVIT. Analysis by western blotting and immunocytochemistry revealed, that the wild type protein was entirely localized in the cytoplasm and completely phosphorylated under resting conditions, whereas the hyperactivable mutants exhibited an increasing degree of nuclear translocation and dephosphorylation (ASILA 20%, HPVIVIT 30–40%, ASILA-HPVIVIT 50–70%). Upon stimulation with PMA and ionomycin, the hyperactivable mutants exhibited an incremental acceleration of nuclear translocation and delay of nuclear export as compared to the wild type protein. Furthermore, T cells expressing hyperactivable NFAT proteins exhibited significantly higher expression rates of different cytokines (IL-2, TNF-α, IFN-γ) upon stimulation with low doses of ionomycin documenting their hyperresponsiveness and biological activity. To provide a new tool for the analysis of the calcineurin-NFAT pathway in vivo and to assess the effect of modulating the affinity of signaling molecules for their upstream regulators, which are consistently kept at low to moderate affinity during evolution, we subsequently generated transgenic mice conditionally expressing different hyperactivable NFAT1 mutants from the ROSA26 locus (NFAT1-HPVIVIT, NFAT1-ASILA-HPVIVIT). Transgene expression in the T cell lineage was achieved by breeding the ROSA26 transgenic mice to CD4-Cre mice, which express the Cre recombinase under the control of the CD4 promoter. CD4 and CD8 T cells from these mice showed significant hyperactivability as assessed by accelerated nuclear translocation and delayed nuclear export of NFAT and substantially increased cytokine expression upon stimulation. Expression of the hyperactivable NFAT proteins early in the germline was achieved by breeding the ROSA26 mice to Cre-Del mice, which express the Cre recombinase under the control of the ACE promoter. While breeding of ROSA26-YFP control mice to Cre-Del mice resulted in transgene expression in all T and B cells, expression of hyperactivable NFAT proteins early in the germline resulted in incremental mosaicism in T and B cells (70% transgene expression in NFAT1-HPVIVIT and 20–30% transgene expression in NFAT1-ASILA-HPVIVIT). This data demonstrate that hyperactivable NFAT1 proteins result in a selective disadvantage for the expressing cells in embryonic development and provide a potential explanation why evolution chose to keep the respective docking sites at moderate affinity.