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Dephosphorylation of the Nuclear Factor of Activated T Cells (NFAT) Transcription Factor Is Regulated by an RNA-Protein Scaffold Complex

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Dephosphorylation of the Nuclear Factor of Activated T Cells (NFAT) Transcription Factor Is Regulated by an RNA-Protein Scaffold Complex Correction BIOCHEMISTRY Correction for “Dephosphorylation of the nuclear factor of ac- B. Sacks, and Anjana Rao, which appeared in issue 28, July 12, tivated T cells (NFAT) transcription factor is regulated by an 2011, of Proc Natl Acad Sci USA (108:11381–11386; first pub- RNA-protein scaffold complex,” by Sonia Sharma, Gregory M. lished June 27, 2011; 10.1073/pnas.1019711108). Findlay, Hozefa S. Bandukwala, Shalini Oberdoerffer, Beate The authors note that, due to a printer’s error, Fig. 1 appeared Baust, Zhigang Li, Valentina Schmidt, Patrick G. Hogan, David incorrectly. The corrected figure and its legend appear below. A C 670 kDa 158 kDa 670 KDa 158 KDa 52 54 56 58 60 62 64 66 68 V IB: NFAT1 Fraction: 5052 54 56 5860 62 64 66 68 70 72 74 76 250 IB: IQGAP1 IB: NFAT1 150 100 IB: IQGAP2 5052 54 56 5860 62 64 66 68 70 72 74 76 250 IB: CaM IB: IQGAP1 150 IB: CK1 ε 100 IB: CnA B 100 bp Fr. 54-60 Fr. 74-80 Fr. 87-93 Load H2O NFAT1: +-- High Low protein protein Fig. 1. NFAT1 coelutes with IQGAP and NRON in resting T cell lysates by size-exclusion chromatography. Hypotonic lysates from (A and B) HA-NFAT1 Jurkat T cells or (C) primary murine CD8+ T cells were fractionated on a Superdex 200 size-exclusion column. (A and C) Individual fractions were analyzed by SDS-PAGE and Western blotting for NFAT1, IQGAP1, IQGAP2, calmodulin (CaM), casein kinase epsilon (CK1ε) and calcineurin A (CnA). Column void volume (V)isin- dicated. (B) Pooled column fractions were analyzed for NRON sequences by RT-PCR. www.pnas.org/cgi/doi/10.1073/pnas.1114486108 17234 | PNAS | October 11, 2011 | vol. 108 | no. 41 www.pnas.org/cgi/doi/10.1073/pnas.1114486108 Downloaded by guest on September 26, 2021 Dephosphorylation of the nuclear factor of activated T cells (NFAT) transcription factor is regulated by an RNA-protein scaffold complex Sonia Sharmaa,1, Gregory M. Findlaya, Hozefa S. Bandukwalaa,1, Shalini Oberdoerffera,2, Beate Bausta, Zhigang Lib, Valentina Schmidtc, Patrick G. Hogana,1, David B. Sacksb, and Anjana Raoa,1,3 aDepartment of Pathology, Harvard Medical School, Immune Disease Institute and Program in Cellular and Molecular Medicine, Children’s Hospital Boston, Boston, MA 02115; bDepartment of Laboratory Medicine, National Institutes of Health, Bethesda, MD 20892-1508; and cDepartment of Medicine, Stony Brook University, Stony Brook, NY 11794 Contributed by Anjana Rao, January 4, 2011 (sent for review December 15, 2010) Nuclear factor of activated T cells (NFAT) proteins are Ca2þ-regu- the cytoplasm of resting cells, from which the kinase dissociated lated transcription factors that control gene expression in many cell after stimulation. types. NFAT proteins are heavily phosphorylated and reside in the To identify NFAT scaffold protein(s), we performed genome- cytoplasm of resting cells; when cells are stimulated by a rise in wide RNAi screens for regulators of NFAT1 nuclear translocation intracellular Ca2þ, NFAT proteins are dephosphorylated by the in Drosophila S2R+ cells (4, 6, 7). A screen for candidates whose Ca2þ∕calmodulin-dependent phosphatase calcineurin and translo- depletion resulted in nuclear accumulation of NFAT1 in resting cate to the nucleus to activate target gene expression. Here we cells yielded two well-known Drosophila scaffold proteins, Homer 2þ show that phosphorylated NFAT1 is present in a large cytoplasmic and Discs Large (4), known to affect Ca signaling upstream of RNA-protein scaffold complex that contains a long intergenic NFAT (8). Homer2 and Homer3 also directly interact with NFAT noncoding RNA (lincRNA), NRON [noncoding (RNA) repressor of (9). We also performed a screen for candidates whose knockdown NFAT]; a scaffold protein, IQ motif containing GTPase activating prevented NFAT1 nuclear translocation in stimulated cells (6, 7). protein (IQGAP); and three NFAT kinases, casein kinase 1, glycogen This screen identified Drosophila Cul4, a component of a Skp, cul- synthase kinase 3, and dual specificity tyrosine phosphorylation lin, F-box E3 ligase complex whose closest human homologue is regulated kinase. Combined knockdown of NRON and IQGAP1 in- Cullin 4B (CUL4B), and two proteins involved in nuclear trans- — creased NFAT dephosphorylation and nuclear import exclusively port Drosophila Fs(2)Ket, an importin-beta whose human homo- after stimulation, without affecting the rate of NFAT rephosphor- logue is karyopherin (importin) beta 1 (KPNB1), and Drosophila ylation and nuclear export; and both NRON-depleted T cells and Cas, a protein that recycles importin-alpha to the cytoplasm, whose T cells from IQGAP1-deficient mice showed increased production human homologue is chromosome segregation 1-like (CSE1L) (7). of NFAT-dependent cytokines. Our results provide evidence that Notably, KPNB1, CSE1L, and CUL4B have been shown to a complex of lincRNA and protein forms a scaffold for a latent tran- bind the long intergenic noncoding RNA (lincRNA) NRON BIOCHEMISTRY scription factor and its regulatory kinases, and support an emer- [noncoding (RNA) repressor of NFAT] (10). Depletion of NRON ging consensus that lincRNAs that bind transcriptional regulators increased NFAT accumulation and transcriptional activity in the have a similar scaffold function. nucleus (10). An NRON-interacting protein that could not have scored in Drosophila screens was IQ motif-containing GTPase- activating protein 1 (IQGAP1), a large scaffold protein repre- ctivation of nuclear factor of activated T cells (NFAT) pro- sented in yeast and vertebrates but not Drosophila (11, 12). teins is regulated by the phosphorylation status of the NFAT A IQGAP proteins possess multiple protein interaction domains: regulatory domain, a conserved region of approximately 300 resi- a calponin homology domain that binds F actin; a WW domain dues N terminal to the DNA-binding domain, which is necessary that may bind proline-rich sequences or sequences containing and sufficient for nuclear transport (1, 2). In resting cells, NFAT phospho-serine or phospho-threonine; an IQ domain that binds proteins are located in the cytoplasm, where they are heavily phos- the ubiquitous Ca2þ sensor calmodulin; a GTPase-activating pro- phorylated through synergistic actions of three different families tein-related domain (GRD) that binds Cdc42 and Rac; and a C- of kinases, casein kinase 1 (CK1), glycogen synthase kinase 3 terminal domain that binds E cadherin, β-catenin, and adenoma- (GSK3), and dual specificity tyrosine phosphorylation regulated tous polyposis coli (APC) (11, 12). IQGAP1 also binds B-Raf, – kinase (DYRK) (2 5). Phosphorylation results in masking of a mitogen-activated protein kinase kinase, and ERK, as a scaffold nuclear localization sequence (NLS), exposure of a nuclear export for MAP kinases (11, 12). sequence (NES), and cytoplasmic localization of NFAT (1, 2). 2þ In this study we investigated the relation between NFAT, the When cells are stimulated to increase intracellular Ca concen- lincRNA NRON, and IQGAP. We find that phosphorylated trations, the calmodulin-dependent phosphatase calcineurin is NFAT1 is present with NRON and IQGAP1 in a cytoplasmic activated and dephosphorylates the NFAT regulatory domain, RNA-protein scaffold complex that also contains all three known causing a conformational change that exposes the NLS and masks the NES (1, 2). This event results in nuclear accumulation of NFAT and transcription of NFAT target genes (1). Author contributions: S.S., P.G.H., D.B.S., and A.R. designed research; S.S., G.M.F., H.S.B., S.O., B.B., and Z.L. performed research; G.M.F. and V.S. contributed new reagents/analytic We previously found, by size-exclusion chromatography of tools; S.S., P.G.H., D.B.S., and A.R. analyzed data; and S.S., P.G.H., and A.R. wrote the paper. hypotonic lysates from resting Cl.7W2 T cells, that fully phos- The authors declare no conflict of interest. phorylated NFAT1 migrated in an unexpectedly high-molecular 1Present address: La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037. weight complex with a substantial fraction of its inhibitory NFAT α ϵ 2Present address: Mouse Cancer Genetics Program, Center for Cancer Research, National kinases CK1 and CK1 (3). Neither calcineurin nor GSK3 Cancer Institute, US National Institutes of Health, Frederick, MD 21702. was detected in this complex. Stimulation with Ca2þ ionophore 3To whom correspondence may be addressed. E-mail: [email protected] or arao@ caused CK1 to dissociate from the complex, with only a slight liai.org. change in the elution properties of NFAT1 (3). These data sug- This article contains supporting information online at www.pnas.org/lookup/suppl/ gested that NFAT1 and CK1 were part of a larger complex within doi:10.1073/pnas.1019711108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1019711108 PNAS ∣ July 12, 2011 ∣ vol. 108 ∣ no. 28 ∣ 11381–11386 670 KDa 158 KDa A C 670 kDa 158 kDa V Fraction: 5052 54 56 5860 62 64 66 68 70 72 74 76 52 54 56 58 60 62 64 66 68 250 IB: NFAT1 IB: NFAT1 150 100 IB: IQGAP1 5052 54 56 5860 62 64 66 68 70 72 74 76 250 IB: IQGAP2 IB: IQGAP1 150 100 IB: CaM B Pooled gel filtration fractions IB: CK1 ε IB: CnA 100 bp 54-60 Fr. 74-80 Fr. Load H2O Fr. 87-93 Fr. Fig. 1. NFAT1 coelutes with IQGAP and NRON in resting T cell lysates by size-exclusion chromatogra- phy. Hypotonic lysates from (A and B) HA-NFAT1 Jurkat T cells or (C) primary murine CD8þ T cells were fractionated on a Superdex 200 size-exclu- sion column. (A and C) Individual fractions were analyzed by SDS-PAGE and Western blotting for NFAT1, IQGAP1, IQGAP2, calmodulin (CaM), casein kinase epsilon (CK1ϵ), and calcineurin A (CnA). NFAT1: + - - Column void volume (V) is indicated.
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