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. 1999 Apr 1;13(7):798-803.
doi: 10.1101/gad.13.7.798.

Yeast calcineurin regulates nuclear localization of the Crz1p transcription factor through dephosphorylation

A Stathopoulos-Gerontides  1 , J J Guo, M S Cyert
Affiliations

Yeast calcineurin regulates nuclear localization of the Crz1p transcription factor through dephosphorylation

A Stathopoulos-Gerontides et al. Genes Dev. .

Abstract

Calcineurin, a Ca2+/calmodulin dependent protein phosphatase, regulates Ca2+-dependent processes in a wide variety of cells. In the yeast, Saccharomyces cerevisiae, calcineurin effects Ca2+-dependent changes in gene expression through regulation of the Crz1p transcription factor. We show here that calcineurin dephosphorylates Crz1p and that this results in translocation of Crz1p to the nucleus. We identify a region of Crz1p that is required for calcineurin-dependent regulation of its phosphorylation, localization, and activity, and show that this region has significant sequence simlarity to a portion of NF-AT, a family of mammalian transcription factors whose localization is also regulated by calcineurin. Thus, the mechanism of Ca2+/calcineurin-dependent signaling shows remarkable conservation between yeast and mammalian cells.

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Figures

Figure 1
Figure 1
Crz1p is dephosphorylated by calcineurin in vitro. HA-Crz1p was purified by immunoprecipitation from crz1Δ strains ASY472 (WT) and ASY475 (cnb1Δ) containing pAMS466 [YEp(HA–CRZ1)], incubated under different conditions as noted, and analyzed by PAGE and Western blotting (see Materials and Methods). (Lanes 1,6) HA–Crz1p incubated with buffer; (lanes 2,7) HA–Crz1p incubated in buffer with 20 mm CaCl2, and calmodulin; (lanes 3,8) HA–Crz1p incubated in buffer with 20 mm CaCl2, 83 units of calmodulin, and 0.8 unit of calcineurin, as described in Materials and Methods; (lanes 4,9) HA–Crz1p incubated in buffer with calcineurin and calmodulin plus 100 mm EGTA; (lanes 5,10) HA–Crz1p incubated in buffer with 20 mm CaCl2 and calmodulin plus calcineurin that was preincubated for 15 min at 30°C with 100 μm cypermethrin.
Figure 2
Figure 2
Translocation of GFP–CRZ1 to the nucleus is induced by Ca2+ addition and requires calcineurin. Living cells of strain ASY472 (WT) or ASY475 (cnb1Δ) containing pAMS463 (GFP–CRZ1) were grown at 21°C, incubated briefly with DAPI to stain DNA, and analyzed by fluorescence microscopy to observe GFP–Crz1p localization (GFP–CRZ1) or nuclear staining (DAPI). (A) Untreated cells; (B) cells incubated with 200 mm CaCl2 for 10 min at 21°C (+ Ca2+).
Figure 3
Figure 3
GFP–Crz1 shuttles from the nucleus to the cytoplasm and nuclear localization requires calcineurin activity. Log-phase cultures of strain ASY472 containing GFP–CRZ1 were grown at 21°C and analyzed by fluorescence microscopy to determine GFP–Crz1p localization. (E–H) Cells incubated for 2 hr with cycloheximide prior to other manipulations described (+chx). (A–D) Cells not incubated with cycloheximide (−chx). (B,F) At t = 0 min, CaCl2 was added to one aliquot of each culture (200 mm) and cells were observed at t = 30 min; (A,E) Equivalent cells not exposed to Ca2+. (D,H) At t = 60 min an aliquot of each Ca2+-treated culture was incubated with 3 μg/ml FK506 and observed at t = 90 min. (C,G) Cells incubated with Ca2+ for 90 min, not treated with FK506.
Figure 4
Figure 4
Localization of GFP–CRZ1 fusions. (A) Diagram of functional domains identified within Crz1p. (Poly-Q) Polyglutamine tract; (SRR), serine-rich region (see text and C); (zinc fingers) sequences that match the consensus for zinc finger DNA-binding motifs (Matheos et al. 1997; Stathopoulos and Cyert 1997). (B) Localization of GFP–Crz1p fusions in crz1Δ strains ASY834 (WT) and ASY835 (cnb1Δ) determined by fluorescence microscopy: (C), predominantly cytoplasmic localization; (N) predominantly nuclear localization; (N/C) localization to both cytosol and nucleus. Localization was observed in resting cells (UT) and cells incubated at 21°C for 10 min with 200 mm CaCl2 (+Ca2+). (C) Crz1p SRR shares sequence similarity with the NF-ATc SRR. Alignment of NF-ATc (Northrop et al. 1994) and Crz1p (Matheos et al. 1997; Stathopoulos and Cyert 1997) amino acid sequences were analyzed by the ALION program (C.G. Nevill-Manning, C. N. Huang, and D. L. Brutlag, pers. comm.; http://motif.stanford.edu/alion/).
Figure 5
Figure 5
The SRR is required for calcineurin-dependent regulation of Crz1p localization, activity, and phosphorylation. (A) GFP–CRZ1SRRΔ displays partial nuclear localization in cnb1Δ cells. The localization of GFP fusions in crz1Δ cells ASY835 (cnb1Δ) containing either plasmids pAMS463 (GFP–Crz1p) or pAMS490 (GFP–CRZ1SRRΔ is shown. (B) GFP–CRZ1SRRΔ directs calcineurin-independent transcription. Cells of crz1Δ strains ASY834 (WT) or ASY835 (cnb1Δ) containing the CDRE–lacZ reporter (pBJ306) and either plasmids pAMS463 (GFP–Crz1p) or pAMS490 (GFP–CRZ1SRRΔ) were grown in synthetic medium at 21°C for 6 hr with or without FK520 (3 μg/ml) and inducing treatments. β-Galactosidase activity is reported for extract of cells that were either untreated (open bars) or treated with 200 mm CaCl2 (solid bars). β-Galactosidase activities were normalized to the amount of protein in extracts from one representative experiment. Each extract was assayed in triplicate and the s.d. is representative of the error between these samples. (C) GFP–CRZ1SRRΔ confers Mn2+ tolerance to calcineurin-deficient cells. Yeast strains were plated onto synthetic medium containing 4 mm MnCl2 and grown at 30°C for 3 days. (WT + GFP). Strain YPH499 containing pGFP–NFUS; (cnb1Δ crz1Δ + GFP–CRZ1SRRΔ, strain ASY835 containing pAMS490; (cnb1Δ crz1Δ + GFP–CRZ1). Strain ASY835 containing pAMS463. (D) GFP–CRZ1SRRΔ does not show a calcineurin-dependent difference in apparent molecular mass. Equal amounts of extracts from crz1Δ strains ASY834 (WT) or ASY835 (cnb1Δ) containing different GFP–Crz1p constructs [pAMS463 (GFP–CRZ1) or pAMS490 (GFP–CRZ1SRRΔ)] were analyzed by Western blotting (see Methods).
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References

    1. Beals CR, Clipstone NA, Ho SN, Crabtree GR. Nuclear localization of NF-ATc by a calcineurin-dependent, cyclosporin-sensitive intramolecular interaction. Genes & Dev. 1997;11:824–834. - PubMed
    1. Clipstone NA, Crabtree GR. Identification of calcineurin as a key signalling enzyme in T-lymphocyte activation. Nature. 1992;357:695–697. - PubMed
    1. Cunningham KW, Fink GR. Calcineurin-dependent growth control in Saccharomyces cerevisiae mutants lacking PMC1, a homolog of plasma membrane Ca2+ ATPases. J Cell Biol. 1994;124:351–363. - PMC - PubMed
    1. ————— Calcineurin inhibits VCX1-dependent H+/Ca2+ exchange and induces Ca2+ -ATPases in Saccharomyces cerevisiae. Mol Cell Biol. 1996;16:2226–2237. - PMC - PubMed
    1. Cyert MS, Thorner J. Regulatory subunit (CNB1 gene product) of yeast Ca2+/calmodulin-dependent phosphoprotein phosphatases is required for adaptation to pheromone. Mol Cell Biol. 1992;12:3460–3469. - PMC - PubMed

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