A conserved family of calcineurin regulators

Abstract

The protein phosphatase calcineurin mediates many cellular responses to calcium signals. Using a genetic screen in yeast, we identified a new family of proteins conserved in fungi and animals that inhibit calcineurin function when overexpressed. Overexpression of the yeast protein Rcn1p or the human homologs DSCR1 or ZAKI-4 inhibited two independent functions of calcineurin in yeast: The activation of the transcription factor Tcn1p and the inhibition of the H(+)/Ca(2+) exchanger Vcx1p. Purified recombinant Rcn1p and DSCR1 bound calcineurin in vitro and inhibited its protein phosphatase activity. Signaling via calmodulin, calcineurin, and Tcn1p induced Rcn1p expression, suggesting that Rcn1p operates as an endogenous feedback inhibitor of calcineurin. Surprisingly, rcn1 null mutants exhibited phenotypes similar to those of Rcn1p-overexpressing cells. This effect may be due to lower expression of calcineurin in rcn1 mutants during signaling conditions. Thus, Rcn1p levels may fine-tune calcineurin signaling in yeast. The structural and functional conservation between Rcn1p and DSCR1 suggests that the mammalian Rcn1p-related proteins, termed calcipressins, will modulate calcineurin signaling in humans and potentially contribute to disorders such as Down Syndrome.

Figure 1

(A) Model of the calcium signaling pathway in yeast. Calcineurin activation by binding calcium and calmodulin inhibits the H⁺/Ca²⁺ exchanger Vcx1p and induces expression of the Ca²⁺ pump Pmc1p through activation of the transcription factor Tcn1p. (B) Multiple sequence alignment of Rcn1p from S. cerevisiae and predicted proteins from other fungi (Candida albicans, Schizosaccharomyces pombe, Neurospora crassa, and Aspergillus nidulans), protozoans (Dictyostelium discoideum), and animals (Caenorhabditus elegans, Drosophila melanogaster, and Homo sapiens) was generated using the Clustal algorithm. Residues identical in at least three of the seven sequences are highlighted and the most conserved central motif corresponding to the DS-24 peptide is underlined. GenBank accession numbers are NP_012763 (S.c.), Q09791 (S.p.), P53806 (C.e.), AAD33987 (D.m.), D83407 (H.s. ZAKI-4), AAF01684 (H.s. DSCR1L), and U85266 (H.s. DSCR1). Other sequences were obtained from ongoing genome sequencing projects (C.a., D.d., and N.c.) or from a compilation of EST sequences at GenBank (A.n. and D.d.). Note that some sequences were truncated at amino- and/or carboxyl termini and three small deletions (●) were introduced into the C. albicans sequence.

Figure 1

(A) Model of the calcium signaling pathway in yeast. Calcineurin activation by binding calcium and calmodulin inhibits the H⁺/Ca²⁺ exchanger Vcx1p and induces expression of the Ca²⁺ pump Pmc1p through activation of the transcription factor Tcn1p. (B) Multiple sequence alignment of Rcn1p from S. cerevisiae and predicted proteins from other fungi (Candida albicans, Schizosaccharomyces pombe, Neurospora crassa, and Aspergillus nidulans), protozoans (Dictyostelium discoideum), and animals (Caenorhabditus elegans, Drosophila melanogaster, and Homo sapiens) was generated using the Clustal algorithm. Residues identical in at least three of the seven sequences are highlighted and the most conserved central motif corresponding to the DS-24 peptide is underlined. GenBank accession numbers are NP_012763 (S.c.), Q09791 (S.p.), P53806 (C.e.), AAD33987 (D.m.), D83407 (H.s. ZAKI-4), AAF01684 (H.s. DSCR1L), and U85266 (H.s. DSCR1). Other sequences were obtained from ongoing genome sequencing projects (C.a., D.d., and N.c.) or from a compilation of EST sequences at GenBank (A.n. and D.d.). Note that some sequences were truncated at amino- and/or carboxyl termini and three small deletions (●) were introduced into the C. albicans sequence.

Figure 2

Rcn1p family members inhibit calcineurin function in yeast. (A) Overexpression of yeast RCN1, human DSCR1, or human ZAKI4 genes in yeast blocked calcineurin-dependent inhibition of Vcx1p, thereby restoring growth to pmc1 mutants but not pmc1 vcx1 mutants in high calcium medium (YPD at pH 5.5 + 200 mm CaCl₂). Photographs of colonies were taken after two days incubation at 30°C. (B) Overexpression of RCN1 inhibited calcineurin-dependent induction of PMC1–lacZ. Reporter gene expression was monitored in pmc1 vcx1 double mutants carrying either empty vector or RCN1 overexpression plasmid after 4 hr growth in medium supplemented with 100 mm CaCl₂ and 0.3 μm FK506 as indicated. Expression was also monitored in wild-type strains containing a constitutively active Tcn1p(C)–Gal4(DB) transcription factor. (C) Overexpression of RCN1, DSCR1, or ZAKI-4 inhibited expression of CDRE–lacZ. Betagalactosidase assays were performed as indicated in B.

Figure 3

Binding and inhibition of calcineurin in vitro by Rcn1p, DSCR1, and DS-24 peptide. (A) GST, GST–Rcn1p, and GST–DSCR1 were immobilized on glutathione-sepharose beads and assayed for the ability to bind purified bovine brain calcineurin. (B) Immobilized GST–DSCR1, but not GST alone bound calcineurin in buffer A, or buffer A supplemented with either 2 mm CaCl₂ or 2 mm EGTA. (C) GST–DSCR1/calcineurin bound to FK506/FKBP12 in the presence of calmodulin and 2 mm CaCl₂. (D–F) Dephosphorylation of RII peptide by calcineurin was inhibited by purified GST–DSCR1, GST–Rcn1p (100 μg/ml), and DS-24 synthetic peptide. Ten-fold excess calmodulin failed to reverse calcineurin inhibition by DSCR1.

Figure 4

Calcineurin regulates RCN1 transcription and Rcn1p stability in yeast. (A) Induction of a RCN1–lacZ reporter gene required calcium, calcineurin, and Tcn1p. A RCN1–lacZ reporter gene was introduced into wild-type and tcn1 mutants. β-Galactosidase activity was assayed in three independent transformants following 4 hr growth at 30°C in YPD at pH 5.5 medium supplemented with 100 mm CaCl₂ and 0.3 μm FK506 as indicated. (B) Rcn1p–HA protein levels increased in response to calcium via the activation of calcineurin and Tcn1p. A low-dosage plasmid containing the epitope-tagged Rcn1p–HA gene was introduced into wild-type yeast and tcn1 mutants. Cells were grown as in A and then total cell protein was fractionated by SDS-PAGE and analyzed by Western blotting. (C) Basal accumulation of Rcn1p–HA required calcineurin but not calcium, calmodulin, or Tcn1p. Total cell extracts were prepared and analyzed as in B using wild-type cells, cnb1 mutants lacking the regulatory B subunit of calcineurin, or cmd1–6 mutants lacking Ca²⁺-binding sites in calmodulin. (D) FK506 destabilized Rcn1p–HA in high calcium conditions. Wild-type yeast expressing Rcn1p–HA were treated with 100 μm cycloheximide for 20 min and then treated with 100 mm CaCl₂ in either the presence or absence of FK506. Total cell protein was extracted at 15 min intervals and analyzed by Western blotting as in B.

Figure 5

Rcn1p promotes calcineurin function and expression. (A) Rcn1p promotes calcineurin-dependent inhibition of Vcx1p. The optical density of pmc1 mutant cultures grown for 20 hr at 30°C was measured at 650 nm and plotted as a function of added CaCl₂. Solid lines represent growth of pmc1 or rcn1 pmc1 mutants and dashed lines represent growth of pmc1 vcx1 or rcn1 pmc1 vcx1 mutants in the presence and absence of 0.3 μm FK506. (B) Calcineurin-dependent induction ratios were calculated for various reporter genes expressed in pmc1 vcx1 double mutants or rcn1 pmc1 vcx1 triple mutants as indicated. Expression of DSCR1 partially complemented the rcn1 defect for PMC1–lacZ expression. (C) Induction of the calcineurin-dependent minimal CDRE–lacZ reporter gene was reduced in the absence of Rcn1p. The CDRE–lacZ reporter gene was introduced into pmc1 vcx1 and rcn1 pmc1 vcx1. β-Galactosidase activity was assayed in three independent transformants following 4 hr growth at 30°C in YPD medium at pH 5.5 supplemented with 100 mm CaCl₂ and 0.3 μm FK506 as indicated. (D) Western blot analysis of Tcn1p–HA extracted from wild type and rcn1 mutants. Total protein was extracted from log phase cells grown in YPD medium at pH 5.5 and analyzed by Western blotting using 12CA5 monoclonal antibody.

Figure 5

Rcn1p promotes calcineurin function and expression. (A) Rcn1p promotes calcineurin-dependent inhibition of Vcx1p. The optical density of pmc1 mutant cultures grown for 20 hr at 30°C was measured at 650 nm and plotted as a function of added CaCl₂. Solid lines represent growth of pmc1 or rcn1 pmc1 mutants and dashed lines represent growth of pmc1 vcx1 or rcn1 pmc1 vcx1 mutants in the presence and absence of 0.3 μm FK506. (B) Calcineurin-dependent induction ratios were calculated for various reporter genes expressed in pmc1 vcx1 double mutants or rcn1 pmc1 vcx1 triple mutants as indicated. Expression of DSCR1 partially complemented the rcn1 defect for PMC1–lacZ expression. (C) Induction of the calcineurin-dependent minimal CDRE–lacZ reporter gene was reduced in the absence of Rcn1p. The CDRE–lacZ reporter gene was introduced into pmc1 vcx1 and rcn1 pmc1 vcx1. β-Galactosidase activity was assayed in three independent transformants following 4 hr growth at 30°C in YPD medium at pH 5.5 supplemented with 100 mm CaCl₂ and 0.3 μm FK506 as indicated. (D) Western blot analysis of Tcn1p–HA extracted from wild type and rcn1 mutants. Total protein was extracted from log phase cells grown in YPD medium at pH 5.5 and analyzed by Western blotting using 12CA5 monoclonal antibody.

Figure 5

Rcn1p promotes calcineurin function and expression. (A) Rcn1p promotes calcineurin-dependent inhibition of Vcx1p. The optical density of pmc1 mutant cultures grown for 20 hr at 30°C was measured at 650 nm and plotted as a function of added CaCl₂. Solid lines represent growth of pmc1 or rcn1 pmc1 mutants and dashed lines represent growth of pmc1 vcx1 or rcn1 pmc1 vcx1 mutants in the presence and absence of 0.3 μm FK506. (B) Calcineurin-dependent induction ratios were calculated for various reporter genes expressed in pmc1 vcx1 double mutants or rcn1 pmc1 vcx1 triple mutants as indicated. Expression of DSCR1 partially complemented the rcn1 defect for PMC1–lacZ expression. (C) Induction of the calcineurin-dependent minimal CDRE–lacZ reporter gene was reduced in the absence of Rcn1p. The CDRE–lacZ reporter gene was introduced into pmc1 vcx1 and rcn1 pmc1 vcx1. β-Galactosidase activity was assayed in three independent transformants following 4 hr growth at 30°C in YPD medium at pH 5.5 supplemented with 100 mm CaCl₂ and 0.3 μm FK506 as indicated. (D) Western blot analysis of Tcn1p–HA extracted from wild type and rcn1 mutants. Total protein was extracted from log phase cells grown in YPD medium at pH 5.5 and analyzed by Western blotting using 12CA5 monoclonal antibody.

Figure 5

Rcn1p promotes calcineurin function and expression. (A) Rcn1p promotes calcineurin-dependent inhibition of Vcx1p. The optical density of pmc1 mutant cultures grown for 20 hr at 30°C was measured at 650 nm and plotted as a function of added CaCl₂. Solid lines represent growth of pmc1 or rcn1 pmc1 mutants and dashed lines represent growth of pmc1 vcx1 or rcn1 pmc1 vcx1 mutants in the presence and absence of 0.3 μm FK506. (B) Calcineurin-dependent induction ratios were calculated for various reporter genes expressed in pmc1 vcx1 double mutants or rcn1 pmc1 vcx1 triple mutants as indicated. Expression of DSCR1 partially complemented the rcn1 defect for PMC1–lacZ expression. (C) Induction of the calcineurin-dependent minimal CDRE–lacZ reporter gene was reduced in the absence of Rcn1p. The CDRE–lacZ reporter gene was introduced into pmc1 vcx1 and rcn1 pmc1 vcx1. β-Galactosidase activity was assayed in three independent transformants following 4 hr growth at 30°C in YPD medium at pH 5.5 supplemented with 100 mm CaCl₂ and 0.3 μm FK506 as indicated. (D) Western blot analysis of Tcn1p–HA extracted from wild type and rcn1 mutants. Total protein was extracted from log phase cells grown in YPD medium at pH 5.5 and analyzed by Western blotting using 12CA5 monoclonal antibody.

Figure 6

Down-regulation of calcineurin during calcium signaling conditions. (A) Cna1p expression correlates with Rcn1p in high calcium conditions. Western blots of Cna1p–MYC in rcn1 mutants, wild type, and Rcn1p-overexpressing strains were performed on total cell protein after 4 hr growth in YPD medium at pH 5.5 supplemented with 100 mm CaCl₂ as indicated. (B) Endogenous Rcn1p increases Cna1p expression in pmc1 vcx1 mutants in high calcium conditions. Experimental conditions were as described in A except 0.3 μm FK506 was added as indicated. (C) Rcn1p is not required to stabilize Cna1p. Cna1p–MYC levels were monitored in pmc1 vcx1 double and rcn1 pmc1 vcx1 triple mutants after a pretreatment with 100 μm cycloheximide for 20 min followed by addition of 100 mm CaCl₂ and 0.3 μm FK506. Total cell protein was extracted at 30 min intervals and analyzed by Western blotting as in Figure 3.

Figure 7

Involvement of Rcn1p in expression of calcineurin structural genes. (A) CNA1–lacZ, (B) CNA2–lacZ, and (C) CNB1–lacZ reporter genes were introduced into wild type and rcn1 deletion mutant. β-Galactosidase activity was assayed in three independent transformants following 4 hr growth at 30°C in YPD medium at pH 5.5 supplemented with 100 mm CaCl₂ and 0.3 μm FK506 as indicated.