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The protein kinase Cmk2 negatively regulates the calcium/calcineurin signalling pathway and expression of calcium pump genes PMR1 and PMC1 in budding yeast

Cell Communication and Signaling volume 17, Article number: 7 (2019) Cite this article

Abstract

Through a genome-wide screen we have identified calcium-tolerant deletion mutants for five genes in the budding yeast Saccharomyces cerevisiae. In addition to CNB1 and RCN1 that are known to play a role in the calcium signalling pathway, the protein kinase gene CMK2, the sphingolipid homeostasis-related gene ORM2 and the gene SIF2 encoding the WD40 repeat-containing subunit of Set3C histone deacetylase complex are involved in the calcium sensitivity of yeast cells to extracellular calcium. Cmk2 and the transcription factor Crz1 have opposite functions in the response of yeast cells to calcium stress. Deletion of CMK2 elevates the level of calcium/calcineurin signalling and increases the expression level of PMR1 and PMC1, which is dependent on Crz1. Effects of Cmk2 on calcium sensitivity and calcium/calcineurin signalling are dependent on its kinase activity. Therefore, Cmk2 is a negative feedback controller of the calcium/calcineurin signalling pathway. Furthermore, the cmk2 crz1 double deletion mutant is more resistant than the crz1 deletion mutant, suggesting that Cmk2 has an additional Crz1-independent role in promoting calcium tolerance.

Background

Calcium ions regulate many cellular processes in both prokaryotes and eukaryotes [1,2,3,4,5]. Regulation of intracellular calcium homeostasis and the calcium/calcineurin signalling pathway are highly conserved in eukaryotic cells [6, 7]. In the budding yeast Saccharomyces cerevisiae, cytosolic calcium homeostasis is regulated by Ca2+ transporters and sequestrators in the plasma and organellar membranes. Transient increases in cytosolic Ca2+ activate the calcium/calcineurin signalling pathway. Sustained Ca2+ accumulation in the cytosol is prevented by a Ca2 sequestration system composed of the calcium pump Pmc1 and the Ca2+/H+ exchanger Vcx1 in the vacuolar membrane as well as the calcium pump Pmr1 and the Ca2+/H+ exchanger Gdt1 in the ER/Golgi secretory pathway [8,9,10]. In our recent studies, we have demonstrated that Rch1 is a novel negative regulator of calcium uptake in the plasma membrane in the budding yeast and the human yeast pathogen Candida albicans [11,12,13,14,15].

As a eukaryotic model organism, S. cerevisiae has been proven to be a valuable genetic tool to elucidate mechanisms regulating calcium homeostasis and calcium signalling in eukaryotic cells as well as to characterize gene-drug and pathway–drug interactions [16,17,18,19,20]. In our previous study, from all nonessential genes in the genome we have identified 120 genes that are involved in the tolerance of the budding yeast to high levels of extracellular calcium [21]. To explore genes involved in the sensitivity of yeast cells to extracellular calcium, we screened the same diploid deletion mutant set for calcium-tolerant gene deletion mutants. In this study, we have identified a total of five genes whose deletion leads to the tolerance of S. cerevisiae cells to high levels of extracellular calcium. Furthermore, we show that the protein kinase Cmk2 is a negative feedback controller of the calcium/calcineurin signalling pathway.

Methods

Strains and reagents

The S. cerevisiae haploid wild type BY4741, the diploid wild type BY4743 strain and their isogenic deletion mutant sets used in this study were purchased from Invitrogen Inc. [21,22,23] (Table 1) Yeast cells were grown at 30 °C in YPD medium (1% yeast extract, 2% peptone, 2% glucose) or SD medium (0.67% yeast nitrogen base without amino acids, 2% glucose, and auxotrophic amino acids as needed). Chemicals were purchased from Sangon Biotech (Shanghai, China), except that cyclosporine A (CsA) and O-nitrophenyl-β-D-galactopyranoside (ONPG) were purchased from Sigma (Beijing, China) and the antibiotic nourseothricin was purchased from Golden Biotechnology Inc. (Missouri, USA).

Table 1 S. cerevisiae strains used in this study
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Genome-wide screen for calcium-tolerant mutations

For primary screens for calcium-tolerant strains, the collection of homozygous diploid deletion mutants of nonessential 4757 genes was replicated onto YPD plates with 0.4 M CaCl2 and YPD plates without supplemented CaCl2 (as controls). respectively. These plates were incubated at 30 °C for 2–3 days. A mutant with a relative colony size increased by more than 30% as compared to the average size of its surrounding mutants on YPD plates containing 0.4 M CaCl2 but not on YPD plates without supplemented CaCl2, was identified as a potential calcium-tolerant mutant. Primary screen was repeated two times. Tolerant mutants were subjected to a secondary screen by a serial dilution assay method as described [22, 24]. The suppressive effects of cyclosporine A on the phenotypes of calcium-tolerant mutants were examined by supplementing 50 μg/ml CsA into YPD plates with or without CaCl2 [25].

Construction of double-gene deletion mutants

To study the interaction between CMK2 and genes encoding components of the calcium/calcineurin signalling pathway in calcium sensitivity, we created their double-gene deletion mutants in the BY4741 genetic background. First, kanMX4 markers in single-gene deletion mutants for CMK2 and VCX1 (cmk2::kanMX4 and vcx1::kanMX4) were replaced by the natMX4 in the plasmid p4339 to generate cmk2::natMX4 and vcx1::natMX4 strains as described previously [26]. The cmk2::natMX4 cassette was PCR amplified from the genomic DNA of the cmk2::natMX4 strain with a pair of primer CMK2-F (5’ CGATATTGTT CAAGATCAGC AG 3)/CMK2-R (5’ ATGCCATGAA GTGTAGCTGC 3′), which locate 100-bp upstream and downstream, respectively, of the open reading frame (ORF) of the CMK2 gene, and used for replacing the CMK2 ORFs in kanMX4 mutants for target genes to generate their double-gene deletion mutants. The deletion of CMK2 gene in these double-gene deletion mutants was confirmed by PCR amplification with detection primer pair CMK2-DF (5’ GAGGCTTATC TTAGAACCC 3′)/CMK2-DR (5’ AGAACCCGTT AGGCAACTAC 3′), which flank the CMK2-F and CMK2-R. Similarly, the double-gene deletion mutant between CNB1 and VCX1 was constructed (Table 1).

Galactosidase activity assay

To measure the calcineurin dependent response element (CDRE)-driven β-galactosidase activity in the wild type and the cmk2/cmk2 mutant, we integrated the StuI-precut plasmid DNA containing the 4 × CDRE-lacZ reporter into the AUR1 locus of these strains as described [12, 27]. The PMC1-lacZ reporter and PMR1-lacZ reporter were described in our previous study [28]. The β-galactosidase activity was determined using the substrate ONPG as described [29,30,31]. Data are mean ± SD from six independent experiments.

DNA manipulation

To clone the full-length gene CMK2 into the centromeric vector pHAC111 [26], a DNA fragment containing the 776-bp promoter, the 1344-bp open reading frame (ORF) and the 341-bp terminator region of CMK2 was amplified with a pair of primers ScCMK2-clonF (5′ gtatgggtag catgcctgca gGACACAATG ATAGGCACAA CGC 3′; lower-case sequence from the pHAC111 vector) and ScCMK2-clonR (5′ aaaacgacgg ccagtgaatt cTGACATTGA CGTTAGCGAT GACT 3′;; lower-case sequence from pHAC111), and cloned between PstI and EcoRI sites in pHAC111 through homologous recombination, which yielded pHAC111-CMK2.

Next, we amplified the DNA fragment CMK2-UP containing the 842-bp promoter and the 225-bp CMK2 ORF region upstream of the AAG codon (lysine) with primers ScCMK2-mutant-F1 (5’ CCTTTCTAGA GCGTTTTCTG TG 3′) and ScCMK2-mutant-R1 (5’ CAATAAGATG GCTATAGCAA CATCTTCATT TGTGG 3′; underlined is the mutated codon of AAG). Similarly, the DNA fragment CMK2-DOWN containing the 1115-bp CMK2 ORF region downstream of the AAG codon and the 414-bp terminator was PCR amplified with primers ScCMK2-mutant-F2 (5′ GAAGATGTTG CTATAGCCAT CTTATTGAAG AAGGCATTGC 3′; underlined is the mutated codon of AAG) and ScCMK2-mutant-R2 (5’ CTCAGAGAAG AACCACGGTG 3′). The two fragments CMK2-UP and CMK2-DOWN were fused together by PCR with primers ScCMK2-clonF and ScCMK2-clonR, and the fused product was cloned between PstI and EcoRI sites in pHAC111 through homologous recombination, yielding pHAC111-CMK2M, expressing a point (K76A) mutant form of Cmk2 under its own promoter. All constructs were confirmed by DNA sequencing.

Statistical analysis

Data are presented as means ± SD. Significant differences were analyzed by GraphPad Prism version 4.00 (USA). P values of < 0.05 were considered to be significant.

Results

Screen for calcium-tolerant gene deletion mutants

In our previous study [21], through a genome-wide approach we have identified 120 genes that are involved in the tolerance of S. cerevisiae cells to high levels of extracellular calcium. To explore genes involved in the sensitivity of yeast cells to extracellular calcium, we screened the same deletion library for calcium-tolerant gene deletion mutants. As a result, we revealed that deletion of CNB1, RCN1, CMK2, ORM2 or SIF2 caused yeast cells to be tolerant to 0.4 M CaCl2, and these phenotypes were more dramatical in the presence of 0.6 M CaCl2 (Fig. 1). The calcium tolerant-phenotypes of yeast cells lacking RCN1, ORM2 or SIF2 on YPD plate containing 0.4 M CaCl2 could be slightly suppressed by the addition of the cyclosporin A (CsA), the specific inhibitor of calcineurin (Fig. 1).

Fig. 1
figure 1

Phenotypes of calcium-tolerant gene deletion mutants. Cells of the wild-type BY4743 and five deletion mutants identified from the library screen were grown at 30 °C in liquid YPD overnight, serially diluted by 10 times and spotted on YPD plates with or without supplemented CaCl2 or cyclosporine A (CsA) as indicated, respectively. Plates were incubated for 2 to 3 days at 30 °C

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RCN1 encodes a protein involved in calcineurin regulation during calcium signalling and has similarity to human DSCR1 that is found in the Down Syndrome candidate region [32]. CNB1 encodes a regulatory subunit of calcineurin, a Ca2+/calmodulin-regulated type 2B protein phosphatase, which dephosphorylates the transcription factor Crz1, and promotes its nuclear localization [9]. Our observation that deletion of CNB1 leads to a calcium-tolerant phenotype in the wild type BY4743 background is consistent with previous studies [33, 34], showing that inactivation of calcineurin including deletion of CNB1 restores the calcium-tolerance of yeast cells lacking PMC1, since calcineurin decreases calcium tolerance of pmc1 cells by inhibiting the function of the vacuolar H+/Ca2+ exchanger Vcx1. Therefore, it is not surprising to note that the difference in calcium sensitivity between the wild type and the cnb1/cnb1 mutant is abolished by CsA, since it suppresses the function of calcineurin in the wild type (Fig. 1). However, interestingly, yeast cells lacking CMK2 were sensitive to calcium stress in the presence of CsA as compared to the wild type (Fig. 1). This suggests that the calcium tolerance due to deletion of CMK2 requires the presence of a functional calcineurin.

Calcium tolerance of cmk2 cells requires the calcium/calcineurin signalling in response to high levels of extracellular calcium

As homologs of mammalian Cam Kinase II, Cmk2 and its paralog Cmk1 are calmodulin-dependent protein kinases (CaMK) that play a role in stress response in S. cerevisiae [35, 36]. The yeast CaMK, encoded by both CMK1 and CMK2, and the calcineurin act independently in promoting survival of pheromone-induced growth arrest [35]. However, deletion of CMK1 did not lead to a calcium-tolerant phenotype of the wild type BY4743 cells as deletion of CMK2, neither did affect the calcium-tolerant phenotype of yeast cells lacking CMK2 (Fig. 2a). This indicates only Cmk2, but not Cmk1, is involved in the response of yeast cells to high levels of external calcium. To examine if the calcium-tolerant phenotype of yeast cells lacking CMK2 is related to the calcium/calcineurin signalling pathway, we constructed double-gene deletion mutants between CMK2 and genes encoding various components of the calcium/calcineurin signalling pathway.

Fig. 2
figure 2

Phenotypes of the wild type BY4741, the single-gene deletion mutant for CMK2 and double-gene deletion mutants between CMK2 and one of other genes indicated. Strains were grown at 30 °C in liquid YPD overnight, serially diluted by 10 times and spotted on YPD plates with or without reagents indicated. Plates were incubated for 2 to 3 days at 30 °C

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Although deletion of CNB1 causes yeast cells to be calcium-tolerant in the haploid BY4741 background as in the diploid BY4743 background, further deletion of CNB1 led to a calcium-sensitive phenotype for yeast cells lacking CMK2 (Fig. 2b). In contrast, deletion of VCX1 did not have an impact on the calcium sensitivity of the wild type BY4741 cells harboring a functional calcineurin, and neither did on the calcium-tolerant phenotype for yeast cells lacking CMK2 (Fig. 2b and c). Like in yeast cells lacking CMK2, further deletion of CNB1 also led to a calcium-sensitive phenotype for yeast cells lacking VCX1 (Fig. 2c). As expected [10], deletion of CRZ1 alone led to a hypersensitivity of BY4741 cells to calcium stress (Fig. 2b). Yeast cells lacking both CRZ1 and CMK2 showed a hypersensitive phenotype on YPD plate containing 0.4 M CaCl2 with a similar degree to cells lacking CRZ1 alone. In contrast, yeast cells lacking both CRZ1 and CMK2 showed a sensitive phenotype on YPD plate containing 0.2 M CaCl2 in a less degree than cells lacking CRZ1 alone (Fig. 2d). However, all these phenotypes could be suppressed by the specific inhibitor of calcineurin, CsA (Fig. 2d). This indicates that Cmk2 and Crz1 have independent and opposite functions in the calcium sensitivity of yeast cells. Taken together, our data suggest that the calcium tolerance due to deletion of CMK2 is dependent on Crz1 under high concentrations of calcium, but is independent of Crz1 under lower concentrations of calcium.

Deletion of CMK2 leads to increased expression of PMR1 and PMC1 due to activation of the calcium/calcineurin signalling

Next, we examined if deletion of CMK2 would activate the calcium/calcineurin signalling. We examined the calcium-dependent response element (CDRE)-lacZ activity in the wild type and the cmk2 mutant. As compared to the wild type, the CDRE-lacZ activity in the cmk2 mutant was significantly increased in the absence or presence of 0.2 M CaCl2 (Fig. 3a). This suggests that deletion of CMK2 elevates the activation levels of the calcium/calcineurin signaling independent of calcium stress.

Fig. 3
figure 3

Deletion of CMK2 increases the activation level of calcium/calcineurin signaling and the expression level of PMR1. Galactosidase activities of 4xCDRE-lacZ (a and d), PMR1-lacZ (b) or PMC1-lacZ (c) in the wild-type (WT), the cmk2 mutant and the crz1 mutant growing in log phase. Symbols # and * show statistically significant differences (P < 0.05) between the wild type and each of the two mutants in the absence or presence of 0.2 M CaCl2, respectively. Values were means of six independent experiments

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Activation of calcium/calcineurin signalling induces the expression of PMR1 and PMC1, but not VCX1 [21, 34, 37]. Next, we examined the expression of PMR1 and PMC1 using their lacZ reporters in the wild type, the crz1 mutant and the cmk2 mutant. The lacZ activities of PMR1 and PMC1 were induced in the wild type in response to 0.2 M CaCl2 (Fig. 3b and c). This is consistent with the previous observation [34]. In the absence of 0.2 M CaCl2, there was no significant difference in the lacZ activities of PMR1 or PMC1 between the wild type and the crz1 mutant, but there was a significant increase in the lacZ activities of PMR1 and PMC1 in the cmk2 mutant as compared to the wild type (Fig. 3b and c).

In the presence of 0.2 M CaCl2, there was a significant decrease in the lacZ activities of both PMR1 or PMC1 in the crz1 mutant as compared to the wild type (Fig. 3b and c). In contrast, there was a significant increase in the lacZ activity of both PMR1 and PMC1, in the cmk2 mutant as compared to the wild type (Fig. 3b and c). Therefore, deletion of CMK2 increase the expression level of both PMR1 and PMC1.

Enhanced CDRE-lacZ activity due to deletion of CMK2 is dependent on Crz1

As we described above, expression of both CDRE-lacZ and PMR1-lacZ reporters was increased in the absence of added calcium in the cmk2 mutant as compared to the wild type (Fig. 3a and b). To examine if this effect is due to enhanced Crz1 activity, we measured the activity of CDRE-lacZ in the crz1, the cmk2 and the crz1/cmk2 mutants. As expected, in the absence or presence of 0.2 M CaCl2, the activity of CDRE-lacZ was almost abolished in both the crz1 mutant and the crz1/cmk2 mutant, albeit increased in the cmk2 mutant (Fig. 3d). In addition, the activity of CDRE-lacZ was significantly lower in either the crz1 mutant or the crz1/cmk2 mutant than the wild type strain in the absence of added calcium, although no significant difference in the activity of CDRE-lacZ was observed between the crz1 mutant and the crz1/cmk2 mutant in the absence or presence of 0.2 M CaCl2 (Fig. 3d). Taken together, these data suggest that the enhanced CDRE-lacZ activity in the cmk2 mutant was dependent on Crz1 in the absence or presence of calcium stress and that a basal level of Crz1 activity is present in yeast cells growing in YPD medium.

Effect of Cmk2 on the CDRE-lacZ activity is dependent on its catalytic activity

We examined if the catalytic activity of the protein kinase Cmk2 is responsible for its suppressive effect on the CDRE-lacZ activity. The critical lysine residue in ScCmk2 was identified from the amino acid alignment between S. cerevisiae ScCmk2, Schizosaccharomyces pombe SpCmk2 [38] and S. cerevisiae ScRck2 [26, 39] (Additional file 1: Figure S1). We first constructed the plasmids pHAC111-CMK2 and pHAC111-CMK2M, expressing the wild type Cmk2 and its kinase-dead point mutant (K76A) variant of Cmk2. Introduction of pHAC111-CMK2, but not pHAC111-CMK2M, partially suppressed the calcium-tolerant phenotype of the cmk2 mutant (Fig. 4a). This suggests that the catalytic activity of Cmk2 is required for the function of Cmk2 in the sensitivity of yeast cells to high levels of extracellular calcium stress.

Fig. 4
figure 4

Effects of Cmk2 on both calcium sensitivity and CDRE-lacZ activity are dependent on its catalytic activity. (a), cells of the wild-type BY4741 and the cmk2 mutant carrying the pHAC111 vector, pHAC111-CMK2 or pHAC111-CMK2M were grown at 30 °C in liquid SD-LEU medium overnight, serially diluted by 10 times and spotted on YPD plates with or without 0.6 M CaCl2, respectively. Plates were incubated for 2 to 3 days at 30 °C. (b), Galactosidase activities of 4xCDRE-lacZ in the wild-type (WT) BY4741and the cmk2 mutant, carrying the pHAC111 vector, pHAC111-CMK2 or pHAC111-CMK2M as indicated. They were grown at 30 °C in liquid SD-LEU medium overnight before they were inoculated to, and grown in, YPD medium to log phase. Cells were further grown in the absence or presence of 0.2 M CaCl2 for 2 h before they were collected for protein extraction and lacZ activity assay. Symbols # and * show statistically significant differences (P < 0.05) between the wild type and each of other strains in the absence or presence of 0.2 M CaCl2, respectively. Values were means of six independent experiments

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Next, we introduced the vector pHAC111 to the wild type BY4741 with the CDRE-lacZ reporter and the cmk2 mutant with the CDRE-lacZ reporter, respectively. In addition, pHAC11-CMK2 and pHAC11-CMK2M were introduced into the cmk2 mutant strain with the CDRE-lacZ reporter, respectively. As expected, in the absence or presence of 0.2 M CaCl2, the cmk2 mutant carrying the pHAC111 vector showed a higher CDRE-lacZ activity than the wild type carrying pHAC111 (Fig. 4b). Introduction of pHAC11-CMK2, but not pHAC11-CMK2M, suppressed the enhanced effect of cmk2 deletion on the CDRE-lacZ activity in the absence or presence of 0.2 M CaCl2 (Fig. 4b). This indicates that the negative effect of Cmk2 on the CDRE-lacZ activity is dependent on its kinase activity.

Discussion

Through a chemical genetic screen, we have identified five genes, CNB1, RCN1, ORM2, SIF2 and CMK2, that are involved in the sensitivity of S. cerevisiae cells to high levels of extracellular calcium. Two genes, both CNB1 and RCN1, encode regulators of calcineurin. Calcineurin not only induces the expression of PMC1 and PMR1 through the action of the transcription factor Crz1, but also inhibits in a Crz1-independent fashion the activity of Vcx1, the high capacity, low affinity vacuolar Ca2+ exchanger [37, 40]. The calcium-tolerance of yeast cells lacking CNB1 is likely due to the release of the inhibitory effect of calcineurin on Vcx1 activity, which is supported by our observation that further deletion of VCX1 reversed the phenotype of cells lacking CNB1 to be hypersensitive to calcium stress with a similar degree to that of cells lacking VCX1 in the presence of CsA (Fig. 2c).

ORM2 encodes a regulator of the sphingolipid homeostasis. Our finding is consistent with previous observations that sphingolipids play roles in calcium homeostasis, protein trafficking/exocytosis, longevity and cellular aging, nutrient uptake, and the interaction of sphingolipids and antifungal drugs in S. cerevisiae [41]. In mammalian cells, sphingosine 1-phosphate is a signalling lipid known to be involved in calcium homeostasis [42,43,44]. SIF2 encodes the WD40 repeat-containing subunit of Set3C histone deacetylase (HDAC) complex [45]. The Set3C binds histone H3 dimethylated at lysine 4 to mediate deacetylation of histones in 5′-transcribed regions [46]. Interestingly, the chaperone mammalian relative of DnaJ (Mrj) decreases the occupancy of nuclear factor of activated T cells (NFAT), the mammalian functional counterpart of yeast Crz1, on the tumor necrosis factor-alpha promoter in cardiomyocytes in an HDAC-dependent manner [47]. A similar role might exist for Set3C on the regulation of Crz1 in gene transcription in response to calcium stress.

CMK2 encodes a calmodulin-dependent protein kinase, and expression of CMK2 is induced by the calcium/calcineurin signalling pathway [48, 49]. Yeast cells lacking CMK2 are tolerant to high levels of external calcium, while cells lacking the CRZ1 are calcium-sensitive. Therefore, Cmk2 and Crz1 have opposite functions in the calcium sensitivity of yeast cells. In addition, we show here that deletion of CMK2 elevates the calcium/calcineurin signalling and increases the expression level of PMR1 and PMC1 in response to calcium stress (Fig. 3). The effects of Cmk2 on calcium sensitivity and calcium/calcineurin signalling are dependent on its catalytical activity (Fig. 4). Taken together, these data suggest that Cmk2 functions as a negative feedback controller for the calcium/calcineurin signalling pathway, which is mediated by Crz1 through calcineurin in response to high levels of extracellular calcium stress (Fig. 5). How Cmk2 regulates the calcium signalling pathway remains to be determined. A plausible possibility would be that Cmk2 directly phosphorylates and negatively regulates calcineurin or Crz1 (Fig. 5). In contrast, Rcn1 is a feedback controller of calcineurin with dual effects [48, 50]. Another possibility is that Cmk2 indirectly affects Set3C, which in turn regulates the activity of Crz1.

Fig. 5
figure 5

Mode of Cmk2 function in the feedback regulation of calcium/calcineurin signaling pathway. In response to high levels of extracellular calcium, Ca2+ enters the cell through unknown pathways, and both calcineurin and Crz1 are activated to induce expression of target genes including CMK2, RCN1, PMR1 and PMC1 [50]. Arrows indicate activation, and T bars represent inhibition. A dashed line means that more data is needed to confirm

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Interestingly, we have observed that the cmk2 crz1 double deletion mutant is more tolerant to 0.2 M CaCl2, albeit not to a higher level of calcium (0.4 M CaCl2), than the crz1 deletion mutant (Fig. 2d). This indicates that Cmk2 has an additional function in calcium tolerance, which is independent of Crz1. However, supplementation of CsA in the medium suppresses the calcium sensitivity of both the crz1 and cmk2/crz1 mutants at both 0.2 M CaCl2 and 0.4 M CaCl2 (Fig. 2d). This indicates that the additional Crz1-independent function of Cmk2 is dependent on calcineurin function (Fig. 5). This additional function could be mediated by the Vcx1, whose activity can be inhibited by calcineurin [34]. Nevertheless, the direct phosphorylation and thereby regulation of Vcx1 activity by Cmk2 could not be excluded. This might also explain what we have observed that higher promoter activities of CDRE-lacZ and PMR1-lacZ in the cmk2 mutant than those in the wild type strain under normal growth conditions (Fig. 3), when the calcium/calcineurin signaling pathway is not activated.

Abbreviations

CaMK:

Calmodulin-dependent protein kinase

CDRE:

Calcineurin dependent response element

CsA:

Cyclosporine A

ER:

Endoplasmic reticulum

NFAT:

Nuclear factor of activated T cells

PCR:

Polymerase chain reaction

YPD:

Yeast peptone dextron

References

  1. Tang RJ, Luan S. Regulation of calcium and magnesium homeostasis in plants: from transporters to signaling network. Curr Opin Plant Biol. 2017;39:97–105.

    Article  CAS  Google Scholar 

  2. Bond R, Ly N, Cyert MS. The unique C terminus of the calcineurin isoform CNAβ1 confers non-canonical regulation of enzyme activity by Ca2+ and calmodulin. J Biol Chem. 2017;292:16709–21.

    Article  CAS  Google Scholar 

  3. Espeso EA. The CRaZy calcium cycle. Adv Exp Med Biol. 2016;892:169–86.

    Article  CAS  Google Scholar 

  4. Plattner H, Verkhratsky A. The ancient roots of calcium signalling evolutionary tree. Cell Calcium. 2015;57:123–32.

    Article  CAS  Google Scholar 

  5. Medler KF. Calcium signaling in taste cells: regulation required. Chem Senses. 2010;35:753–65.

    Article  CAS  Google Scholar 

  6. Serra-Cardona A, Canadell D, Arino J. Coordinate responses to alkaline pH stress in budding yeast. Microb Cell. 2015;2:182–96.

    Article  CAS  Google Scholar 

  7. Cui J, Kaandorp JA, Sloot PM, Lloyd CM, Filatov MV. Calcium homeostasis and signaling in yeast cells and cardiac myocytes. FEMS Yeast Res. 2009;9:1137–47.

    Article  CAS  Google Scholar 

  8. Colinet AS, Thines L, Deschamps A, Flemal G, Demaegd D, Morsomme P. Acidic and uncharged polar residues in the consensus motifs of the yeast Ca2+ transporter Gdt1p are required for calcium transport. Cell Microbiol. 2017;19(7). https://doi.org/10.1111/cmi.12729.

  9. Cyert MS, Philpott CC. Regulation of cation balance in Saccharomyces cerevisiae. Genetics. 2013;193:677–713.

    Article  CAS  Google Scholar 

  10. Cyert MS. (2003) Calcineurin signaling in Saccharomyces cerevisiae: how yeast go crazy in response to stress. Biochem. Biophys. Res. Commun. 2003;311:1143–50.

    CAS  Google Scholar 

  11. Jiang L, Xu D, Hameed A, Fang T, Bakr Ahmad Fazili A, Asghar F. The plasma membrane protein Rch1 and the Golgi/ER calcium pump Pmr1 have an additive effect on filamentation in Candida albicans. Fung Genet Biol. 2018;115(1–8).

  12. Zhao Y, Yan H, Happeck R, Peiter-Volk T, Xu H, Zhang Y, Peiter E, van Oostende Triplet C, Whiteway M, Jiang L. The plasma membrane protein Rch1 is a negative regulator of cytosolic calcium homeostasis and positively regulated by the calcium/calcineurin signaling pathway in budding yeast. Eur J Cell Biol. 2016;95:164–74.

    Article  CAS  Google Scholar 

  13. Xu D, Cheng J, Cao C, Wang L, Jiang L. Genetic interactions between Rch1 and the high-affinity calcium influx system Cch1/Mid1/Ecm7 in the regulation of calcium homeostasis, drug tolerance, hyphal development and virulence in Candida albicans. FEMS Yeast Res 2015;15(7). pii: fov079.

  14. Alber J, Jiang L, Geyer J. CaRch1p does not functionally interact with the high-affinity Ca2+ influx system (HACS) of Candida albicans. Yeast. 2013;30:449–57.

    Article  CAS  Google Scholar 

  15. Jiang L, Alber J, Wang J, Du W, Li X, Geyer J. The Candida albicans plasma membrane protein Rch1p a member of the vertebrate SLC10 carrier family, is a novel regulator of cytosolic Ca2+ homoeostasis. Biochem J. 2012;444:497–502.

    Article  CAS  Google Scholar 

  16. Jiang L, Wang L, Fang T, Papadopoulos V. Disruption of ergosterol and tryptophan biosynthesis, as well as cell wall integrity pathway and the intracellular pH homeostasis, lead to mono-(2-ethylhexyl)-phthalate toxicity in budding yeast. Chemosphere. 2018;206:643–54.

    Article  CAS  Google Scholar 

  17. Jiang L, Cao C, Zhang L, Lin W, Xia J, Xu H, Zhang Y. Cadmium-induced activation of high osmolarity glycerol pathway through its Sln1 branch is dependent on the MAP kinase kinase kinase Ssk2, but not its paralog Ssk22, in budding yeast. FEMS Yeast Res. 2014;14:1263–72.

    Article  CAS  Google Scholar 

  18. Luo, C, Cao C, Jiang L. The endosomal sorting complex required for transport (ESCRT) is required for the sensitivity of yeast cells to nickel ions in Saccharomyces cerevisiae. FEMS Yeast Res 2016;16(3). pii: fow028.

  19. Du J, Cao C, Jiang L. Genome-scale genetic screen of lead ion-sensitive gene deletion mutations in Saccharomyces cerevisiae. Gene. 2015;563:155–9.

    Article  CAS  Google Scholar 

  20. Martin DC, Kim H, Mackin NA, Maldonado-Báez L, Evangelista CC Jr, Beaudry VG, Dudgeon DD, Naiman DQ, Erdman SE, Cunningham KW. New regulators of a high affinity Ca2+ influx system revealed through a genome-wide screen in yeast. J Biol Chem. 2011;286:10744–54.

    Article  CAS  Google Scholar 

  21. Zhao Y, Du J, Zhao G, Jiang L. Activation of calcineurin is mainly responsible for the calcium sensitivity of gene deletion mutations in the genome of budding yeast. Genomics. 2013;101:49–56.

    Article  Google Scholar 

  22. Zhang L, Liu N, Ma X, Jiang L. The transcriptional control machinery as well as the cell wall integrity and its regulation are involved in the detoxification of the organic solvent dimethyl sulfoxide in Saccharomyces cerevisiae. FEMS Yeast Res. 2013;13:200–18.

    Article  Google Scholar 

  23. Zhao J, Lin W, Ma X, Lu Q, Ma X, Bian G, Jiang L. The protein kinase Hal5p is the high-copy suppressor of lithium-sensitive mutations of genes involved in sporulation and meiosis as well as ergosterol biosynthesis in Saccharomyces cerevisiae. Genomics. 2010;95:290–8.

    Article  CAS  Google Scholar 

  24. Xiong B, Zhang L, Xu H, Yang Y, Jiang L. Cadmium induces the activation of cell wall integrity pathway in budding yeast. Chem Biol Interact. 2015;240:316–23.

    Article  CAS  Google Scholar 

  25. Xu H, Whiteway H, Jiang L. The tricarboxylic acid cycle, cell wall integrity pathway, cytokinesis and intracellular pH homeostasis are involved in the sensitivity of Candida albicans cells to high levels of extracellular calcium. Genomics. 2018. https://doi.org/10.1016/j.ygeno.2018.08.001.

  26. Jiang L, Niu S, Clines KL, Burke DJ, Sturgill TW. Analyses of the effects of Rck2p mutants on Pbs2pDD-induced toxicity in Saccharomyces cerevisiae identify a MAP kinase docking motif, and unexpected functional inactivation due to acidic substitution of T379. Mol Genet Genomics. 2004;271:208–19.

    Article  CAS  Google Scholar 

  27. Araki Y, Wu H, Kitagaki H, Akao T, Takagi H, Shimoi H. Ethanol stress stimulates the Ca2+-mediated calcineurin/Crz1 pathway in Saccharomyces cerevisiae. J Biosci Bioeng. 2009;10:71–6.

    Google Scholar 

  28. Zhao Y, Du J, Xiong B, Xu H, Jiang L. ESCRT components regulate the expression of the ER/Golgi calcium pump gene PMR1 through the Rim101/Nrg1 pathway in budding yeast. J Mol Cell Biol. 2013;5:336–44.

    Article  CAS  Google Scholar 

  29. Jiang L, Wang J, Asghar F, Snyder N, Cunningham KW. CaGdt1 plays a compensatory role for the calcium pump CaPmr1 in the regulation of calcium signaling and cell wall integrity signaling in Candida albicans. Cell Commun Signal. 2018c;16:33.

    Article  Google Scholar 

  30. Zhao Y, Xiong B, Xu H, Jiang L. Expression of NYV1 encoding the negative regulator of Pmc1 activity is repressed by two transcriptional repressors Nrg1 and Mig1. FEBS Lett. 2014;588:3195–201.

    Article  CAS  Google Scholar 

  31. Yan H, Zhao Y, Jiang L. The putative transcription factor CaRtg3 is involved in tolerance to cations and antifungal drugs as well as serum-induced filamentation in Candida albicans. FEMS Yeast Res. 2014;14:614–23.

    Article  CAS  Google Scholar 

  32. Hilioti Z, Gallagher DA, Low-Nam ST, Ramaswamy P, Gajer P, Kingsbury TJ, Birchwood CJ, Levchenko A, Cunningham KW. GSK-3 kinases enhance calcineurin signaling by phosphorylation of RCNs. Genes Dev. 2004;18:35–47.

    Article  CAS  Google Scholar 

  33. 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;124:351–63.

    Article  CAS  Google Scholar 

  34. Cunningham KW, Fink GR. Calcineurin inhibits VCX1-dependent H+/Ca2+ exchange and induces Ca2+ ATPases in Saccharomyces cerevisiae. Mol Cell Biol. 1996;16:2226–22237.

    Article  CAS  Google Scholar 

  35. Moser MJ, Geiser JR, Davis TN. Ca2+-calmodulin promotes survival of pheromone-induced growth arrest by activation of calcineurin and Ca2+-calmodulin-dependent protein kinase. Mol Cell Biol. 1996;16:4824–31.

    Article  CAS  Google Scholar 

  36. Pausch MH, Kaim D, Kunisawa R, Admon A, Thorner J. Multiple Ca2+/calmodulin-dependent protein kinase genes in a unicellular eukaryote. EMBO J. 1991;10:1511–22.

    Article  CAS  Google Scholar 

  37. Stathopoulos AM, Cyert MS. Calcineurin acts through the CRZ1/TCN1-encoded transcription factor to regulate gene expression in yeast. Genes Dev. 1997;11:3432–44.

    Article  CAS  Google Scholar 

  38. Alemany V, Sanchez-Piris M, Bachs O, Aligue R. Cmk2, a novel serine/threonine kinase in fission yeast. FEBS Lett. 2002;524:79–86.

    Article  CAS  Google Scholar 

  39. Li X, Huang X, Zhao J, Zhao J, Wei Y, Jiang L. The MAP kinase-activated protein kinase Rck2p plays a role in rapamycin sensitivity in Saccharomyces cerevisiae and Candida albicans. FEMS Yeast Res. 2008;8:715–24.

    Article  CAS  Google Scholar 

  40. Matheos DP, Kingsbury TJ, Ahsan US, Cunningham KW. Tcn1p/Crz1p, a calcineurin-dependent transcription factor that differentially regulates gene expression in Saccharomyces cerevisiae. Genes Dev. 1997;11:3445–58.

    Article  CAS  Google Scholar 

  41. Dickson RC, Sumanasekera C, Lester RL. Functions and metabolism of sphingolipids in Saccharomyces cerevisiae. Prog Lipid Res. 2006;45:447–65.

    Article  CAS  Google Scholar 

  42. Rahar B, Chawla S, Pandey S, Bhatt AN, Saxena S. Sphingosine-1-phosphate pretreatment amends hypoxia-induced metabolic dysfunction and impairment of myogenic potential in differentiating C2C12 myoblasts by stimulating viability, calcium homeostasis and energy generation. J Physiol Sci. 2018;68:137–51.

    Article  CAS  Google Scholar 

  43. Keul P, van Borren MM, Ghanem A, Müller FU, Baartscheer A, Verkerk AO, Stümpel F, Schulte JS, Hamdani N, Linke WA, van Loenen P, Matus M, Schmitz W, Stypmann J, Tiemann K, Ravesloot JH, Alewijnse AE, Hermann S, Spijkers LJ, Hiller KH, Herr D, Heusch G, Schäfers M, Peters SL, Chun J, Levkau B. Sphingosine-1-phosphate receptor 1 regulates cardiac function by modulating Ca2+ sensitivity and Na+/H+ exchange and mediates protection by ischemic preconditioning. J Am Heart Assoc 2016;5(5). pii: e003393.

  44. Ihlefeld K, Claas RF, Koch A, Pfeilschifter JM, Meyer Zu Heringdorf D. Evidence for a link between histone deacetylation and Ca2+ homoeostasis in sphingosine-1-phosphate lyase-deficient fibroblasts. Biochem J. 2012;447:457–64.

    Article  CAS  Google Scholar 

  45. Pijnappel WW, Schaft D, Roguev A, Shevchenko A, Tekotte H, Wilm M, Rigaut G, Séraphin B, Aasland R, Stewart AF. The S. cerevisiae SET3 complex includes two histone deacetylases, Hos2 and Hst1, and is a meiotic-specific repressor of the sporulation gene program. Genes Dev. 2001;15:2991–3004.

    Article  CAS  Google Scholar 

  46. Kim T, Xu Z, Clauder-Münster S, Steinmetz LM, Buratowski S. Set3 HDAC mediates effects of overlapping noncoding transcription on gene induction kinetics. Cell. 2012;150:1158–69.

    Article  CAS  Google Scholar 

  47. Dai YS, Xu J, Molkentin JD. The DnaJ-related factor Mrj interacts with nuclear factor of activated T cells c3 and mediates transcriptional repression through class II histone deacetylase recruitment. Mol Cell Biol. 2005;25:9936–48.

    Article  CAS  Google Scholar 

  48. Cunningham KW. Acidic calcium stores of Saccharomyces cerevisiae. Cell Calcium. 2011;50:129–38.

    Article  CAS  Google Scholar 

  49. Dudgeon DD, Zhang N, Ositelu OO, Kim H, Cunningham KW. Nonapoptotic death of Saccharomyces cerevisiae cells that is stimulated by Hsp90 and inhibited by calcineurin and Cmk2 in response to endoplasmic reticulum stresses. Eukaryot Cell. 2008;7:2037–51.

    Article  CAS  Google Scholar 

  50. Mehta S, Li H, Hogan PG, Cunningham KW. Domain architecture of the regulators of calcineurin (RCANs) and identification of a divergent RCAN in yeast. Mol Cell Biol. 2009;29:2777–93.

    Article  CAS  Google Scholar 

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Acknowledgements

The author is grateful to Yoshio Araki for kindly providing reagents. This work was carried out with technical support from Yunying Zhao, Yan Zhang, Jingcai Du, Jingwen Zhao, Bing Xiong and Chunlei Cao, and financially supported by grants from the National Natural Science Foundation of China to LJ (No. 81571966 and No. 81371784).

Funding

This work was funded by the National Natural Science Foundation of China to LJ (No. 81571966 and No. 81371784).

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  1. Huihui Xu, Tianshu Fang and Hongbo Yan contributed equally to this work.

Authors and Affiliations

  1. Laboratory for Yeast Molecular and Cell Biology, School of Agricultural Engineering and Food Science, Shandong University of Technology, Zibo, Shandong, China

    Huihui Xu, Tianshu Fang, Hongbo Yan & Linghuo Jiang

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  1. Huihui Xu
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HX, TF and HY carried out experiments and analyzed the data, LJ designed the study and wrote the manuscript and analyzed the data. All authors read and approved the final manuscript.

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Additional file

Additional file 1:

Figure S1. Amino acid sequence comparison between ScCMK2, SpCMK2 and ScRCK2. (PDF 88 kb)

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Xu, H., Fang, T., Yan, H. et al. The protein kinase Cmk2 negatively regulates the calcium/calcineurin signalling pathway and expression of calcium pump genes PMR1 and PMC1 in budding yeast. Cell Commun Signal 17, 7 (2019). https://doi.org/10.1186/s12964-019-0320-z

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Keywords

Cell Communication and Signaling

ISSN: 1478-811X