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

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.

Keywords: Calcineurin; Calcium signalling; Cmk2; Crz1; Pmr1; Saccharomyces cerevisiae.

Fig. 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

Fig. 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

Fig. 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

Fig. 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

Fig. 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