To identify other potential Cas5 phosphorylation sites, we analyzed its amino acid sequence using NetPhos and followed up on S769, a highly conserved residue in one of the zinc finger domains with a phosphorylation site-prediction score of 0.980 (Fig. 6a)38, 39. To assess the functional significance of this residue, we engineered strains in which one allele of CAS5 was deleted and the other was replaced with a wild-type allele, the phosphomimetic S769E allele, or the phosphodeficient S769A allele. Introduction of the phosphomimetic S769E allele as the only source of CAS5 phenocopied homozygous deletion of CAS5 in terms of hypersensitivity to caspofungin, whereas introduction of the phosphodeficient S769A allele conferred a wild-type phenotype (Fig. 6b). Consistent with our hypothesis, the S769E mutation blocked the upregulation of ECM331 and PGA13 in response to caspofungin, whereas the S769A mutation did not (Fig. 6c). Notably, these mutations did not affect Cas5 induction or dephosphorylation in response to caspofungin (Fig. 6d and Supplementary Fig. 2f), suggesting that the S769E substitution does not affect the capacity of Cas5 to sense cell wall stress, and that additional phosphorylation sites remain to be identified. Our data supports a model in which Cas5 is activated by dephosphorylation in response to cell wall stress.Fig. 6The S769E substitution in the Cas5 DNA-binding domain phenocopies CAS5 deletion. a Schematic showing the position of S769 in the Cas5 zinc finger domain and the alignment of Cas5 orthologs in related fungal species. The alignment was performed using the Candida Genome Database (CGD), and the phosphorylation site-prediction score was generated using NetPhos. b The phosphomimetic S769E substitution in Cas5 confers hypersensitivity to caspofungin. Caspofungin susceptibility assays were conducted in YPD medium. Growth was measured by absorbance at 600 nm after 48 h at 30 °C. Optical densities were averaged for duplicate measurements. Data was quantitatively displayed with color using Treeview (see color bar in Fig. 2). c The S769E substitution blocks induction of cell wall genes in response to caspofungin treatment. The transcript levels of ECM331 and PGA13 were monitored by qRT-PCR and normalized to GPD1. Plotted are the fold-changes in transcript levels of ECM331 or PGA13 in response to caspofungin relative to untreated. Error bars represent standard deviation (s.d.) from the mean of triplicate samples. The fold change in gene expression for each mutant strain was compared with the wild-type strain using one-way ANOVA in GraphPad Prism (**P < 0.01). d Phosphomutations in CAS5 do not affect band shifts associated with activation of the cell wall stress response, as observed upon caspofungin treatment. Cas5 was monitored by western blot and detected using an α-HA antibody. Full blots are shown in Supplementary Fig. 2f