When monitoring Cas5 protein levels, we observed that the Cas5 protein demonstrated a downward band shift when cells were exposed to cell wall antagonists, indicative of a post-translational modification (Fig. 5e and Supplementary Fig. 2c). To confirm this shift was a consequence of cell wall stress, we employed a strain in which we could specifically inhibit the kinase activity of the cell wall integrity regulator Pkc135. In this strain, the only allele of PKC1 carries an M850G gatekeeper mutation, rendering the kinase susceptible to inhibition by ATP analog 1-naphthyl-PP1 (1-NA-PP1), without affecting its kinase activity in the absence of the inhibitor35, 36. In the presence of 1-NA-PP1, the gatekeeper mutant was hypersensitive to caspofungin, similar to a pkc1Δ/pkc1Δ mutant (Supplementary Fig. 3b). Inhibition of Pkc1 kinase activity induced Cas5 protein levels and resulted in a downward mobility shift (Fig. 5f and Supplementary Fig. 2d), similar to what was observed in a wild-type strain treated with caspofungin (Fig. 5d). Given that a change in protein phosphorylation is a common mechanism to regulate transcription factor activity37, we assessed whether Cas5 was modified by phosphorylation. We performed two-dimensional gel electrophoresis coupled to western blot analysis. In the absence of stress, Cas5-HA resolved into a heterogeneous population of differentially charged species (Fig. 5g). In response to caspofungin, Cas5 collapsed into a less electronegative species, consistent with protein dephosphorylation. To confirm that Cas5 was phosphorylated under basal conditions, we monitored Cas5 mobility by western blot analysis following lambda phosphatase treatment. Phosphatase treatment led to a faster migrating Cas5 band, consistent with Cas5 being a phosphoprotein (Fig. 5h and Supplementary Fig. 2e). Finally, to determine if dephosphorylation of Cas5 is coupled to the activation of gene expression, we monitored transcript levels of ECM331 and PGA13, two Cas5-dependent caspofungin-responsive cell wall genes (Supplementary Data 4)22. ECM331 and PGA13 were upregulated in wild-type cells with the same caspofungin exposure required to induce Cas5 dephosphorylation, and this upregulation was partially blocked by deletion of CAS5 (Fig. 5i). Thus, Cas5 is regulated by dephosphorylation and governs the expression of caspofungin-responsive cell wall genes in response to cell wall stress.