On the basis of our observations that Cas5 function is regulated by phosphorylation, we hypothesized that dephosphorylation of key residues is important for Cas5 activation. To map the phosphorylation residues on Cas5 important for its activity, we performed immunoprecipitation coupled to mass spectrometry with HA-tagged Cas5. Phosphorylation was detected at serine residues S462 and S476 (Supplementary Fig. 4a). To test whether these serine residues along with two other adjacent residues, S464 and S472, were functionally important for Cas5 function in regulating cell wall integrity, we mutagenized all four serines to either glutamic acid, to mimic a constitutively phosphorylated state, or alanine, to mimic a constitutively unphosphorylated state. The resulting mutants displayed wild-type tolerance to caspofungin (Supplementary Fig. 4b), with no appreciable difference in the band shift upon caspofungin treatment (Supplementary Fig. 4c), suggesting that the sites identified by mass spectrometry were not sufficient to control Cas5 function.