FIG. 3.

Phenotypic analysis of the CBK1-T743E allele indicated that the kinase has increased activity in vitro but did not hyperactivate Ace2. (A) Cbk1-T743E was expressed at levels comparable to wild-type Cbk1. Total cell lysates prepared from wild-type, CBK1-T743E, or cbk1Δ strains were probed with an anti-Cbk1 antibody. PGK1 was used as a loading control (bottom panel). (B) Cbk1-T743E exhibited increased kinase activity in vitro. Cbk1 was immunoprecipitated from wild-type (CBK1), CBK1-T743E, and cbk1Δ strains and used in an in vitro kinase assay with a fragment of Ace2. The top panel is an immunoblot of total Cbk1. The bottom panels are autoradiographs of Cbk1 autophosphorylation (middle panel) and phosphorylation of the Ace2 fragment (bottom panel). (C) The CBK1-T743E strain has a wild-type cell separation phenotype. Wild-type (CBK1), cbk1Δ, and CBK1-T743E strains were grown to mid-log phase and imaged by differential interference contrast (DIC). The number of connected cells in a group was determined for wild-type and CBK1-T743E strains. Standard deviations are shown for three independent trials. (D) The CBK1-T743E strain does not exhibit hyperactivation of Ace2 target transcription. QPCR was performed on RNA isolated from the indicated strains. Transcription of CTS1 relative to cbk1Δ is shown. Error bars indicate standard errors of the means of five experiments. There was no significant difference between CTS1 transcript levels in CBK1 and CBK1-T743E strains (P = 0.43).