Figure 4
Caspofungin controls PKA activity in a Wsc1-dependent manner. (a) Wild-type (WT) and wsc1∆ cells harboring pRS423-prCUP-6xMYC-cki1 2_200(S125/130A) plasmid were grown in YPD medium in the absence and presence of CAS for 2 h. PKA-dependent phosphorylation of Cki1 was analyzed by whole cell extraction and Western blot analysis using an anti c-myc antibody. A representative Western blot is shown (upper panel). Ratios of phosphorylated (Cki1-P) and non-phosphorylated (Cki1) forms of Cki1 in the presence of CAS relative to untreated cells are shown for each strain in the lower panel. The means and SD of three (n = 3) independent experiments are indicated. (b) The PKA-dependent phosphorylation of Pat1 was lost upon CAS treatment. Wild-type cells expressing a myc-tagged Pat1 were grown to mid-log phase in YPD medium and then treated or not with CAS (15 ng/ml) or transferred to a medium lacking glucose (YP) for the indicated time. The level of PKA phosphorylation (α-Sub) was assessed by Western blotting with the anti-PKA substrate antibody. Numbers indicate the relative amounts (quantified by densitometric analysis) of Phospho-Pat1 with respect to time zero normalized with respect to total Pat1 (α-myc). (c) Glycogen accumulation is induced in the presence of CAS. Cells of the indicated yeast strains (RAS2 val19 corresponds to the wild-type strain expressing this RAS2 allele) were grown on YPD medium in the absence or presence of CAS (15 ng/ml), Calcofluor white (CFW, 5 µg/ml), or aminocandin (AMC, 15 ng/ml) for 2 h. Cells were then collected and resuspended in a solution of 0.2% iodine/0.4% potassium iodide and, after 3 min., were spotted onto YPD plates and photographs were taken. The darker the color, the higher the amount of glycogen that was accumulated. Glycogen provides an indirect measurement of cAMP/PKA activity.
