FIG. 3.

PKA regulates Crz1p activity in vivo. (A) Mutation of serines 409, 410, 423, 427, and 429 to alanine results in increased Crz1p activity. KWY251 cells (crz1Δ 4xCDRE::LacZ) expressing wild-type GFP-Crz1p (pKK249), GFP-Crz1p^{S409,410,423A} (pKK251), or GFP-Crz1p^{S409,410,423,427,429A} (pKK267) were treated with 100 mM CaCl₂ where indicated. β-Galactosidase activity was measured and is reported as the maximum rate OD₄₁₅ change/minute/microgram of protein. (B) PKA activation reduces Ca²⁺-dependent Crz1p activation. KKY436 cells (cdc35Δ pde2Δ yak1Δ 4xCDRE::LacZ) were incubated with 100 mM CaCl₂ in the absence or presence of 3 mM cAMP as indicated, and the β-galactosidase activity was determined. (C) Constitutive PKA activity results in decreased Crz1p activity. Strains ASY832 (4xCDRE::LacZ) and KKY385 (bcy1Δ 4xCDRE::LacZ) were treated with 100 mM CaCl₂ as indicated, and the β-galactosidase activity was determined.