Figure 5. Activation of Cek1p in the presence of calcium ionophore A23187 and calcium.

C. albicans cells growing exponentially were treated as described below for 30 minutes. Numbers below the blots in Panel A show the fold change in dually phosphorylated Cek1p in the presence of A23187 relative to the vehicle control for each strain. Lower numbers in Panels B and C indicate relative amount of dually phosphorylated Cek1p normalized to actin and shown relative to lane 3 (panel B), or cells treated with FK506 only in the absence of calcium (panel C, lane 1). Experiments were repeated 3 times; representative blots are shown. Panel A, Mutations in the calmodulin binding motif of Dfi1p affect A23187-dependent Cek1p activation. Dfi1p complemented and mutant strains were treated with 4 µM A23187 (+) or 100% ethanol (−) as a vehicle control. DFI1, DFI1-TAP; dfi1, Δdfi1 null; RK, dfi1-RKAA-TAP; ER, dfi1-EERR-TAP; WQ, dfi1-WWQQ- TAP. Panels B and C, Activation of Cek1p is calcium dependent and independent of calcineurin. Wild type cells were grown in the presence of 1 mM CaCl₂, 2 µg/mL FK506, 4 µM A23187 and/or the appropriate vehicle controls. Panel B, all lanes were from the same gel, and irrelevant lanes were removed.