Fig. 5.

Phosphorylation of S392 in TASK-1 inhibits 14-3-3 binding. (A) In vitro phosphorylation of recombinant GST–TASK-1 wild-type, GST–TASK-1 S392A and GST–TASK-1 S393A fusion proteins with PKA catalytic subunit. Samples were resolved on an SDS-PAGE gel containing 75 µM Phostag reagent and 75 µM MnCl₂. ‘P’ suffixes denote the number of phosphorylated residues. The gel is representative of three independent experiments. (B) 14-3-3 protein binding to the TASK-1 C-terminal trafficking control region is abolished by double phosphorylation. GST-TASK-1 or GST-TASK-3 C-terminal fusion proteins were in vitro phosphorylated using recombinant PKA catalytic subunit and an ATP regeneration system (energy mix; EM). Samples were resolved on SDS-PAGE gels (upper three panels) and on an SDS-PAGE gel containing 100 µM Phostag reagent and 100 µM MnCl₂ (bottom). The upper two panels show western blots using antibodies against the indicated proteins, and the lower two panels show Coomassie-stained gels. TASK-CT15, the last 15 amino acids of the TASK C-terminus.