Fig. 7.

Phosphorylation of casp-9 by PKA at S183 results in destabilization similar to S183E. a Mature, cleaved forms of casp-9 unphosphorylatable alanine variants were incubated with PKA and ATP to direct phosphorylation specifically to S99 and S195 or to S183. No impact on mobility was observed upon Coomassie-stained denaturing SDS PAGE. b Mobility shifts of unphosphorylated (no ATP) and phosphorylated (+ ATP) S183A or S99A/S195A casp-9 following non-denaturing native agarose gel electrophoresis after 15 min or 1 h of phosphorylation. After 1 h of phosphorylation, S99A/S195A (conditions promoting S183 phosphorylation) exhibited a drastic shift in migration, similar to what was observed in cleaved S183E, indicating that S183 phosphorylation destabilizes casp-9 leading to a shift in the conformation