PKA shapes mitochondria through phosphorylation of Drp1 at SerPKA.

(A, B) PC12 cells expressing mitochondrial GFP (green) and either wild-type or SPKAA-mutant Drp1 instead of endogenous Drp1 were treated ± forskolin/rolipram (25/2 µM, 3 h), fixed, and epifluorescence micrographs (representatives in A) were subjected to digital morphometry (means ± s.e.m. of ∼300 cells per condition from a representative experiment). (C–D) HeLa cells co-expressing the indicated constructs (om, outer mitochondrial) were fixed and processed for immunofluorescence for mitochondrial cytochrome oxidase II (mito, red) and GFP (green). Shown are representative images (C) and mitochondrial morphology analysis (D, means ± s.e.m. of ∼200–300 cells per condition from a representative experiment). (E–G) HeLa cells expressing WT or SPKAD-mutant GFP-Drp1 were incubated for up to 4 h with the PKA inhibitor H89 (20 µM) and analyzed by quantitative immunoblotting for phosphorylated (pDrp1, SerPKA, and SerCDK) and total Drp1 (E) or by immunofluorescence for mitochondrial morphology (F, representative image; G, means ± s.e.m. of ∼200 cells/condition from a representative experiment). For immunoblot analysis only (E), trace amounts (5% plasmid) of PKA catalytic subunit were cotransfected to increase the signal strength with the phospho-SerPKA Drp1 antibody.