Fig. 3. PKAc^W196G is differentially compartmentalized in adrenal cells.

(A) Diagram depicts biotin-labeled proteins surrounding PKAc-miniTurbo in live cells and the final isolated peptides after streptavidin capture and trypsin digest. Labels: C, PKAc; Trb, miniTurbo. (B) Immunoblot of lysates from stable PKAc-miniTurbo H295R cell lines upon 48 hours of doxycycline induction and 2 hours of biotin incubation. NeutrAvidin-HRP labels biotinylated proteins. Expression of each PKAc-miniTurbo variant is indicated above each lane. Representative of four biological replicates. (C and D) Volcano plots of proximity proteomics for PKAc-W196G versus WT PKAc (C) and PKAc-W196G versus PKAc-L205R (D). Proteins underrepresented (left) and enriched (right) in the PKAc^W196G proximity proteome. Gray dots indicate proteins with a corrected P value lower than 0.05. Colored dots indicate RII-selective (teal) and RI-recruiting (yellow) complex components. Data from four biological replicates. (E) STRING network depiction of proteins with increased PKAc^W196G association versus WT PKAc. (F) Selected gene ontology cell component enrichment scores for highly disparate categories between WT PKAc (gray) and PKAc-W196G (green). (G) Left: PKAc-W196G gene ontology enrichment for major cell compartments and organelles relative to WT PKAc. Right: Schematic depiction of results in a prototypic cell. (H) PKAc-W196G association with PKA regulatory components relative to WT PKAc. (I) PKAc-W196G association with AKAPs relative to WT PKAc. Colors indicate likely RII-selective (teal) and RI-recruiting (yellow) AKAPs.