Figure 3.

Rab32 can interact with the holoenzyme of PKA in mammalian cells. HEK-293 cells were transiently transfected with Flag–Rab32 or Flag–Rab32L188P cDNA. Triton X-100–soluble extracts were immunoprecipitated with Sepharose-conjugated antiFlag antibody. (A) Copurification of the PKA holoenzyme was measured by assaying for PKA catalytic subunit activity stimulated by exogenous cAMP. PKA activity was measured as pmol/min/mg of ³²P incorporated into the PKA substrate kemptide using a filter paper binding assay (Corbin and Reimann, 1974). Specific PKA activity was blocked by 10 μM of the inhibitor PKI (5–24) peptide. (B) AntiFlag immunoprecipitates were separated on SDS-PAGE after the catalytic subunit had been eluted from the complex with exogenous cAMP and electrotransferred to nitrocellulose. RII overlay assays (top) were performed to determine if Rab32 was the only AKAP present in these fractions. Control experiments confirmed that equal levels of protein were immunoprecipitated in these experiments (bottom). A representative example of three independent experiments is shown.