Phosphorylation of Rap1GAP2 by cyclic nucleotide–regulated kinases. (A) COS-1 cells were transiently transfected with VSV-tagged wild-type (wt) Rap1GAP2a and mutants of Rap1GAP2a containing serine-to-alanine mutations of serine 7 and serine 549, either singly (S7A and S549A) or in combination (S7A/S549A). Expressed proteins were precipitated with anti-VSV antibody, and in vitro kinase assays were performed using γ-32P]ATP and purified cGKI or a catalytic subunit of cAK. To detect 32P incorporation, samples were separated by SDS-PAGE, blotted onto nitrocellulose, and exposed to film (32P). Then protein amounts were determined using anti-VSV antibodies and ECL detection (WB). Each kinase strongly phosphorylated Rap1GAP2a, and the mutation of serine 7 to alanine abolished Rap1GAP2a phosphorylation by cGKI. (B) Rap1-GTP levels were analyzed in COS-1 cells transiently transfected with Rap1, CalDAG-GEFIII (CD-GEF III), wild-type Rap1GAP2a, and mutants of Rap1GAP2a containing mutations of serine 7 to alanine (S7A), aspartate (S7D) or glutamate (S7E), as described in the legend to Figure 6. Mutation of serine 7 did not change Rap1GAP2 activity in COS-1 cells. Shown are representative results from at least 3 independent experiments.