Effect of phosphorylation on activities of Rap1A to interact with Raf-1 and to suppress Ras-dependent Raf-1 activation

Rap1A is phosphorylated by cAMP-dependent protein kinase (PRA), and this ph osphorylation has been shown to modulate its interaction with other protein s. However, it is not known whether Rap1A phosphorylation is involved in re gulation of its cellular functions, including suppression of Ras-dependent Raf-l activation. We have previously shown that this suppressive activity o f Rap1A is attributable to its greatly enhanced ability to bind to the cyst eine-rich region (CRR, residues 152-184) of Raf-l compared with that of Ras , Here, we show that phosphorylation of Rap1A by PKA abolished its binding activity to CRR, Furthermore, a mutant Rap1A(S180E), whose sole PKA phospho rylation residue, Ser-180, was substituted by an acidic residue, Glu, to mi mic its phosphorylated form, failed to suppress Ras-dependent Raf-l activat ion in COS-7 cells. These results indicate that the CRR binding activity an d the Ras-suppressive function of Rap1A can be modulated through phosphoryl ation and suggest that Rap1A may function as a PKA-dependent regulator of R af-l activation, not merely as a suppressor.