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Involvement of Rap1A Phosphorylation in Regulation of Raf-1 Kinase Activity: Chang-Deng HU, Ken-ichi KARIYA, Tomoyo OKADA, and Tohru KATAOKA (Dept. Physiol. II, Kobe Univ. Sch. Med.)

Phosphorylation of Rap1A has been thought to be a physiological event contributing to its cellular functions. However, it is not known whether phosphorylation of Rap1A is involved in its suppressive activity on Ras-dependent Raf-1 activation. Recently, we identified the cysteine-rich region (CRR, residues 152-184) of Raf-1 as a novel Ras-binding domain required for Raf-1 activation, in addition to the Ras binding domain (RBD, residues 51-131). Also we found that Rap1A has very high binding affinity for CRR and forms a ternary complex with Ras and Raf-1, through independent binding of Rap1A and Ras to CRR and RBD, respectively. These findings not only suggest that the suppression of Ras-dependent Raf-1 activation by Rap1A may be due to the block of Ras-CRR association but also imply that the Ras-suppressive activity of Rap1A may be regulated through altering Rap1A-CRR association. Here, we report that in vitro phosphorylation of Rap1A by cAMP-dependent protein kinase A (PKA) greatly attenuated the binding ability of Rap1A to CRR. Contrary to unphosphorylated Rap1A, phosphorylated Rap1A no longer formed the ternary complex with Ras and Raf. Furthermore, a mutant Rap1A(S180E), whose in vivo and in vitro PKA-phosphorylation site S180 was substituted with acidic amino acid glutamate to mimic the function of in vivo phosphorylated Rap1A, failed to suppress Raf-1 activation by Ras when cotransfected into COS7 cells. The loss of Ras suppressive activity of Rap1A(S180E) was likely due to the reduced CRR binding ability as indicated by the observation that Rap1A(S180E) also exhibited reduced binding ability to CRR in vitro. Considering the direct inhibitory effect of PKA on Raf-1 kinase activity, our results suggest that PKA may have dual effects on Raf-1 kinase activity through direct phosphorylation of Raf-1 and phosphorylation of Rap1A.