INHIBITION OF RAF-1 SIGNALING BY A MONOCLONAL-ANTIBODY, WHICH INTERFERES WITH RAF-1 ACTIVATION AND WITH MEK SUBSTRATE-BINDING

Raf-1 is a serine/threonine specific kinase that integrates signaling by a large number of mitogens to elicit a transcriptional response in the nucleus, Activated Raf-1 phosphorylates and activates MAPK/ERK kin ase (Mek), thus initiating the Mek-->MAP kinase cascade, which ultimat ely results in the phosphorylation and activation of transcription fac tors by MAP kinase. Here we have characterized the mechanism by which monoclonal antibody URP26K, which binds to an epitope in the Raf-1 kin ase domain, inhibits intracellular signal transduction. This antibody preferentially immunoprecipitated the underphosphorylated, non-activat ed form of Raf-1 from quiescent cells. Baculovirus-expressed Raf-1 imm unoprecipitated with URP26K was Largely refractory to. phosphorylation and activation mediated by protein kinase C (PKC)alpha or the tyrosin e kinase Lck. In addition, URP26K reduced the binding of Raf-1 to its vitro, but did not disturb the Ras. Microinjection of with cells block ed DIVA synthesis initiated by serum, insulin and various purified gro wth factors, but it did not block DNA synthesis initiated by v-ras. Mi croinjected URP26K also impaired the expression of stably transfected beta-galactosidase reporter genes regulated by minimal promoter elemen ts. These results demonstrate, (i) that the URP26K monoclonal antibody inhibits Raf-1 by preventing activating Raf-1 phosphorylation and/or association with its substrate Mek, (ii) that inhibition of Raf-1 by U RP26K does not interfere with Ras-induced DNA synthesis. In contrast t o dominant negative Raf-1 mutants, which also block Ras signaling by b inding to the Ras effector domain, antibody mediated Raf-1 inhibition thus reveals branchpoint of mitogenic signaling at the level of Ras.