B-RAF PROTEIN ISOFORMS INTERACT WITH AND PHOSPHORYLATE MEK-1 ON SERINE RESIDUE-218 AND RESIDUE-222

The B-raf/c-Rmil proto-oncogene belongs to the raf/mil family of serin e/threonine protein kinases, Tt encodes multiple protein isoforms resu lting from alternative splicing of two exons located upstream of the k inase domain, Recent studies suggested that B-Raf could be the interme diate molecule between Ras and Mek-1 (MAP Kinase Kinase) in signalling pathways specific of neural cells. However, there has been no evidenc e for a direct interaction between B-Raf and Mek-1, We report here tha t different B-Raf isoforms can be co-immunoprecipitated with anti-Mek- 1 antisera in COS-1 cells and that the kinase activity of B-Raf is not required for its interaction with Mek-1. We also show that all B-Raf isoforms tested phosphorylate Mek-1 in a time-dependent manner, wherea s kinase defective mutants fail to do so. Finally, we demonstrate that the constitutively activated S218D, S222D and S218D/S222D mutants of Mek-1 interact similarly with B-Raf. However, only the S218D and S222D mutants, and not the S218D/S222D double mutant, can be phosphorylated by B-Raf isoforms. Therefore, serine residues 218 and 222, previously shown to regulate Mek-1 activity, appear to be the major phosphorylat ion sites by B-Raf in vitro.