B-Raf protein isoforms interact with and phosphorylate Mek-1 on serine residues 218 and 222.
The B-raf/c-Rmil proto-oncogene belongs to the raf/mil family of serine/threonine protein kinases. It encodes multiple protein isoforms resulting from alternative splicing of two exons located upstream of the kinase domain. Recent studies suggested that B-Raf could be the intermediate molecule between Ras and Mek-1 (MAP Kinase Kinase) in signalling pathways specific of neural cells. However, there has been no evidence for a direct interaction between B-Raf and Mek-1. We report here that 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, whereas 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 phosphorylation sites by B-Raf in vitro.
Papin
C
C
Unité Mixte de Recherche 146 du CNRS, Institut Curie, ORSAY, France.
Eychène
A
A
Brunet
A
A
Pagès
G
G
Pouysségur
J
J
Calothy
G
G
Barnier
J V
JV
D000818:Animals; D002460:Cell Line; D048369:MAP Kinase Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases; D010766:Phosphorylation; D011233:Precipitin Tests; D017346:Protein Serine-Threonine Kinases / Q000276:immunology / Q000378:metabolism* / Q000502:physiology*; D011505:Protein-Tyrosine Kinases / Q000276:immunology / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000276:immunology / Q000502:physiology*; D019908:Proto-Oncogene Proteins c-raf; D012694:Serine / Q000378:metabolism*
D011518:Proto-Oncogene Proteins; D012694:Serine; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D048369:MAP Kinase Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases