Oncogenic B-RafV600E inhibits apoptosis and promotes ERK-dependent inactivation of Bad and Bim.
Recent studies have revealed that B-Raf mutations are very common in malignant melanoma and are required for tumor growth and maintenance. The majority of melanoma-associated B-Raf mutations involve a single point mutation, V600E, which results in greatly elevated B-Raf kinase activity and constitutive activation of MAPK/ERK downstream. Here we show that B-Raf(V600E) increases resistance to apoptosis induced by chemotherapeutic drugs and promotes ERK-dependent phosphorylation of the BH3-only proteins Bim and Bad that are involved in setting thresholds for apoptosis. ERK-dependent phosphorylation of Bim resulted in degradation of this BH3-only protein, whereas phosphorylation of Bad has previously been shown to result in its sequestration by 14-3-3 proteins. Consistent with this, inhibition of ERK activity in a panel of melanoma cell lines resulted in stabilization of Bim and dephosphorylation of Bad. Furthermore, apoptosis induced through overexpression of Bad or Bim was efficiently blocked by coexpression of mutant B-Raf(V600E). However, small interfering RNA-mediated silencing of Bim and Bad expression conferred only modest protection against cytotoxic drugs, whereas oncogenic B-Raf strongly protected against the same stimuli. These observations suggest that B-Raf-initiated inactivation of Bad and Bim only partly contributes to the anti-apoptotic activities of this oncogene and that other points within the cell death machinery are also targeted by deregulated ERK signaling.
Molecular Cell Biology Laboratory, Department of Genetics, The Smurfit Institute, Trinity College, Dublin 2, Ireland.
Clare
C
Sheridan
Gabriela
G
Brumatti
Seamus J
SJ
Martin
D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism*; D000072224:Bcl-2-Like Protein 11; D045744:Cell Line, Tumor; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008545:Melanoma / Q000503:physiopathology; D008565:Membrane Proteins / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism; D009857:Oncogenes / Q000235:genetics*; D010766:Phosphorylation; D017354:Point Mutation; D021381:Protein Transport; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics* / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000378:metabolism; D051025:bcl-Associated Death Protein / Q000378:metabolism*
D051017:Apoptosis Regulatory Proteins; C494819:BAD protein, human; C000606775:BCL2L11 protein, human; D000072224:Bcl-2-Like Protein 11; D008565:Membrane Proteins; D011518:Proto-Oncogene Proteins; D051028:bcl-2-Associated X Protein; D051025:bcl-Associated Death Protein; D012694:Serine; D048493:Proto-Oncogene Proteins B-raf; D048049:Extracellular Signal-Regulated MAP Kinases