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.
Sheridan
Clare
C
Molecular Cell Biology Laboratory, Department of Genetics, The Smurfit Institute, Trinity College, Dublin 2, Ireland.
Brumatti
Gabriela
G
Martin
Seamus J
SJ
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