B-Raf(V600E) signaling deregulates the mitotic spindle checkpoint through stabilizing Mps1 levels in melanoma cells.
The B-Raf(V600E) mutant, found in 65% of human melanomas, drives constitutive activation of the extracellular signal-regulated kinase (ERK) pathway and is implicated in tumorigenesis. Recently, we showed that B-Raf is important for spindle formation and the mitotic spindle checkpoint arrest. In this study, we demonstrate that B-Raf(V600E) signaling deregulates the spindle checkpoint as a consequence of stabilizing monopolar spindle 1 (Mps1) levels in human melanoma cells. Upon introducing the B-Raf(V600E) mutant into wild-type B-Raf melanoma cells, Mps1 protein and activity increased 3- and 10-fold, respectively. In addition, Mps1 became hyperphosphorylated, which correlated with stabilization of Mps1 protein levels. In contrast, reduction of B-Raf by RNAi or inactivation of ERK by the MEK inhibitor U0126 resulted in a precipitous decline in Mps1 levels. Together, these results suggest that B-Raf signaling through ERK regulates the stability of Mps1. Finally, B-Raf(V600E) expression induces a mitotic delay due to promoting robust activation of the mitotic spindle checkpoint. These effects were dependent on the induction of Mps1 levels by oncogenic B-Raf(V600E) as shown by depleting Mps1 with short interfering RNA. Collectively, our findings implicate a new mechanism through which B-Raf(V600E) exerts its oncogenic effects in melanoma.
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Molecular Oncology Program, Cutaneous Oncology Division, H Lee Moffitt Cancer Center & Research Institute, Tampa, FL 33612, USA.
Guadagno
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D019943:Amino Acid Substitution / Q000502:physiology; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002455:Cell Division / Q000235:genetics; D045744:Cell Line, Tumor; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D018816:Genes, cdc; D018698:Glutamic Acid / Q000235:genetics; D006801:Humans; D008545:Melanoma / Q000235:genetics* / Q000378:metabolism; D010766:Phosphorylation; D020641:Polymorphism, Single Nucleotide / Q000502:physiology; D011485:Protein Binding; D011499:Protein Processing, Post-Translational / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology; D008941:Spindle Apparatus / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D014633:Valine / Q000235:genetics
D018797:Cell Cycle Proteins; D018698:Glutamic Acid; D011505:Protein-Tyrosine Kinases; C482119:BRAF protein, human; D017346:Protein Serine-Threonine Kinases; D048493:Proto-Oncogene Proteins B-raf; D048049:Extracellular Signal-Regulated MAP Kinases; C075840:TTK protein, human; D014633:Valine