title: Knockdown of B-Raf impairs spindle formation and the mitotic checkpoint in human somatic cells.
abstract: It is well established that B-Raf signaling through the MAP kinase (ERK) pathways plays a prominent role in regulating cell proliferation but how it does this is not completely understood. Here, we show that B-Raf serves a physiological role during mitosis in human somatic cells. Knockdown of B-Raf using short interfering RNA (siRNA) resulted in pleiotropic spindle abnormalities and misaligned chromosomes in over 80% of the mitotic cells analyzed. A second B-Raf siRNA gave similar results suggesting these effects are specific to downregulating B-Raf protein. In agreement with these findings, a portion of B-Raf was detected at the spindle structures including the spindle poles and kinetochores. Knockdown of C-Raf (Raf-1) had no detectable effects on spindle formation or chromosome alignment. Activation of the spindle assembly checkpoint was found to be dependent on B-Raf as evident by the inability of checkpoint proteins Bub1 and Mad2 to localize to unattached kinetochores in HeLa cells treated with B-Raf siRNA. Consistent with this, live-cell imaging microscopy showed that B-Raf-depleted cells exited mitosis earlier than control non-depleted cells. Finally, we provide evidence that B-Raf signaling promotes phosphorylation and kinetochore localization of the mitotic checkpoint kinase Mps1. Blocking B-Raf expression, ERK activity, or phosphorylation at Ser-821 residue perturbed Mps1 localization at unattached kinetochores. Thus, our data implicates a mitotic role for B-Raf in regulating spindle formation and the spindle checkpoint in human somatic cells.
authors.affiliation: Molecular Oncology Program, H. Lee Moffitt Cancer Center & Research Institute, Tampa, Florida 33612, USA.
authors.forename: M K
authors.initials: MK
authors.lastname: Borysova
authors.forename: Y
authors.initials: Y
authors.lastname: Cui
authors.forename: M
authors.initials: M
authors.lastname: Snyder
authors.forename: T M
authors.initials: TM
authors.lastname: Guadagno
mesh_terms: D018797:Cell Cycle Proteins / Q000378:metabolism; D002877:Chromosomes, Human / Q000201:enzymology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology* / Q000201:enzymology*; D006367:HeLa Cells; D006801:Humans; D018386:Kinetochores / Q000201:enzymology; D008938:Mitosis; D009154:Mutation / Q000235:genetics; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D021381:Protein Transport; D011505:Protein-Tyrosine Kinases; D048493:Proto-Oncogene Proteins B-raf / Q000172:deficiency* / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D015398:Signal Transduction; D008941:Spindle Apparatus / Q000201:enzymology*
chemical_list: D018797:Cell Cycle Proteins; D010750:Phosphoproteins; D034741:RNA, Small Interfering; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D048493:Proto-Oncogene Proteins B-raf; D019908:Proto-Oncogene Proteins c-raf; D048049:Extracellular Signal-Regulated MAP Kinases; C075840:TTK protein, human