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  • Original Article
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B-RafV600E signaling deregulates the mitotic spindle checkpoint through stabilizing Mps1 levels in melanoma cells

Abstract

The B-RafV600E 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-RafV600E signaling deregulates the spindle checkpoint as a consequence of stabilizing monopolar spindle 1 (Mps1) levels in human melanoma cells. Upon introducing the B-RafV600E 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-RafV600E 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-RafV600E as shown by depleting Mps1 with short interfering RNA. Collectively, our findings implicate a new mechanism through which B-RafV600E exerts its oncogenic effects in melanoma.

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Accession codes

Accessions

GenBank/EMBL/DDBJ

Abbreviations

APC:

anaphase-promoting complex

CHX:

cycloheximide

ERK:

extracellular signal-regulated kinase

IP:

immunoprecipitation

Mps1:

monopolar spindle 1

SCR:

scrambled siRNA

siRNA:

short interfering RNA

WB:

western blot

WT:

wild type

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Acknowledgements

We thank John Kyriakis, Daniel Peeper and Jiandong Chen for providing valuable reagents and Mark Winey for providing a protocol for immunostaining kinetochore-associated proteins. We also thank members of the Guadagno laboratory for their support and helpful suggestions. We acknowledge the support of the Analytical Microscopy (Joseph Johnson and Mark Lloyd), Molecular Biology and Flow Cytometry core facilities at the H. Lee Moffitt Cancer Center. This work was supported in part by the National Institutes of Health (grant GM62542) and a DOD-sponsored National Functional Genomics and Proteomics programmatic grant at Moffitt (to TMG). TM Guadagno was supported as a V Scholar by the Jimmy V Foundation.

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Correspondence to T M Guadagno.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc).

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Cui, Y., Guadagno, T. B-RafV600E signaling deregulates the mitotic spindle checkpoint through stabilizing Mps1 levels in melanoma cells. Oncogene 27, 3122–3133 (2008). https://doi.org/10.1038/sj.onc.1210972

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