PD01-01: VHL-mediated ubiquitination of the kinase Mps1 regulates the mitotic checkpoint in clear cell renal cell carcinoma

Mark Woodford*, Sarah Backe, Rebecca Sager, Oleg Shapiro, Imad Nsouli, Laura Wengert, Gennady Bratslavsky, Dimitra Bourboulia, Mehdi Mollapour, Syracuse, NY

Introduction: Abnormal chromosome segregation during mitosis causes aneuploidy, a hallmark of cancers associated with high risk for tumorigenesis, which is normally regulated by the mitotic checkpoint. Mps1 kinase activity is essential for spindle checkpoint signaling. Mps1 is over-expressed in clear cell renal cell carcinoma (ccRCC) and requires the molecular chaperone Hsp90 for its activity. Mps1 phosphorylates Hsp90, regulating chaperone function of numerous oncogenic client proteins, including Mps1, and conferring tumor selectivity of Hsp90 inhibitors in ccRCC. The most frequent alteration leading to ccRCC is loss of von Hippel Lindau (VHL), the recognition subunit of an E3-ubiquitin ligase complex that targets proteins for degradation. As Mps1 is over-expressed in ccRCC, the objective of the current study was to determine whether Mps1 gets targeted for degradation by VHL and whether this regulates the mitotic checkpoint.

Methods: VHL-mediated ubiquitination of Mps1 was examined in vitro and VHL-dependent degradation of Mps1 was examined in ccRCC cell lines by Western blot. Site-directed mutagenesis was utilized to determine Mps1 ubiquitination sites and the effect of increased Mps1 stability on mitotic checkpoint was determined using flow cytometry.

Results: Mps1 kinase is ubiquitinated by a VHL containing E3-ubiquitin ligase complex and Mps1 activity is essential for its ubiquitination. Re-expression of VHL in VHL-null ccRCC cell lines leads to proteasomal degradation of Mps1. VHL degrades Mps1 in an oxygen independent manner by ubiquitination of Mps1-K86, K827, and K848. Mps1 ubiquitination regulates cell cycle progression via exit from the mitotic checkpoint.

Conclusions: Mps1 is targeted for degradation by the tumor suppressor VHL in a hypoxia-independent manner and Mps1 is over-expressed in VHL-null ccRCC. VHL-mediated ubiquitination of Mps1 regulates mitotic checkpoint progression. Mps1 stability additionally mediates Hsp90 post-translational modification and tumor selectivity of Hsp90 inhibitors.

Source of Funding: This work was also supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number, R01GM139932 (D.B.), R35GM139584 (M.M.), and R01GM124256 (M.M.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This work was also supported by the Urology Care Foundation-American Urological Association (M.M.).