The Molecular Mechanism Through Which B-RAF^V600E Regulating Mps1 Protein Stability, Contributing Its Excess Accumulation At Centrosomes

Background B-RAF, a serine-threonine kinase, is one of three RAF protein kinase family members thattransduces the signals from RAS to ERK, regulating a variety of biological events, such as cell growth,differentiation and aoptosis. The B-RAF mutation have been found in many human cancers, such as 70% ofMelanoma, 50% of thyroid carcinoma, 20% of colon carcinoma. More than 90% of the oncogenic B-RAFmutations occur as V600E. Dr Cui has found that the B-RAF plays a vital role in mitosis. The B-RAF mutationwas sufficient to induce spindle abnormalities and chromosomal instability, resulting in euploidy in humanmelanocytes or in immortalized human mammary epithelial cells. Then, Mps1 was identified as a new target ofB-RAF^V600Eby MS. B-RAF^V600Esignaling prolongs activation of the spindle checkpoint through stabilizingMps1 levels in melanoma cells. However, the molecular mechanism through which B-RAF^V600Eregulating theMps1 protein stability is poorly understood.Objective To understand the molecular mechanism through which B-RAF^V600Eregulates the Mps1 proteinstability.Methods In order to elucidate the molecular mechanism through which B-RAF^V600Eregulates the Mps1protein stability, we mainly study the following aspects: （1） To test the effect of B-RAF^V600Esignaling on theubiquitination of Mps1, in vivo ubiquitin analysis was performed in 293T cells. （2） To further identify theB-RAF^V600Esignaling-dependent phosphorylation sites within human Mps1 by LC-MS/MS. （3） To evaluatethe effect of B-RAF^V600Esignaling-dependent phosphorylation sites within Mps1 on the Mps1 stability.Result （1） In vivo ubiquitin analysis show that the level of ubiquitin-Mps1 complex was significantlyreduced in B-RAF^V600Eexpressing cells that compared with non- B-RAF^V600Eexpressing cells, allowing theaccumulation of excess Mps1 at centrosomes. （2） Three B-RAF^V600Esignaling-dependent phosphorylation siteswithin Mps1 （S281、S436、S821） were identified by LC-MS/MS. （3） In this report, we focus on B-RAF^V600Esignaling-dependent phosphorylation sites at S281 and S436 because the study about the phosphorylation ofS821 has been reported previously. To understand how B-RAF^V600Esignaling regulates Mps1 stability, pointmutations of the phosphorylation sites were made in the pBabe-puro-HA-Mps1 expression vector. Results areas follows:①As expected, Mps1-WT protein was quickly degraded within half an hour. Interestingly,mutation of S281 to glutamic acid results in stabilization of the Mps1 protein while the other mutants have nosignificant effect on degradation, indicating that S281 is responsible for Mps1 stability. Next, we sought toevaluate the possibility that S281 phosphorylation suppresses APC/C-mediated degradation by examining Mps1 levels in cells overexpressing Cdc20. Mps1-WT protein was significantly decreased when Cdc20 wasoverexpressed. Similarly, Cdc20 up-regulation results in a decrease in the levels of Mps1 S436 and S821mutants. However, the levels of Mps1 S281E did not change when Cdc20 was overexpressed suggesting thatphosphorylation of S281 attenuates Mps1 degradation by the APC/CCdc20pathway.②To further evaluatewhether phosphorylation of S281 affects the interaction of Mps1 with Cdc27 （an integral subunit of APC/C）,we overexpressed wild-type and mutated HA-Mps1 in Sbcl2 cells, and subjected their protein extracts toimmunoprecipitation with anti-HA （or anti-Cdc27） antibody and Western blot with anti-Cdc27 （or anti-HA）antibody. Cdc27 was readily detected in Mps1 immunoprecipitates from cells expressing wild type, S436, orS821 mutant Mps1, The only mutant that lost the ability to bind Cdc27 was S281E. Likewise, HA-Mps1protein was hardly detected in Cdc27 immunoprecipitates from Mps1 S281E expressing cells. Hence, thesedata indicated that phosphorylation of Mps1 at S281 abolished its ability to bind Cdc27. Furthermore, weexamined whether phosphorylation of S281 affects the ubiquitination level of Mps1 protein. The results showsan accumulation of polyubiquitinated wild-type Mps1. Similar results were obtained when cells weretransfected with S281A, S436A/E, and S821A/E. However, the ability of S281E to bind ubiquitin wassignificantly compromised, suggesting that phosphorylation of S281 prevents polyubiquination of Mps1.③We further determined whether phosphorylation of S281 regulates the accumulation of Mps1 at centrosomes,We estimated the centrosomal levels of Mps1 by IIF following the local background correction methoddescribed by Kasbek et al. We observed that the Mps1 signal at centrosomes in cells expressing GFP-S436E orGFP-S821A/E has no significant change compared with wild-type. In contrast, GFP-S281A could notefficiently accumulate at centrosomes. Interestingly, the centrosomal signal was markedly stronger in cellsexpressing S281E. Together, these observations suggest that phosphorylation of S281 suppresses Mps1degradation thereby increasing the ability of Mps1 to accumulate at centrosomes.Conclusion Based on our findings, we propose that phosphorylation of Mps1 at residue S281 by B-RAF^V600Esignaling prevents the degradation of Mps1 by the anaphase promoting complex/cyclosome （APC/C）, thusallowing the Mps1 protein to accumulate in excess at centrosomes.