title: [Study of negative feedback between wild-type BRAF or RAFV600E and Mps1 in melanoma].
abstract: OBJECTIVE
  To study the effect of Mps1 on BRAFWT/MEK/ERK pathway in the presence of wild type BRAF or BRAFV600E in melanoma.
  
  
  METHODS
  Melanoma cells harboring BRAFWT genotype were transfected either with pBabe-puro-GST-BRAF-WT and/or pBabe-puro-GFP-Mps1-WT or pBabe-puro-GST-BRAFV600E and/or pBabe-puro-GFP-Mps1-WT, followed by Western blot to detect Mps1 and p-ERK expression. The melanoma cells harboring BRAFWT and BRAFV600E genotype were infected with pSUPER-Mps1 retrovirus to knockdown the endogenous Mps1 protein, followed by Western blot to detect Mps1 and p-ERK expression. Meanwhile, melanoma cells harboring BRAFV600E genotype were infected with pBabe-puro-GFP-Mps1 and Western blot was performed to detect Mps1 and p-ERK expression.
  
  
  RESULTS
  In melanoma cells harboring BRAFWT genotype and transfected with pBabe-puro-GST-BRAF-WT and pBabe-puro-GFP-Mps1-WT, phospho-ERK levels were notably reduced as compared to either negative control or empty vector. However, cells transfected with pBabe-puro-GST-BRAFV600E and pBabe-puro-GFP-Mps1-WT, phospho-ERK levels did not change significantly compared with either negative control or empty vector. Knockout of Mps1 in BRAF wild-type cell lines led to an increased ERK activity. However, there was no significant change of ERK activity in BRAFV600E cell lines in the absence of Mps1. The expression of p-ERK in BRAFV600E mutant cell lines infected with pBabe-puro-GFP-Mps1-WT did not show any significant difference from either negative control or empty vector.
  
  
  CONCLUSIONS
  Based on these findings, it suggests that there exists an auto-regulatory negative feedback loop between the Mps1 kinase and BRAFWT/ERK signaling. Oncogenic BRAFV600E abrogates the regulatory negative feedback loop of Mps1 on the MAPK pathway.
authors.affiliation: Department of Pathology, Shanxi Medical University, Taiyuan 030001, China.
authors.forename: Ling
authors.initials: L
authors.lastname: Zhang
authors.forename: Chanting
authors.initials: C
authors.lastname: He
authors.forename: Yanghui
authors.initials: Y
authors.lastname: Bi
authors.forename: Feng
authors.initials: F
authors.lastname: Liu
authors.forename: Heyang
authors.initials: H
authors.lastname: Cui
authors.forename: Juan
authors.initials: J
authors.lastname: Wang
authors.forename: Bin
authors.initials: B
authors.lastname: Song
authors.forename: Ruyi
authors.initials: R
authors.lastname: Shi
authors.forename: Bin
authors.initials: B
authors.lastname: Yang
authors.forename: Fang
authors.initials: F
authors.lastname: Wang
authors.forename: Zhiwu
authors.initials: Z
authors.lastname: Jia
authors.forename: Zhenxiang
authors.initials: Z
authors.lastname: Zhao
authors.affiliation: Key Laboratory of Cellular Physiology, Ministry of Education, Shanxi Medical University, Taiyuan 030001, China; E-mail:liujing5585@163.com.
authors.forename: Jing
authors.initials: J
authors.lastname: Liu
mesh_terms: D018797:Cell Cycle Proteins / Q000378:metabolism*; D045744:Cell Line, Tumor; D006801:Humans; D020935:MAP Kinase Signaling System; D008545:Melanoma / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010641:Phenotype; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D048493:Proto-Oncogene Proteins B-raf / Q000378:metabolism*; D015398:Signal Transduction; D014162:Transfection
chemical_list: D018797:Cell Cycle Proteins; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D048493:Proto-Oncogene Proteins B-raf; C075840:TTK protein, human