Study of negative feedback between wild-type BRAF or RAFV600E and Mps1 in melanoma
10.3760/cma.j.issn.0529-5807.2015.04.014
- VernacularTitle:BRAF 野生型及 BRAFV600 E 突变型的黑色素瘤细胞中 Mps1基因对 BRAF WT／MEK／ERK 通路的影响
- Author:
Ling ZHANG
1
;
Chanting HE
;
Yanghui BI
;
Feng LIU
;
Heyang CUI
;
Juan WANG
;
Bin SONG
;
Ruyi SHI
;
Bin YANG
;
Fang WANG
;
Zhiwu JIA
;
Zhenxiang ZHAO
;
Jing LIU
Author Information
1. 山西医科大学基础医学院病理学教研室
- Keywords:
Melanoma;
Transfection;
Blotting,western
- From:
Chinese Journal of Pathology
2015;(4):274-277
- CountryChina
- Language:Chinese
-
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.
