Nuclear phosphoproteomics analysis reveals that CDK1/2 are involved in EGF-regulated constitutive pre-mRNA splicing in MDA-MB-468 cells.
The epidermal growth factor (EGF) receptor (EGFR) pathway is one of the most dysregulated and extensively investigated signaling pathways in human cancers and plays important roles in the regulation of nuclear functions through both cytoplasmic and nuclear EGFR pathways. However, the current understanding of the nuclear phosphorylation responses to activated EGFR pathways remains limited. In the present study, phosphoproteomics analysis revealed the increased phosphorylation of 90 nuclear proteins, primarily involved in RNA processing, pre-mRNA splicing and cell cycle regulation, upon EGF stimulation in MDA-MB-468 cells. Cellular splicing assays of the β-globin (HBB) minigene confirmed that EGF induced constitutive pre-mRNA splicing. Further analysis of phosphoproteomics data identified multiple CDK1/2 substrates in pre-mRNA splicing-related proteins, and both CDK1/2 inhibitors and CDK1/2 knockdowns reduced EGF-regulated pre-mRNA splicing. In conclusion, the results of the present study provide evidence that CDK1/2 participate in the regulation of constitutive pre-mRNA splicing by EGF stimulation in MDA-MB-468 cells.



In this study, we successfully carried out a survey of nuclear phosphorylation changes in response to EGF stimulation. The results from the functional category analysis and pre-mRNA splicing assay strongly indicated that EGFR activation increased constitutive pre-mRNA splicing in MDA-MB-468 cells, revealing additional role of EGFR on regulation of mRNA maturation beyond alternative pre-mRNA splicing reported by previous studies. Furthermore, we found that CDK1/2 participated in constitutive pre-mRNA splicing regulation by EGF in MDA-MB-468 cells. Our study provides new knowledge for understanding the regulation of constitutive pre-mRNA splicing by EGF stimulation.
CAS Key Laboratory of Genome Sciences and Information, China Gastrointestinal Cancer Research Center, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China. Electronic address: chenxw@big.ac.cn.
Xianwei
X
Chen
CAS Key Laboratory of Genome Sciences and Information, China Gastrointestinal Cancer Research Center, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, 100101, China. Electronic address: guodan@big.ac.cn.
Dan
D
Guo
CAS Key Laboratory of Genome Sciences and Information, China Gastrointestinal Cancer Research Center, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China. Electronic address: yhzhu@big.ac.cn.
Yinghui
Y
Zhu
CAS Key Laboratory of Genome Sciences and Information, China Gastrointestinal Cancer Research Center, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China. Electronic address: xianfeng@big.ac.cn.
Feng
F
Xian
CAS Key Laboratory of Genome Sciences and Information, China Gastrointestinal Cancer Research Center, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China. Electronic address: siqiliu@big.ac.cn.
Siqi
S
Liu
CAS Key Laboratory of Genome Sciences and Information, China Gastrointestinal Cancer Research Center, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China. Electronic address: wul@big.ac.cn.
Lin
L
Wu
CAS Key Laboratory of Genome Sciences and Information, China Gastrointestinal Cancer Research Center, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China. Electronic address: louxm@big.ac.cn.
Xiaomin
X
Lou
D016203:CDC2 Protein Kinase / Q000502:physiology*; D045744:Cell Line, Tumor; D051357:Cyclin-Dependent Kinase 2 / Q000502:physiology*; D004815:Epidermal Growth Factor / Q000502:physiology*; D066246:ErbB Receptors / Q000378:metabolism; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D012322:RNA Precursors; D012326:RNA Splicing
D009687:Nuclear Proteins; D012322:RNA Precursors; D004815:Epidermal Growth Factor; C512478:EGFR protein, human; D066246:ErbB Receptors; D016203:CDC2 Protein Kinase; C528899:CDK1 protein, human; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2
CDK1/2; EGF; MDA-MB-468 cells; Nuclear phosphoproteomics; Pre-mRNA splicing