Effects of Activating Mutations on EGFR Cellular Protein Turnover and Amino Acid Recycling Determined Using SILAC Mass Spectrometry

Q3 Biochemistry, Genetics and Molecular Biology International Journal of Cell Biology Pub Date : 2015-11-24 DOI:10.1155/2015/798936
M. Greig, Sherry L. Niessen, S. Weinrich, J. Feng, Manli Shi, Ted Johnson
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引用次数: 19

Abstract

Rapid mutations of proteins that are targeted in cancer therapy often lead to drug resistance. Often, the mutation directly affects a drug's binding site, effectively blocking binding of the drug, but these mutations can have other effects such as changing the protein turnover half-life. Utilizing SILAC MS, we measured the cellular turnover rates of an important non-small cell lung cancer target, epidermal growth factor receptor (EGFR). Wild-type (WT) EGFR, EGFR with a single activating mutant (Del 746–750 or L858R), and the drug-resistant double mutant (L858R/T790M) EGFR were analyzed. In non-small cell lung cancer cell lines, EGFR turnover rates ranged from 28 hours in A431 cells (WT) to 7.5 hours in the PC-9 cells (Del 746–750 mutant). The measurement of EGFR turnover rate in PC-9 cells dosed with irreversible inhibitors has additional complexity due to inhibitor effects on cell viability and results were reported as a range. Finally, essential amino acid recycling (K and R) was measured in different cell lines. The recycling was different in each cell line, but the overall inclusion of the effect of amino acid recycling on calculating EGFR turnover rates resulted in a 10–20% reduction in rates.
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激活突变对EGFR细胞蛋白质周转和氨基酸再循环的影响
癌症治疗中靶向蛋白的快速突变常常导致耐药性。通常,突变直接影响药物的结合位点,有效地阻断药物的结合,但这些突变可以产生其他影响,如改变蛋白质的周转半衰期。利用SILAC质谱,我们测量了一个重要的非小细胞肺癌靶点表皮生长因子受体(EGFR)的细胞周转率。分析野生型(WT) EGFR、单激活突变体(Del 746-750或L858R) EGFR和耐药双突变体(L858R/T790M) EGFR。在非小细胞肺癌细胞系中,EGFR周转率从A431细胞(WT)的28小时到PC-9细胞(Del 746-750突变体)的7.5小时不等。使用不可逆抑制剂的PC-9细胞中EGFR周转率的测量具有额外的复杂性,因为抑制剂对细胞活力有影响,报告的结果是一个范围。最后,测定不同细胞系的必需氨基酸再循环(K和R)。每个细胞系的再循环是不同的,但氨基酸再循环对计算EGFR周转率的总体影响导致了10-20%的速率降低。
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来源期刊
International Journal of Cell Biology
International Journal of Cell Biology Biochemistry, Genetics and Molecular Biology-Cell Biology
CiteScore
3.30
自引率
0.00%
发文量
4
审稿时长
20 weeks
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