Effects of Activating Mutations on EGFR Cellular Protein Turnover and Amino Acid Recycling Determined Using SILAC Mass Spectrometry
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 S...
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| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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Wiley
2015-01-01
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| Series: | International Journal of Cell Biology |
| Online Access: | http://dx.doi.org/10.1155/2015/798936 |
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| _version_ | 1832565230371078144 |
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| author | Michael J. Greig Sherry Niessen Scott L. Weinrich Jun Li Feng Manli Shi Ted O. Johnson |
| author_facet | Michael J. Greig Sherry Niessen Scott L. Weinrich Jun Li Feng Manli Shi Ted O. Johnson |
| author_sort | Michael J. Greig |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-4cdd757837e142f3bc96408a4ac0c80a |
| institution | Kabale University |
| issn | 1687-8876 1687-8884 |
| language | English |
| publishDate | 2015-01-01 |
| publisher | Wiley |
| record_format | Article |
| series | International Journal of Cell Biology |
| spelling | doaj-art-4cdd757837e142f3bc96408a4ac0c80a2025-02-03T01:08:50ZengWileyInternational Journal of Cell Biology1687-88761687-88842015-01-01201510.1155/2015/798936798936Effects of Activating Mutations on EGFR Cellular Protein Turnover and Amino Acid Recycling Determined Using SILAC Mass SpectrometryMichael J. Greig0Sherry Niessen1Scott L. Weinrich2Jun Li Feng3Manli Shi4Ted O. Johnson5Worldwide Medicinal Chemistry, Pfizer, Inc., La Jolla Laboratories, 10770 Science Center Drive, San Diego, CA 92121, USAWorldwide Medicinal Chemistry, Pfizer, Inc., La Jolla Laboratories, 10770 Science Center Drive, San Diego, CA 92121, USAOncology Research Unit, Pfizer, Inc., La Jolla Laboratories, 10770 Science Center Drive, San Diego, CA 92121, USAWorldwide Medicinal Chemistry, Pfizer, Inc., La Jolla Laboratories, 10770 Science Center Drive, San Diego, CA 92121, USAOncology Research Unit, Pfizer, Inc., La Jolla Laboratories, 10770 Science Center Drive, San Diego, CA 92121, USAWorldwide Medicinal Chemistry, Pfizer, Inc., La Jolla Laboratories, 10770 Science Center Drive, San Diego, CA 92121, USARapid 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.http://dx.doi.org/10.1155/2015/798936 |
| spellingShingle | Michael J. Greig Sherry Niessen Scott L. Weinrich Jun Li Feng Manli Shi Ted O. Johnson Effects of Activating Mutations on EGFR Cellular Protein Turnover and Amino Acid Recycling Determined Using SILAC Mass Spectrometry International Journal of Cell Biology |
| title | Effects of Activating Mutations on EGFR Cellular Protein Turnover and Amino Acid Recycling Determined Using SILAC Mass Spectrometry |
| title_full | Effects of Activating Mutations on EGFR Cellular Protein Turnover and Amino Acid Recycling Determined Using SILAC Mass Spectrometry |
| title_fullStr | Effects of Activating Mutations on EGFR Cellular Protein Turnover and Amino Acid Recycling Determined Using SILAC Mass Spectrometry |
| title_full_unstemmed | Effects of Activating Mutations on EGFR Cellular Protein Turnover and Amino Acid Recycling Determined Using SILAC Mass Spectrometry |
| title_short | Effects of Activating Mutations on EGFR Cellular Protein Turnover and Amino Acid Recycling Determined Using SILAC Mass Spectrometry |
| title_sort | effects of activating mutations on egfr cellular protein turnover and amino acid recycling determined using silac mass spectrometry |
| url | http://dx.doi.org/10.1155/2015/798936 |
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