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Encoding optical control in LCK kinase to quantitatively investigate its activity in live cells
LCK is a tyrosine kinase essential for initiating T-cell antigen receptor (TCR) signaling. A complete understanding of LCK function is constrained by a paucity of methods to quantitatively study its function within live cells. To address this limitation, we generated LCK*, in which a key active site...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5736103/ https://www.ncbi.nlm.nih.gov/pubmed/29083415 http://dx.doi.org/10.1038/nsmb.3492 |
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author | Liaunardy-Jopeace, Ardiyanto Murton, Ben L Mahesh, Mohan Chin, Jason W James, John R |
author_facet | Liaunardy-Jopeace, Ardiyanto Murton, Ben L Mahesh, Mohan Chin, Jason W James, John R |
author_sort | Liaunardy-Jopeace, Ardiyanto |
collection | PubMed |
description | LCK is a tyrosine kinase essential for initiating T-cell antigen receptor (TCR) signaling. A complete understanding of LCK function is constrained by a paucity of methods to quantitatively study its function within live cells. To address this limitation, we generated LCK*, in which a key active site lysine is replaced by a photo-caged equivalent, using genetic code expansion. This enabled fine temporal and spatial control over kinase activity, allowing us to quantify phosphorylation kinetics in situ using biochemical and imaging approaches. We find that auto-phosphorylation of the LCK active site loop is indispensable for its catalytic activity and that LCK can stimulate its own activation by adopting a more open conformation, which can be modulated by point mutations. We then show that CD4 and CD8, the T cell coreceptors, can enhance LCK activity, helping to explain their effect in physiological TCR signaling. Our approach also provides general insights into SRC-family kinase dynamics. |
format | Online Article Text |
id | pubmed-5736103 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
record_format | MEDLINE/PubMed |
spelling | pubmed-57361032018-04-30 Encoding optical control in LCK kinase to quantitatively investigate its activity in live cells Liaunardy-Jopeace, Ardiyanto Murton, Ben L Mahesh, Mohan Chin, Jason W James, John R Nat Struct Mol Biol Article LCK is a tyrosine kinase essential for initiating T-cell antigen receptor (TCR) signaling. A complete understanding of LCK function is constrained by a paucity of methods to quantitatively study its function within live cells. To address this limitation, we generated LCK*, in which a key active site lysine is replaced by a photo-caged equivalent, using genetic code expansion. This enabled fine temporal and spatial control over kinase activity, allowing us to quantify phosphorylation kinetics in situ using biochemical and imaging approaches. We find that auto-phosphorylation of the LCK active site loop is indispensable for its catalytic activity and that LCK can stimulate its own activation by adopting a more open conformation, which can be modulated by point mutations. We then show that CD4 and CD8, the T cell coreceptors, can enhance LCK activity, helping to explain their effect in physiological TCR signaling. Our approach also provides general insights into SRC-family kinase dynamics. 2017-10-30 2017-12 /pmc/articles/PMC5736103/ /pubmed/29083415 http://dx.doi.org/10.1038/nsmb.3492 Text en http://www.nature.com/authors/editorial_policies/license.html#terms Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#terms |
spellingShingle | Article Liaunardy-Jopeace, Ardiyanto Murton, Ben L Mahesh, Mohan Chin, Jason W James, John R Encoding optical control in LCK kinase to quantitatively investigate its activity in live cells |
title | Encoding optical control in LCK kinase to quantitatively investigate its activity in live cells |
title_full | Encoding optical control in LCK kinase to quantitatively investigate its activity in live cells |
title_fullStr | Encoding optical control in LCK kinase to quantitatively investigate its activity in live cells |
title_full_unstemmed | Encoding optical control in LCK kinase to quantitatively investigate its activity in live cells |
title_short | Encoding optical control in LCK kinase to quantitatively investigate its activity in live cells |
title_sort | encoding optical control in lck kinase to quantitatively investigate its activity in live cells |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5736103/ https://www.ncbi.nlm.nih.gov/pubmed/29083415 http://dx.doi.org/10.1038/nsmb.3492 |
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