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The CDC42 effector protein MRCKβ autophosphorylates on Threonine 1108
The CDC42 small GTPase is a major influence on actin-myosin cytoskeleton organization and dynamics, signalling via effector proteins including the Myotonic dystrophy related CDC42-binding protein kinases (MRCK) α and β. We previously identified Serine 1003 of MRCKα as a site of autophosphorylation,...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Taylor & Francis
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7549636/ https://www.ncbi.nlm.nih.gov/pubmed/30667325 http://dx.doi.org/10.1080/21541248.2018.1564472 |
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author | Unbekandt, Mathieu Lilla, Sergio Zanivan, Sara Olson, Michael F. |
author_facet | Unbekandt, Mathieu Lilla, Sergio Zanivan, Sara Olson, Michael F. |
author_sort | Unbekandt, Mathieu |
collection | PubMed |
description | The CDC42 small GTPase is a major influence on actin-myosin cytoskeleton organization and dynamics, signalling via effector proteins including the Myotonic dystrophy related CDC42-binding protein kinases (MRCK) α and β. We previously identified Serine 1003 of MRCKα as a site of autophosphorylation, and showed that a phosphorylation-sensitive antibody raised against this site could be used as a surrogate indicator of kinase activity. In this study, a kinase-dead version of MRCKβ was established by mutation of the conserved Lysine 105 to Methionine (K105M), which was then used for mass spectrometry analysis to identify phosphorylation events that occurred in catalytically-competent MRCKβ but not in the kinase-dead form. A total of ten phosphorylations were identified on wild-type MRCKβ, of which the previously undescribed Threonine 1108 (Thr1108) was not found on kinase-dead MRCKβ K105M, consistent with this being due to autophosphorylation. Mutation of Thr1108 to non-phosphorylatable Alanine (T1108A) or phosphomimetic Glutamate (T1108E) did not affect the ability of MRCKβ to phosphorylate recombinant myosin light chain in vitro, or observably alter the subcellular localization of green fluorescent protein (GFP)-tagged MRCKβ expressed in MDA MB 231 human breast cancer cells. Although phosphorylation of Thr1108 did not appear to contribute to MRCKβ function or regulation, the identification of this phosphorylation does make it possible to characterize whether this site could be used as a surrogate biomarker of kinase activity and inhibitor efficacy as we previously demonstrated for Ser 1003 in MRCKα. |
format | Online Article Text |
id | pubmed-7549636 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | Taylor & Francis |
record_format | MEDLINE/PubMed |
spelling | pubmed-75496362020-10-27 The CDC42 effector protein MRCKβ autophosphorylates on Threonine 1108 Unbekandt, Mathieu Lilla, Sergio Zanivan, Sara Olson, Michael F. Small GTPases Research Paper The CDC42 small GTPase is a major influence on actin-myosin cytoskeleton organization and dynamics, signalling via effector proteins including the Myotonic dystrophy related CDC42-binding protein kinases (MRCK) α and β. We previously identified Serine 1003 of MRCKα as a site of autophosphorylation, and showed that a phosphorylation-sensitive antibody raised against this site could be used as a surrogate indicator of kinase activity. In this study, a kinase-dead version of MRCKβ was established by mutation of the conserved Lysine 105 to Methionine (K105M), which was then used for mass spectrometry analysis to identify phosphorylation events that occurred in catalytically-competent MRCKβ but not in the kinase-dead form. A total of ten phosphorylations were identified on wild-type MRCKβ, of which the previously undescribed Threonine 1108 (Thr1108) was not found on kinase-dead MRCKβ K105M, consistent with this being due to autophosphorylation. Mutation of Thr1108 to non-phosphorylatable Alanine (T1108A) or phosphomimetic Glutamate (T1108E) did not affect the ability of MRCKβ to phosphorylate recombinant myosin light chain in vitro, or observably alter the subcellular localization of green fluorescent protein (GFP)-tagged MRCKβ expressed in MDA MB 231 human breast cancer cells. Although phosphorylation of Thr1108 did not appear to contribute to MRCKβ function or regulation, the identification of this phosphorylation does make it possible to characterize whether this site could be used as a surrogate biomarker of kinase activity and inhibitor efficacy as we previously demonstrated for Ser 1003 in MRCKα. Taylor & Francis 2019-01-22 /pmc/articles/PMC7549636/ /pubmed/30667325 http://dx.doi.org/10.1080/21541248.2018.1564472 Text en © 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. https://creativecommons.org/licenses/by/4.0/This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Research Paper Unbekandt, Mathieu Lilla, Sergio Zanivan, Sara Olson, Michael F. The CDC42 effector protein MRCKβ autophosphorylates on Threonine 1108 |
title | The CDC42 effector protein MRCKβ autophosphorylates on Threonine 1108 |
title_full | The CDC42 effector protein MRCKβ autophosphorylates on Threonine 1108 |
title_fullStr | The CDC42 effector protein MRCKβ autophosphorylates on Threonine 1108 |
title_full_unstemmed | The CDC42 effector protein MRCKβ autophosphorylates on Threonine 1108 |
title_short | The CDC42 effector protein MRCKβ autophosphorylates on Threonine 1108 |
title_sort | cdc42 effector protein mrckβ autophosphorylates on threonine 1108 |
topic | Research Paper |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7549636/ https://www.ncbi.nlm.nih.gov/pubmed/30667325 http://dx.doi.org/10.1080/21541248.2018.1564472 |
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