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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,...

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Autores principales: Unbekandt, Mathieu, Lilla, Sergio, Zanivan, Sara, Olson, Michael F.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Taylor & Francis 2019
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.
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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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