Unbekandt, M., Lilla, S., Zanivan, S. R. and Olson, M. F. (2020) The CDC42 effector protein MRCKß autophosphorylates on Threonine 1108. Small GTPases, 11(6), pp. 451-460. (doi: 10.1080/21541248.2018.1564472) (PMID:30667325) (PMCID:PMC7549636)
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Abstract
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.
Item Type: | Articles |
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Additional Information: | Also funded by Cancer Research UK (A18276) and Worldwide Cancer Research (14-0223 to M.F. Olson). |
Status: | Published |
Refereed: | Yes |
Glasgow Author(s) Enlighten ID: | Lilla, Dr Sergio and Zanivan, Professor Sara and Unbekandt, Dr Mathieu and Olson, Professor Michael |
Authors: | Unbekandt, M., Lilla, S., Zanivan, S. R., and Olson, M. F. |
College/School: | College of Medical Veterinary and Life Sciences > School of Cancer Sciences |
Journal Name: | Small GTPases |
Publisher: | Taylor and Francis |
ISSN: | 2154-1248 |
ISSN (Online): | 2154-1256 |
Published Online: | 22 January 2019 |
Copyright Holders: | Copyright © 2019 The Authors |
First Published: | First published in Small GTPases 11(6): 451-460 |
Publisher Policy: | Reproduced under a Creative Commons License |
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