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De-palmitoylation of tissue factor regulates its activity, phosphorylation and cellular functions

Ettelaie, Camille; Featherby, Sophie; Rondon, Araci M. R.; Greenman, John; Versteeg, Henri H.; Maraveyas, Anthony

Authors

Camille Ettelaie

Sophie Featherby

Araci M. R. Rondon

Henri H. Versteeg

Anthony Maraveyas



Abstract

In this study, the role of de-palmitoylation of tissue factor (TF) in the decryption of its activity was explored. TF-tGFP constructs were prepared by mutagenesis-substitution at Cys245 to prevent or mimic palmitolyation. Additionally, to reduce TF de-palmitoylation, the expression of palmitoyl-protein thioesterases (PPT) was suppressed. Other TF mutants were prepared with altered flexibility, hydrophobicity or length of the transmembrane domain. The outcome of these alterations on fXa-generation, fVIIa binding, Ser253 phosphorylation and TF-microvesicle release were assessed in endothelial cells, and the influence on endothelial and MCF-7 cell proliferation and apoptosis was analysed. Preventing TF palmitoylation (TFSer245-tGFP), increasing the hydropho-bicity (TFPhe241-tGFP) or lengthening (TFLongTM-tGFP) of the transmembrane domain enhanced fXa-generation in resting cells compared to cells expressing TFWt-tGFP, but fXa-generation was not further increased following PAR2 activation. Extending the available length of the transmembrane domain enhanced the TF-tGFP release within microvesicles and Ser253 phosphorylation and increased cell proliferation. Moreover, prevention of PKCα-mediated Ser253 phosphorylation with Gö6976 did not preclude fXa-generation. Conversely, reducing the hydrophobicity (TFSer242-tGFP), shortening (TFShortTM-tGFP) or reducing the flexibility (TFVal225-tGFP) of the transmembrane domain suppressed fXa-generation, fVIIa-HRP binding and Ser253 phosphorylation following PAR2 activa-tion. PPT knock-down or mimicking palmitoylation (TFPhe245-tGFP) reduced fXa-generation without affecting fVIIa binding. This study has for the first time shown that TF procoagulant activity is regulated through de-palmitoylation, which alters the orientation of its transmembrane domain and is independent of TF phosphorylation. However, Ser253 phosphorylation is facilitated by changes in the orientation of the transmembrane domain and can induce TF-cellular signalling that influences cellular proliferation/apoptosis.

Citation

Rondon, A. M., Ettelaie, C., Featherby, S., Rondon, A. M. R., Greenman, J., Versteeg, H. H., & Maraveyas, A. (2021). De-palmitoylation of tissue factor regulates its activity, phosphorylation and cellular functions. Cancers, 13(15), https://doi.org/10.3390/cancers13153837

Journal Article Type Article
Acceptance Date Jul 26, 2021
Online Publication Date Jul 30, 2021
Publication Date Aug 1, 2021
Deposit Date Aug 3, 2021
Publicly Available Date Aug 5, 2021
Journal Cancers
Electronic ISSN 2072-6694
Publisher MDPI
Peer Reviewed Peer Reviewed
Volume 13
Issue 15
Article Number 3837
DOI https://doi.org/10.3390/cancers13153837
Keywords Tissue factor; Factor VIIa; Encryption; Palmitoylation; Transmembrane-domain; Palmitoyl-protein thioesterase
Public URL https://hull-repository.worktribe.com/output/3815655
Publisher URL https://www.mdpi.com/2072-6694/13/15/3837

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Publisher Licence URL
https://creativecommons.org/licenses/by/4.0/

Copyright Statement
Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/)





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