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Oligoubiquitination of tissue factor on Lys255 promotes Ser253-dephosphorylation and terminates TF release

Ettelaie, Camille; Collier, Mary E.W.; Featherby, Sophie; Greenman, John; Maraveyas, Anthony


Mary E.W. Collier

Sophie Featherby


Restriction of tissue factor (TF) activity at the cell surface and TF release are critical for prevention of excessive coagulation. This study examined the regulation of TF dephosphorylation and its release through ubiquitination. A plasmid containing the sequence to express the tandem protein TF-tGFP was mutated to include an arginine-substitution at Lys255 within TF. MDA-MB-231 cell line, and HCAEC endothelial cells were transfected and subsequently activated with PAR2-agonist peptide. The wild-type and mutant TF-tGFP were immunoprecipitated from the cell lysates and the ubiquitination and phosphorylation state of TF examined. Analysis of the proteins showed that arginine-substitution of Lys255 within TF prevented its ubiquitination while the wild-type TF-tGFP was oligoubiquitinated. The TF-associated oligoubiquitin chain was estimated to contain up to 4 ubiquitin units, with the linkage formed between Lys63 of one ubiquitin unit, and the C-terminus of the next unit. The Lys255 → Arg substitution of TF-tGFP prolonged the phosphorylation of Ser253 within TF, compared to the wild-type TF-tGFP, lengthened the presence of TF-tGFP at the cell surface and extended the duration of TF-tGFP release from cells following PAR2 activation. A biotinylated 19-mer peptide corresponding to the C-terminus of TF (TFc) was used as substrate to show that the ubiquitination of TF was mediated by the Ube2D family of E2-enzymes and involved Mdm2. Moreover, double-phosphorylation of TFc was prerequisite for ubiquitination, with subsequent dephosphorylation of Ser253 by phosphatase PP2A. In conclusion, oligoubiquitination of Lys255 within TF permits PP2A to bind and dephosphorylate Ser253 and occurs to terminate TF release and contain its activity.


Ettelaie, C., Collier, M. E., Featherby, S., Greenman, J., & Maraveyas, A. (2016). Oligoubiquitination of tissue factor on Lys255 promotes Ser253-dephosphorylation and terminates TF release. BBA - Molecular Cell Research, 1863(11), 2846-2857.

Journal Article Type Article
Acceptance Date Sep 2, 2016
Online Publication Date Sep 3, 2016
Publication Date 2016-11
Deposit Date Oct 19, 2016
Publicly Available Date Oct 19, 2016
Journal Biochimica et biophysica acta (BBA) - Molecular cell research
Print ISSN 0167-4889
Electronic ISSN 1879-2596
Publisher Elsevier
Peer Reviewed Peer Reviewed
Volume 1863
Issue 11
Pages 2846-2857
Keywords Tissue factor, Ubiquitination, Phosphorylation, Ubiquitin-conjugating enzymes, Protein phosphatase 2 A, Microvesicles
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Additional Information This article is maintained by: Elsevier; Article Title: Oligoubiquitination of tissue factor on Lys255 promotes Ser253-dephosphorylation and terminates TF release; Journal Title: Biochimica et Biophysica Acta (BBA) - Molecular Cell Research; CrossRef DOI link to publisher maintained version:; Content Type: article; Copyright: © 2016 Elsevier B.V.