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

Biochim Biophys Acta. 2016 Nov;1863(11):2846-2857. doi: 10.1016/j.bbamcr.2016.09.005. Epub 2016 Sep 4.


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.

Keywords: Microvesicles; Phosphorylation; Protein phosphatase 2A; Tissue factor; Ubiquitin-conjugating enzymes; Ubiquitination.

MeSH terms

  • Blood Coagulation* / drug effects
  • Cell Line, Tumor
  • Endothelial Cells / drug effects
  • Endothelial Cells / metabolism*
  • Female
  • Humans
  • Lysine
  • Oligopeptides / pharmacology
  • Phosphorylation
  • Protein Binding
  • Protein Phosphatase 2 / metabolism
  • Proto-Oncogene Proteins c-mdm2 / metabolism
  • Receptor, PAR-2 / agonists
  • Receptor, PAR-2 / metabolism
  • Serine
  • Thromboplastin / chemistry
  • Thromboplastin / genetics
  • Thromboplastin / metabolism*
  • Time Factors
  • Transfection
  • Ubiquitination


  • 2-furoyl-LIGRLO-amide
  • Oligopeptides
  • Receptor, PAR-2
  • Serine
  • Thromboplastin
  • MDM2 protein, human
  • Proto-Oncogene Proteins c-mdm2
  • Protein Phosphatase 2
  • Lysine