Abstract

We have previously shown that phosphorylation of tissue factor (TF) at Ser253 increases the incorporation of TF into microvesicles (MVs) following protease-activated receptor 2 (PAR2) activation through a process involving filamin A, whereas phosphorylation of TF at Ser258 suppresses this process. Here, we examined the contribution of the individual phosphorylation of these serine residues to the interaction between filamin A and TF, and further examined how filamin A regulates the incorporation of TF into MVs. In vitro binding assays using recombinant filamin A C-terminal repeats 22-24 with biotinylated phospho-TF cytoplasmic domain peptides as bait showed that filamin A had the highest binding affinities for phospho-Ser253 and double-phosphorylated TF peptides, while the phospho-Ser258 TF peptide had the lowest affinity. Analysis of MDA-MB-231 cells using an in situ proximity ligation assay revealed increased proximity between the C-terminus of filamin A and TF following PAR2 activation, which was concurrent with Ser253 phosphorylation and TF-positive MV release from these cells. Knock-down of filamin A expression suppressed PAR2-mediated increases in cell surface TF procoagulant activity without reducing cell surface TF antigen expression. Disrupting lipid rafts by pre-incubation with methyl-β-cyclodextrin prior to PAR2 activation reduced TF-positive MV release and cell surface TF procoagulant activity to the same extent as filamin A knock-down. In conclusion, this study shows that the interaction between TF and filamin A is dependent on the differential phosphorylation of Ser253 and Ser258. Furthermore, the interaction of TF with filamin A may translocate cell surface TF to cholesterol-rich lipid rafts, increasing cell surface TF activity as well as TF incorporation and release into MVs.

Highlights

  • We have demonstrated that the presence of filamin-A is crucial for tissue factor (TF) to be incorporated into MVs in response to PAR2 activation [9]

  • The phosphoSer258 TF peptide exhibited the lowest affinity for filamin-A (Kd = 7.34±0.79 μM), with the non-phosphorylated TF peptide having an intermediate affinity (Kd = 3.63±0.93 μM) (n=8)

  • Antibody competed out the binding of the phospho-Ser253 TF peptide, indicating that the binding was mediated through repeats 22-24 of filamin-A (Figure 1D)

Read more

Summary

Objectives

The aim of this study was to determine whether the individual phosphorylation of TF at. Since filamin-A binds to the cytoplasmic domain of TF in a phosphorylationdependent manner [12] the main aim of this study was to examine the individual role of. In future experiments we aim to examine the lipid raft distribution of cell surface TF following suppression of filamin-A expression

Methods
Results
Discussion
Conclusion
Full Text
Paper version not known

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call

Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.