Spectroscopic Characterization of Successive Phosphorylation of the Tissue Factor Cytoplasmic Region

Abstract

Tissue Factor (TF) is well known for its role during the activation of the coagulation pathway, but it is also critical for tumor biology and inflammation through protease activated receptor (PAR) 2 signaling. This signaling function is modulated by the successive phosphorylation of residues Ser253 and Ser258 within the TF cytoplasmic region (TFCR). This paper reports how we used NMR and spectroscopic methods to investigate the structural propensities of the unphosphorylated and phosphorylated forms of the TFCR. When unphosphorylated, the TFCR forms a local hydrophobic collapse around Trp254 and an electropositive patch from the membrane proximal basic block (Arg246-Lys247) to the conserved PKCα consensus residue Lys255. Phosphorylation of Ser253 alters the charge characteristics of this membrane proximal region, thereby strengthening the interaction between residue Ala248 and the Trp254 aromatic group. Phosphorylation of the Ser258-Pro259 motif destabilizes a turn at the C-terminus to form an extended polyproline helical motif. Our data suggests that by changing both its charge and local structural propensity, covalent modifications of the TFCR can potentially regulate its association with the cellular membrane and its signaling partners.

Regulation of the Incorporation of Tissue Factor into Microparticles by Serine Phosphorylation of the Cytoplasmic Domain of Tissue Factor

“…4E). Therefore, these data indicate that the suppression of TF release by Ser 258 phosphorylation involves the coordination of signaling mechanisms by TF itself, although we cannot dismiss possible additional contributions from conformational changes within the TF protein (28).…”

“…Therefore, we suggest that as well as Ser 258 phosphorylation, dephosphorylation of Ser 253 also requires the induction of signaling mechanisms arising from PAR2 activation and may take up to 90 min to become effective. Such mechanisms may also involve alterations in the structure of the cytoplasmic domain of TF by proteins such as Pin1 (28) leading to dephosphorylation events (29) and acting as a timedependent switch (30). Overexpression of TF Ala258 in HCAEC did not lead to the suppression of TF release (Fig.…”

“…The phosphorylation of Ser 258 appears to either enhance the activity of phosphatases 1/2A (Fig. 4) or alternatively permit the dephosphorylation of Ser 253 through structural alterations within the cytoplasmic domain of TF (28). In support of this hypothesis, the phospho-Ser 258 -Pro 259 motif is a potential recognition site for the peptidylprolyl cis/trans isomerase-1 Pin1 (29), which may be capable of inducing conformational changes within proteins that contain the consensus motif (30).…”

“…Phosphorylation of Ser 258 may become possible as a consequence of structural alterations in the cytoplasmic domain of TF induced by the phosphorylation of Ser 253 (28). Similarly, dephosphorylation of phospho-Ser 253 may become permissible as a consequence of structural alterations brought about by Ser 258 phosphorylation.…”

Targeting clotting proteins in cancer therapy – progress and challenges

“…The human TF cytoplasmic domain is sequentially phosphorylated by protein kinase C at Ser 253 and by Pro-directed kinases, including p38 MAP kinase (74), at Ser 258 (75;76) and adopts a more compact structure upon phosphorylation (77). PAR2 activation induces TF phosphorylation (78), and in primary breast cancer biopsies Ser 258 phosphorylation status of TF is correlated with PAR2 expression and only found in patients with relapse after initial therapy (79).…”