Tissue factor cytoplasmic domain peptide is multiply phosphorylated in vitro

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Signaling

Background:

• Tissue factor (TF) is a key initiator of the extrinsic coagulation pathway.
• The cytoplasmic domain of TF plays a role in its cellular functions, including signaling.
• Post-translational modifications like phosphorylation can regulate protein activity.

Purpose of the Study:

• To investigate the phosphorylation of human tissue factor (TF) in vitro.
• To identify the specific sites and extent of TF phosphorylation.
• To characterize the phosphorylation of the TF cytoplasmic domain using a synthetic peptide.

Main Methods:

• Incubation of human TF with U87-MG cell lysates and fractions.
• Preparative isoelectric focusing (IEF) for peptide separation.
• Synthesis of a carboxyl-terminal cytoplasmic domain peptide (TF245-263).
• In vitro phosphorylation assays using radiolabeled ATP and cell lysates.
• Analysis of phosphorylation patterns, including diphosphate forms.

Main Results:

• Human TF was phosphorylated when incubated with U87-MG cell lysates.
• A synthetic peptide of the TF cytoplasmic domain (TF245-263) was identified as a protein kinase substrate.
• The TF245-263 peptide was phosphorylated at three serine residues, with a diphosphate form also detected.
• Phosphorylation occurred independently of calcium and protein kinase C.

Conclusions:

• Tissue factor can be multiply phosphorylated in vitro.
• The synthetic TF245-263 cytoplasmic domain peptide serves as a valuable model substrate for studying TF phosphorylation.
• These findings contribute to understanding the regulation of tissue factor activity through phosphorylation.