title: p38α phosphorylates serine 258 within the cytoplasmic domain of tissue factor and prevents its incorporation into cell-derived microparticles.
abstract: We previously showed that the phosphorylation of Ser253 within the cytoplasmic domain of human tissue factor (TF) initiates the incorporation and release of this protein into cell-derived microparticles. Furthermore, subsequent phosphorylation of Ser258 terminates this process. However, the identity of the kinase responsible for the phosphorylation of Ser258 and mode of action of this enzyme remain unknown. In this study, p38α was identified as the proline-directed kinase capable of phosphorylating Ser258 specifically, and without any detectable activity towards Ser253. Furthermore, using synthetic peptides, it was shown that the Km for the reaction decreased by approximately 10 fold on substitution of Ser253 with phospho-Ser253. Either inhibition of p38 using SB202190 or knockdown of p38α expression in coronary artery endothelial cells overexpressing wild-type TF, resulted in decreased phosphorylation of Ser258, following activation of cells with PAR2-agonist peptide (PAR2-AP). In agreement with our previous data, inhibition of phosphorylation of this residue maintained the release of TF. Activation of PAR2 in cells transfected to overexpress TF, resulted in two separate peaks of p38 activity at approximately 40 and 120 min post-activation. Furthermore, overexpression of Ala253-substituted TF enhanced the second p38 activation peak. However, the second peak was absent in cells devoid of TF or in cells overexpressing the Asp253-substituted TF. Our data clearly identifies p38α as a kinase capable of phosphorylating Ser258 within the cytoplasmic domain of TF. Moreover, it appears that the presence of TF within the cells regulates the late activation of p38 and consequently the termination of TF release into microparticles.
authors.affiliation: Biomedical Section, Department of Biological Sciences, University of Hull, Hull, HU6 7RX, UK. C.Ettelaie@hull.ac.uk
authors.forename: Camille
authors.initials: C
authors.lastname: Ettelaie
authors.forename: Azza M
authors.initials: AM
authors.lastname: Elkeeb
authors.forename: Anthony
authors.initials: A
authors.lastname: Maraveyas
authors.forename: Mary Elizabeth W
authors.initials: ME
authors.lastname: Collier
mesh_terms: D000595:Amino Acid Sequence; D015153:Blotting, Western; D055252:Cell-Derived Microparticles / Q000378:metabolism*; D002478:Cells, Cultured; D003331:Coronary Vessels / Q000166:cytology / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D048308:Mitogen-Activated Protein Kinase 14 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000494:pharmacology; D010766:Phosphorylation; D011487:Protein Conformation; D034741:RNA, Small Interfering / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000378:metabolism*; D015398:Signal Transduction; D013925:Thromboplastin / Q000378:metabolism*
chemical_list: C487404:2-furoyl-LIGRLO-amide; D009842:Oligopeptides; D034741:RNA, Small Interfering; D012694:Serine; D013925:Thromboplastin; D048308:Mitogen-Activated Protein Kinase 14