title: Phosphorylation of protein S by platelet kinases enhances its activated protein C cofactor activity.
abstract: Protein S (PS) is a multifunctional plasma protein of the hemostatic and inflammatory pathways, although mechanisms for its regulation are poorly understood. Since certain plasma proteins are regulated through extracellular phosphorylation, we investigated whether the anticoagulant activity of PS is regulated through phosphorylation by platelet-secreted kinases. PS was phosphorylated on exposure to activated platelets or their releasates, as judged by immunoblotting for phospho-amino acids and PS. PS phosphorylation was reduced by specific inhibitors of casein kinase 1 (CK1) and casein kinase 2 (CK2) (10 μM D4476, 100 μM CK2-inhibitory peptide YNLKSKSSEDIDESS). Involvement of CKs in PS phosphorylation was confirmed using purified CK1/CK2. Phosphorylation of PS by purified CK1 did not affect its activated protein C (APC) cofactor activity in activated partial thromboplastin time assays in PS-depleted plasma. However, phosphorylation of PS by CK2 or by CK1/CK2 increased PS cofactor activity ∼1.5-fold (158.7±4.8%, P<0.01) or ∼2-fold (191.5±6.4%, P<0.0001), respectively. The APC cofactor activity of PS in PS-depleted plasma exposed to platelet-secreted kinases was enhanced, while CK2 but not CK1 inhibitors reduced APC cofactor activity. Mass spectrometry revealed a phosphorylated CK2 site at Thr37 within the N-terminal Gla-domain. Thus, platelet-mediated extracellular phosphorylation of PS is a potential mechanism by which its activity is regulated.
authors.affiliation: Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 N. Torrey Pines Rd, La Jolla, CA 92037, USA.
authors.forename: Fabian
authors.initials: F
authors.lastname: Stavenuiter
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Gale
authors.forename: Mary J
authors.initials: MJ
authors.lastname: Heeb
mesh_terms: D000595:Amino Acid Sequence; D001549:Benzamides / Q000494:pharmacology; D001665:Binding Sites / Q000235:genetics; D001792:Blood Platelets / Q000201:enzymology* / Q000378:metabolism; D047389:Casein Kinase I / Q000037:antagonists & inhibitors / Q000097:blood / Q000378:metabolism; D047390:Casein Kinase II / Q000037:antagonists & inhibitors / Q000097:blood / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D015151:Immunoblotting; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000494:pharmacology; D010314:Partial Thromboplastin Time; D010766:Phosphorylation; D011486:Protein C / Q000378:metabolism*; D011494:Protein Kinases / Q000097:blood / Q000378:metabolism*; D017293:Protein S / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013912:Threonine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: C493177:4-(4-(2,3-dihydrobenzo(1,4)dioxin-6-yl)-5-pyridin-2-yl-1H-imidazol-2-yl)benzamide; D001549:Benzamides; D007093:Imidazoles; D009842:Oligopeptides; D011486:Protein C; D017293:Protein S; D013912:Threonine; D011494:Protein Kinases; D047389:Casein Kinase I; D047390:Casein Kinase II
keywords: blood coagulation regulation; vitamin K-dependent protein