Endogenous phosphorylation of the lipoprotein-associated coagulation inhibitor at serine-2.
Lipoprotein-associated coagulation inhibitor (LACI) inhibits activated Factor X (Xa) directly and, in an Xa-dependent fashion, inhibits Factor VIIa-tissue factor (TF), presumably by forming a quaternary Xa-LACI-VIIa-TF complex. LACI isolated from the conditioned media of HepG2 cells grown in the presence of [32P]orthophosphate was observed to be covalently phosphorylated. Dephosphorylation of 32P-LACI with phosphatase resulted in an almost complete removal of the radiolabel. Phosphoamino acid analysis of the purified 32P-LACI established that the phosphorylation occurred on (a) serine residue(s). At its N-terminus, LACI contains a cluster of acidic residues C-terminal to the serine-2 residue. Such a site is characteristic of the sites phosphorylated by casein kinase II (CKII) in protein substrates. Edman degradation of endogenously labelled 32P-LACI revealed that the serine-2 residue was a major site of phosphorylation. Phosphorylation of purified LACI by bovine CKII was observed to occur in vitro; amino acid sequence analysis demonstrated that CKII phosphorylated LACI at the serine-2 residue. Recombinant LACI expressed from mouse C127 fibroblasts transfected using a bovine-papilloma-virus expression vector was found to be endogenously phosphorylated. By using site-directed mutagenesis, an altered form of LACI was produced in which the serine-2 residue had been changed to alanine. This altered LACI, although expressed in similar quantity to the wild-type LACI, was not detectably phosphorylated. Using the altered LACI in functional studies demonstrated that a serine residue at position 2, and thus the phosphorylation of this site, was not essential for LACI's inhibition of Xa and VIIa-TF activities.
Girard
T J
TJ
Division of Hematology/Oncology, Washington University School of Medicine, Jewish Hospital, St. Louis, MO 63110.
McCourt
D
D
Novotny
W F
WF
MacPhail
L A
LA
Likert
K M
KM
Broze
G J
GJ
D015153:Blotting, Western; D047388:Casein Kinases; D005167:Factor VII / Q000037:antagonists & inhibitors* / Q000378:metabolism; D015942:Factor VIIa / Q000037:antagonists & inhibitors; D015951:Factor Xa / Q000378:metabolism; D065427:Factor Xa Inhibitors; D006801:Humans; D066298:In Vitro Techniques; D008074:Lipoproteins / Q000378:metabolism*; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D013329:Structure-Activity Relationship; D013925:Thromboplastin / Q000037:antagonists & inhibitors* / Q000378:metabolism; D014407:Tumor Cells, Cultured
D065427:Factor Xa Inhibitors; D008074:Lipoproteins; C051928:lipoprotein-associated coagulation inhibitor; D010768:Phosphoserine; D012694:Serine; D005167:Factor VII; D013925:Thromboplastin; D011494:Protein Kinases; D047388:Casein Kinases; D015942:Factor VIIa; D015951:Factor Xa