Phosphorylation of urokinase is not a limiting step in biosynthesis, but results in a reduced PAI-1 sensitivity

We have previously described that human pro-urokinase (pro-uPA) and urokinase (uPA) synthesized by A431 carcinoma and HT1080 fibrosarcoma cell lines are phosphorylated in serine. To increase the level of phosphorylated uPA produced in culture, we have transfected A431 cells with a plasmid containing the entire uPA gene driven by a constitutive viral promoter and screened the resulting clones by both fibrinolytic assay and immunoprecipitation with a monoclonal antibody (5B4) to uPA. Immunoprecipitation of the 32P labeled conditioned medium from A431-P1 clone yielded about 10 fold more Pser-uPA than the parental cells. To test whether phosphorylation affects the sensitivity of uPA to plasminogen activator type 1 (PAI-1), immunoaffinity purified 35S-uPA and 32P-uPA were incubated with PAI-1 and the complex formation was analysed by SDS-PAGE. We observed that a large proportion of the 35S-B chain was complexed with PAI-1 whereas very little 32P-B chain was associated with PAI-1, suggesting that phosphorylation may impair the ability of uPA to interact with PAI-1.