title: Separation and characterization of nonphosphorylated and serine-phosphorylated urokinase. Catalytic properties and sensitivity to plasminogen activator inhibitor type 1.
abstract: Urokinase synthesized by human A431 epidermoid carcinoma cells is phosphorylated on serine (Mastronicola, M. R., Stoppelli, M. P., Migliaccio, A., Auricchio, F., and Blasi, F. (1990) FEBS Lett. 266, 109-114). To test the possibility that phosphorylation may have specific effects on urokinase function, the phosphorylated and nonphosphorylated forms of urokinase were separated by Fe(3+)-Sepharose chromatography. Both forms exhibit indistinguishable Km and kcat for plasminogen activation. On the other hand, their sensitivity toward the specific plasminogen activator inhibitor type 1 is different as assessed by measuring both the stability of the covalent complex and the residual enzymatic activity. Phosphorylated urokinase was 50% inhibited at a concentration of plasminogen activator inhibitor type 1 4-fold higher than nonphosphorylated urokinase (0.7 versus 0.15 nM). Furthermore about 10% of phosphorylated urokinase was resistant to plasminogen activator inhibitor type 1 at a concentration as high as 20 nM. Thus, phosphorylation affects urokinase sensitivity to plasminogen activator inhibitor type 1, therefore resulting in a net, although indirect, increase of urokinase activity. These results suggest the existence of a novel cellular regulatory mechanism of extracellular proteolysis.
authors.affiliation: International Institute of Genetics and Biophysics, Consiglio Nazionale delle Ricerche, Naples, Italy.
authors.forename: P
authors.initials: P
authors.lastname: Franco
authors.forename: M R
authors.initials: MR
authors.lastname: Mastronicola
authors.forename: D
authors.initials: D
authors.lastname: De Cesare
authors.forename: M L
authors.initials: ML
authors.lastname: Nolli
authors.forename: T C
authors.initials: TC
authors.lastname: Wun
authors.forename: P
authors.initials: P
authors.lastname: Verde
authors.forename: F
authors.initials: F
authors.lastname: Blasi
authors.forename: M P
authors.initials: MP
authors.lastname: Stoppelli
mesh_terms: D002384:Catalysis; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D015849:Plasminogen Inactivators / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014568:Urokinase-Type Plasminogen Activator / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D015849:Plasminogen Inactivators; D010768:Phosphoserine; D014568:Urokinase-Type Plasminogen Activator