Separation and characterization of nonphosphorylated and serine-phosphorylated urokinase. Catalytic properties and sensitivity to plasminogen activator inhibitor type 1.
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.
Franco
P
P
International Institute of Genetics and Biophysics, Consiglio Nazionale delle Ricerche, Naples, Italy.
Mastronicola
M R
MR
De Cesare
D
D
Nolli
M L
ML
Wun
T C
TC
Verde
P
P
Blasi
F
F
Stoppelli
M P
MP
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
D015849:Plasminogen Inactivators; D010768:Phosphoserine; D014568:Urokinase-Type Plasminogen Activator