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BIOTECHNOLOGICAL AND PHYSIOLOGICAL STUDIES OF PRO-UROKINASE PHOSPHORYLATION

Objective


The urokinase plasminogen activation (uPA) system is composed of the activator itself, urokinase (uPA) and by several specific inhibitors (PAI-1 and PAI-2). In addition, a specific receptor has been identified for uPA. UPa is synthesized as a 431 amino acids inactive zymogen, pro-uPA, which can be bound to surface receptors (uPAR) and here activated by a single proteolytic cleavage. In the malignant A431 cell line pro-uPA is phosphorylated in serine.

Pro-uPA is phosphorylated in at least 2 sites, one on the amino terminal, low molecular weight 17 kD A chain, the other on the carboxy terminal 30 kDa B chain. Phosphorylation of pro-uPA occurs intracellularly, and the phosphorylated pro uPA can be chased into the medium. Binding to the pro-uPA receptor is not required for phosphorylation, as incubation with uPA receptor antagonist peptides does not inhibit accumulation of the phosphorylated pro-uPA in the culture medium.

Phosphorylation of uPA occurs at least in human cell lines, and this process is in part modulated by protein kinase C. Phosphorylation results in a constitutive activation of u-PA, which cannot be regulated by PAI-1 serine 137 and serine 303 have been identified as major phosphorylation sites in vivo.
Plasminogen activation is involved in the maintenance of the blood fluidity and in a series of events which are related and required for the migration and invasiveness of normal and tumor cells. We have recently discovered a novel feature of one of the two plasminogen activators, pro-urokinase, i.e. its ability to be phosphorylated in a serine residue. We propose to study this phenomenon in detail with the goal of identifying the structural features, the enzymes involved, the physiological role of the phosphorylation. Finally, we plan to mutagenize the pro-urokinase molecule with the aim of producing novel variants which may have important properties both for the fibrinolytic and for the invasion required activity of this enzyme.

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

UNIVERSITY OF COPENHAGEN
Address
Noerregade 10
1017 Koebenhavn K/copenhaegen
Denmark

Participants (2)

CONSEJO SUPERIOR DE INVESTIGACIONES CIENTIFICAS
Spain
Address
Calle Serrano 117
Madrid
NATIONAL RESEARCH COUNCIL OF ITALY
Italy
Address
Via Guglielmo Marconi 10
80125 Napoli