Serine phosphorylation of biosynthetic pro‐urokinase from human tumor cells

Abstract

Phosphorylation is a potent mechanism regulating the activity of many intracellular enzymes. We have discovered that the product of the human urokinase plasminogen activator gene, pro-uPA, is phosphorylated in serine in at least two human cell lines. Phosphorylation occurs within the cell during biosynthesis, and phosphorylated intracellular pro-uPA is secreted into the medium. Of the secreted pro-uPA molecules, 20-50% are phosphorylated in serine, thus representing a meaningful fraction of the total biosynthetic pro-uPA. Although the sites of phosphorylation have not yet been determined, at least two such sites must exist; in fact plasmin cleavage of phosphorylated single chain pro-uPA yields a two chain uPA in which both chains are phosphorylated. A specific function for pro-uPA phosphorylation has not yet been identified; however, it is tempting to speculate that, as in many other cases, phosphorylation may affect the activity of the enzyme, its response to inhibitors or the conversion of pro-uPA zymogen to active two-chain uPA. This would represent an additional way of regulating extracellular proteolysis, an important pathway involved in both intra-and extravascular phenomena like fibrinolysis, cell migration and invasiveness.

Phosphorylation of human plasminogen activators and plasminogen

“…The results obtained are in line with those obtained by Mastronicola et al [12] in the case of P-Ser of u-PA where two phosphorylation sites have been demonstrated.…”

“…From the quantitative analysis on the level of phosphorylation obtained by HPLC, it can be concluded that only a limited number (1 or 2) of the Ser and Thr residues, present in the molecules analyzed, should be susceptible to phosphorylation in agreement with previous data on Ser-phosphorylation of u-PA molecules secreted by tumor cells [12].…”

“…Recent studies, performed in our [ 131 and other laboratories [12,, have shown that human urokinase-type PA can be phosphorylated in Tyr and/or Ser residues. In this paper, we have shown by two different approaches, HPLC and immunoblotting, that both PAS contain P-Ser residues, that u-PA and PG contain P-Thr residues and that, in addition to u-PA, also t-PA and PG contain P-Tyr residues.…”

Protein Kinase C-dependent in VivoPhosphorylation of Prourokinase Leads to the Formation of a Receptor Competitive Antagonist

“…Urokinase function is also subjected to post-translational control; we found that the human proenzyme undergoes intra-cellular serine phosphorylation in A431 human carcinoma cells, resulting in a reduction of its sensitivity to the inhibitor PAI-1 (22)(23)(24). According to other reports, phosphorylated urinary urokinase activates plasminogen with a greater catalytic efficiency and is neither inhibited by PAI-1 nor by PAI-2 (25).…”

“…It is possible that the conformation attained by the mature protein, either recombinant or secreted from A431 cells may not totally resemble that of intracellular pro-uPA (22). Alternatively, an unknown kinase may be responsible for the modification of Ser 303 in vivo, provided its activity is regulated by PKC.…”