J. Cell Biol.
© The Rockefeller University Press
0021-9525/97/05/779/13 $2.00
Volume 137, Number 3, May 5, 1997 779-791

Phosphorylation of Human Pro-Urokinase on Ser^138/303 Impairs Its Receptor-dependent Ability to Promote Myelomonocytic Adherence and Motility

Paola Franco,^* Ciro Iaccarino,^* Ferdinando Chiaradonna,^* Anna Brandazza, Carlo Iavarone,^* M. Rosaria Mastronicola,^* M. Luisa Nolli,^§ and M. Patrizia Stoppelli^*

^* International Institute of Genetics and Biophysics, Consiglio Nazionale delle Ricerche, Via Marconi, 10, Napoli, Italy;  Farmitalia Carlo Erba, Nerviano (Milano), Italy; and ^§ Lepetit Spa Research Center, Gerenzano (Varese), Italy

Serine phosphorylation of human pro-urokinase (pro-uPA) by A431 human carcinoma cells results in a catalytically active molecule with reduced sensitivity to plasminogen activator inhibitor type 1. We mapped the phosphorylated seryl residues by analyzing the in vivo phosphorylation state of engineered prouPA variants carrying a COOH-terminal poly-histidine tag. Stably transfected A431 cells do not incorporate radioactive phosphate into tagged pro-uPA in which the serines 138 and 303 have been replaced with glutamic residues, although endogenous nontagged pro-uPA is ³²P-labeled on A and B chains. Moreover, the catalyticindependent ability of the mono- and di-substituted "phosphorylation-like" variants to bind to the GPIanchored urokinase receptor (uPAR) and promote adherence of differentiating U937, HL-60, and THP-1 myelomonocytic cells was examined. We found that glutamic residues as well as the naturally occurring phosphoserines at positions 138 and 303 abolish proadhesive ability, although they do not interfere with receptor binding. In addition, pro-uPA carrying Glu^138/303 lacks the capability to induce a chemotactic response of THP-1 cells. The exclusive presence of Glu¹³⁸ reduces pro-uPA proadhesive and chemotactic ability by 70- 80%, indicating that a phosphoserine residue at the same position plays a major inhibitory role of myeloid cell response to pro-urokinase. The di-substitution does not affect pro-uPA ability to interact with vitronectin or to enhance binding of urea-denatured vitronectin to uPAR. However, unlike wild-type tagged pro-uPA, the di-substituted variant does not induce receptor polarization in pre-adherent U937 cells. Taken together, the data support the possibility that pro-uPA phosphorylation on Ser^138/303 can modulate uPAR transducing ability.

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