Phosphorylation of Human Pro-Urokinase on Ser138/303 Impairs Its Receptor-dependent Ability to Promote Myelomonocytic Adherence and Motility

doi:10.1083/jcb.137.3.779

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© The Rockefeller University Press, 0021-9525/1997//779 $5.00
The Journal of Cell Biology, Volume 137, Number 3, , 1997 779-791

Article

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 A431human carcinoma cells results in a catalytically active moleculewith reduced sensitivity to plasminogen activator inhibitortype 1. We mapped the phosphorylated seryl residues by analyzing the in vivo phosphorylation state of engineered prouPA variantscarrying a COOH-terminal poly-histidine tag. Stably transfectedA431 cells do not incorporate radioactive phosphate into taggedpro-uPA in which the serines 138 and 303 have been replacedwith glutamic residues, although endogenous nontagged pro-uPAis ³²P-labeled on A and B chains. Moreover, the catalyticindependentability 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 glutamicresidues as well as the naturally occurring phosphoserinesat positions 138 and 303 abolish proadhesive ability, althoughthey do not interfere with receptor binding. In addition, pro-uPAcarrying Glu^138/303 lacks the capability to induce a chemotacticresponse 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 positionplays a major inhibitory role of myeloid cell response to pro-urokinase.The di-substitution does not affect pro-uPA ability to interactwith vitronectin or to enhance binding of urea-denatured vitronectinto uPAR. However, unlike wild-type tagged pro-uPA, the di-substitutedvariant does not induce receptor polarization in pre-adherentU937 cells. Taken together, the data support the possibilitythat pro-uPA phosphorylation on Ser^138/303 can modulate uPARtransducing ability.

1. Abbreviations used in this paper: DFP, diisopropylfluorophosphate; GPI, glycosylphosphatidylinositol; His-pro-uPA^wt, histidine-taggedwildtype pro-uPA; PAI-1, plasminogen activator inhibitor type1; Pro-uPA, pro-urokinase; Pro-uPA^wt, wild-type pro-urokinase;Pser-uPA, serinephosphorylated uPA; Ser-uPA, nonphosphorylateduPA; uPA, urokinase.

The work was supported by Progetto Finalizzato ApplicazioniCliniche della Ricerca Oncologica (CNR), XI Progetto AIDS (ISS),and Associazione Italiana per la Ricerca sul Cancro (AIRC).

First authorship is equally shared by P. Franco and C. Iaccarino.

Please address all correspondence to M.P. Stoppelli, InternationalInstitute of Genetics and Biophysics, Via Marconi, 10-80125,Napoli, Italy. Tel.: 39 81 7257260. Fax: 39 81 5936123. E-mail:stoppelli{at}iigbna.iigb.na.cnr.it

The current address of M.R. Mastronicola is Institute of Biology,2nd University of Naples, Via Arena, 18-81100 Caserta, Italy.

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