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Phosphorylation of human pro-urokinase on Ser138/303 impairs its receptor-dependent ability to promote myelomonocytic adherence and motility.

Franco P, Iaccarino C, Chiaradonna F, Brandazza A, Iavarone C, Mastronicola MR, Nolli ML, Stoppelli MP - J. Cell Biol. (1997)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: International Institute of Genetics and Biophysics, Consiglio Nazionale delle Ricerche, Napoli, Italy.

ABSTRACT
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 pro-uPA 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 catalytic-independent ability of the mono- and di-substituted "phosphorylation-like" variants to bind to the GPI-anchored 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 Glu138/303 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 Ser138/303 can modulate uPAR transducing ability.

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uPAR binding ability of phosphorylated and  “phosphorylation-like” prouPA variants. (A) 30 × 106  subconfluent A431 cells  were metabolically labeled  with [32P]orthophosphate for  24 h, the conditioned medium was then removed, and  receptor-bound pro-uPA was  extracted by treating the cells  with a total of 10 ml of acidic  buffer for 5 min. Half of the  neutralized acid wash was incubated with 5B4-agarose  (lane 2) or glycine-blocked  agarose (lane 1), and the resulting matrix-bound proteins were analyzed by 12.5%  SDS-PAGE under reducing  conditions. (B) Serine phosphorylated pro-uPA was purified from A431 cell line by  the Fe3+ chromatography  procedure, whereas the histidine-tagged proteins were purified by Ni-NTA chromatography from the conditioned medium of HeLa stable transfectants. The result of a competition between 125I-ATF (105 cpm/sample) and the indicated nanomolar concentrations of unlabeled urinary uPA (•), Pser-uPA (▴), His-pro-uPAwt (□ ), His-pro-uPA138E(▵), His-pro-uPA138E/303E (○) to U937 cell uPARs is  shown. Cell-bound radioactivity is reported as a percentage of the maximal binding in the absence of competitor (125I-ATF specific  binding to control cells in the absence of competitor, 3,000 dpm). Data are shown as the mean of three independent experiments performed in duplicate; standard deviations are indicated by error bars.
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Figure 4: uPAR binding ability of phosphorylated and “phosphorylation-like” prouPA variants. (A) 30 × 106 subconfluent A431 cells were metabolically labeled with [32P]orthophosphate for 24 h, the conditioned medium was then removed, and receptor-bound pro-uPA was extracted by treating the cells with a total of 10 ml of acidic buffer for 5 min. Half of the neutralized acid wash was incubated with 5B4-agarose (lane 2) or glycine-blocked agarose (lane 1), and the resulting matrix-bound proteins were analyzed by 12.5% SDS-PAGE under reducing conditions. (B) Serine phosphorylated pro-uPA was purified from A431 cell line by the Fe3+ chromatography procedure, whereas the histidine-tagged proteins were purified by Ni-NTA chromatography from the conditioned medium of HeLa stable transfectants. The result of a competition between 125I-ATF (105 cpm/sample) and the indicated nanomolar concentrations of unlabeled urinary uPA (•), Pser-uPA (▴), His-pro-uPAwt (□ ), His-pro-uPA138E(▵), His-pro-uPA138E/303E (○) to U937 cell uPARs is shown. Cell-bound radioactivity is reported as a percentage of the maximal binding in the absence of competitor (125I-ATF specific binding to control cells in the absence of competitor, 3,000 dpm). Data are shown as the mean of three independent experiments performed in duplicate; standard deviations are indicated by error bars.

Mentions: The localization of pro-uPA phosphorylation sites does not suggest functional consequences on uPAR binding, as the growth factor-like domain does not include any phosphoserine. However, given the possibility that negatively charged side chains may trigger conformational changes even at distant sites, we analyzed the uPAR binding ability of phosphorylated pro-uPA. Previous work has shown that A431 human carcinoma cells synthesize pro-uPA and bind it to cell surface receptors in an autocrine fashion. According to a previously published protocol, receptor-bound pro-uPA can be extracted by a short acidic treatment of A431 cells (Stoppelli et al., 1986). Here we demonstrate that acid-released pro-uPA from the surface of A431 cells, which have been metabolically labeled with [32P]orthophosphate, is indeed 32P-labeled. Fig. 4 A shows the immunoprecipitated 47-kD protein from neutralized acid wash of 32P-labeled A431 cells with 5B4 anti-uPA antibody (lane 2), analyzed onto a 12.5% SDS-PAGE under reducing conditions. Incubation of the same extract with an irrelevant antibody confirms the specificity of the reaction (lane 1); however, this experiment could only detect a severe impairment of receptor binding ability, but it does not draw any conclusion on the affinity of phosphorylated prouPA for uPAR. To address this question, conditioned medium from A431 cells was fractionated with the Fe3+ Sepharose chromatography. As previously published, this matrix only retains phosphorylated uPA (Pser-uPA) which can be subsequently eluted by raising the pH and washing the column with a phosphate buffer (Franco et al., 1992). A further immunoaffinity step allows purified phosphorylated uPA to recover and to test its affinity for uPAR. No major differences in the relative Kds for uPAR were detected in binding assays in which unlabeled Pser-uPA or human urinary uPA were used as competitors of 125I-ATF binding to monocyte-like U937 cells, at the concentrations reported in Fig. 4 B.


Phosphorylation of human pro-urokinase on Ser138/303 impairs its receptor-dependent ability to promote myelomonocytic adherence and motility.

Franco P, Iaccarino C, Chiaradonna F, Brandazza A, Iavarone C, Mastronicola MR, Nolli ML, Stoppelli MP - J. Cell Biol. (1997)

uPAR binding ability of phosphorylated and  “phosphorylation-like” prouPA variants. (A) 30 × 106  subconfluent A431 cells  were metabolically labeled  with [32P]orthophosphate for  24 h, the conditioned medium was then removed, and  receptor-bound pro-uPA was  extracted by treating the cells  with a total of 10 ml of acidic  buffer for 5 min. Half of the  neutralized acid wash was incubated with 5B4-agarose  (lane 2) or glycine-blocked  agarose (lane 1), and the resulting matrix-bound proteins were analyzed by 12.5%  SDS-PAGE under reducing  conditions. (B) Serine phosphorylated pro-uPA was purified from A431 cell line by  the Fe3+ chromatography  procedure, whereas the histidine-tagged proteins were purified by Ni-NTA chromatography from the conditioned medium of HeLa stable transfectants. The result of a competition between 125I-ATF (105 cpm/sample) and the indicated nanomolar concentrations of unlabeled urinary uPA (•), Pser-uPA (▴), His-pro-uPAwt (□ ), His-pro-uPA138E(▵), His-pro-uPA138E/303E (○) to U937 cell uPARs is  shown. Cell-bound radioactivity is reported as a percentage of the maximal binding in the absence of competitor (125I-ATF specific  binding to control cells in the absence of competitor, 3,000 dpm). Data are shown as the mean of three independent experiments performed in duplicate; standard deviations are indicated by error bars.
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Related In: Results  -  Collection

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Figure 4: uPAR binding ability of phosphorylated and “phosphorylation-like” prouPA variants. (A) 30 × 106 subconfluent A431 cells were metabolically labeled with [32P]orthophosphate for 24 h, the conditioned medium was then removed, and receptor-bound pro-uPA was extracted by treating the cells with a total of 10 ml of acidic buffer for 5 min. Half of the neutralized acid wash was incubated with 5B4-agarose (lane 2) or glycine-blocked agarose (lane 1), and the resulting matrix-bound proteins were analyzed by 12.5% SDS-PAGE under reducing conditions. (B) Serine phosphorylated pro-uPA was purified from A431 cell line by the Fe3+ chromatography procedure, whereas the histidine-tagged proteins were purified by Ni-NTA chromatography from the conditioned medium of HeLa stable transfectants. The result of a competition between 125I-ATF (105 cpm/sample) and the indicated nanomolar concentrations of unlabeled urinary uPA (•), Pser-uPA (▴), His-pro-uPAwt (□ ), His-pro-uPA138E(▵), His-pro-uPA138E/303E (○) to U937 cell uPARs is shown. Cell-bound radioactivity is reported as a percentage of the maximal binding in the absence of competitor (125I-ATF specific binding to control cells in the absence of competitor, 3,000 dpm). Data are shown as the mean of three independent experiments performed in duplicate; standard deviations are indicated by error bars.
Mentions: The localization of pro-uPA phosphorylation sites does not suggest functional consequences on uPAR binding, as the growth factor-like domain does not include any phosphoserine. However, given the possibility that negatively charged side chains may trigger conformational changes even at distant sites, we analyzed the uPAR binding ability of phosphorylated pro-uPA. Previous work has shown that A431 human carcinoma cells synthesize pro-uPA and bind it to cell surface receptors in an autocrine fashion. According to a previously published protocol, receptor-bound pro-uPA can be extracted by a short acidic treatment of A431 cells (Stoppelli et al., 1986). Here we demonstrate that acid-released pro-uPA from the surface of A431 cells, which have been metabolically labeled with [32P]orthophosphate, is indeed 32P-labeled. Fig. 4 A shows the immunoprecipitated 47-kD protein from neutralized acid wash of 32P-labeled A431 cells with 5B4 anti-uPA antibody (lane 2), analyzed onto a 12.5% SDS-PAGE under reducing conditions. Incubation of the same extract with an irrelevant antibody confirms the specificity of the reaction (lane 1); however, this experiment could only detect a severe impairment of receptor binding ability, but it does not draw any conclusion on the affinity of phosphorylated prouPA for uPAR. To address this question, conditioned medium from A431 cells was fractionated with the Fe3+ Sepharose chromatography. As previously published, this matrix only retains phosphorylated uPA (Pser-uPA) which can be subsequently eluted by raising the pH and washing the column with a phosphate buffer (Franco et al., 1992). A further immunoaffinity step allows purified phosphorylated uPA to recover and to test its affinity for uPAR. No major differences in the relative Kds for uPAR were detected in binding assays in which unlabeled Pser-uPA or human urinary uPA were used as competitors of 125I-ATF binding to monocyte-like U937 cells, at the concentrations reported in Fig. 4 B.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: International Institute of Genetics and Biophysics, Consiglio Nazionale delle Ricerche, Napoli, Italy.

ABSTRACT
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 pro-uPA 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 catalytic-independent ability of the mono- and di-substituted "phosphorylation-like" variants to bind to the GPI-anchored 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 Glu138/303 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 Ser138/303 can modulate uPAR transducing ability.

Show MeSH
Related in: MedlinePlus