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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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Phosphorylation state of His-pro-uPAwt and His-prouPA138E/303E vs untagged pro-uPAwt in A431 stable clones. Subconfluent A431 cells, stably overexpressing His-pro-uPAwt (A) or  His-pro-uPA138E/303E (B), have been metabolically labeled with  either [35S]methionine or with [32P]phosphate for 18 h. The resulting conditioned medium was subjected to Ni-NTA chromatography to recover the histidine-tagged pro-uPAs. The Ni-NTA  excluded proteins were incubated with 5B4 anti-uPA antibody to  isolate untagged pro-uPAwt. Each sample, deriving from 0.5 ×  106 cells, has been analyzed onto a 12.5% SDS-PAGE under reducing conditions.
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Figure 3: Phosphorylation state of His-pro-uPAwt and His-prouPA138E/303E vs untagged pro-uPAwt in A431 stable clones. Subconfluent A431 cells, stably overexpressing His-pro-uPAwt (A) or His-pro-uPA138E/303E (B), have been metabolically labeled with either [35S]methionine or with [32P]phosphate for 18 h. The resulting conditioned medium was subjected to Ni-NTA chromatography to recover the histidine-tagged pro-uPAs. The Ni-NTA excluded proteins were incubated with 5B4 anti-uPA antibody to isolate untagged pro-uPAwt. Each sample, deriving from 0.5 × 106 cells, has been analyzed onto a 12.5% SDS-PAGE under reducing conditions.

Mentions: PcDNAneoI vector carrying either pro-uPA gene encoding His-pro-uPAwt or the mini-gene encoding His-prouPA138E/303E was stably transfected into A431 epidermoid carcinoma cell line. Two geneticin-resistant clones, producing 1.5–2 μg pro-uPA/106 cells in 18 h, were selected for further studies. To rule out the possibility that His-tagging may affect pro-uPA in vivo phosphorylation, a preliminary experiment was designed in which the phosphorylation level of His-pro-uPAwt was compared to that of untagged endogenous pro-uPAwt. In agreement with the previously reported phosphorylation pattern, in both cases, three bands can be observed under reducing conditions corresponding to the single (47 kD) and two-chain uPA (30 kD and 17 kD, respectively), following metabolic labeling with [35S]methionine or [32P]orthophosphate (Fig. 3 A).


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)

Phosphorylation state of His-pro-uPAwt and His-prouPA138E/303E vs untagged pro-uPAwt in A431 stable clones. Subconfluent A431 cells, stably overexpressing His-pro-uPAwt (A) or  His-pro-uPA138E/303E (B), have been metabolically labeled with  either [35S]methionine or with [32P]phosphate for 18 h. The resulting conditioned medium was subjected to Ni-NTA chromatography to recover the histidine-tagged pro-uPAs. The Ni-NTA  excluded proteins were incubated with 5B4 anti-uPA antibody to  isolate untagged pro-uPAwt. Each sample, deriving from 0.5 ×  106 cells, has been analyzed onto a 12.5% SDS-PAGE under reducing conditions.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139889&req=5

Figure 3: Phosphorylation state of His-pro-uPAwt and His-prouPA138E/303E vs untagged pro-uPAwt in A431 stable clones. Subconfluent A431 cells, stably overexpressing His-pro-uPAwt (A) or His-pro-uPA138E/303E (B), have been metabolically labeled with either [35S]methionine or with [32P]phosphate for 18 h. The resulting conditioned medium was subjected to Ni-NTA chromatography to recover the histidine-tagged pro-uPAs. The Ni-NTA excluded proteins were incubated with 5B4 anti-uPA antibody to isolate untagged pro-uPAwt. Each sample, deriving from 0.5 × 106 cells, has been analyzed onto a 12.5% SDS-PAGE under reducing conditions.
Mentions: PcDNAneoI vector carrying either pro-uPA gene encoding His-pro-uPAwt or the mini-gene encoding His-prouPA138E/303E was stably transfected into A431 epidermoid carcinoma cell line. Two geneticin-resistant clones, producing 1.5–2 μg pro-uPA/106 cells in 18 h, were selected for further studies. To rule out the possibility that His-tagging may affect pro-uPA in vivo phosphorylation, a preliminary experiment was designed in which the phosphorylation level of His-pro-uPAwt was compared to that of untagged endogenous pro-uPAwt. In agreement with the previously reported phosphorylation pattern, in both cases, three bands can be observed under reducing conditions corresponding to the single (47 kD) and two-chain uPA (30 kD and 17 kD, respectively), following metabolic labeling with [35S]methionine or [32P]orthophosphate (Fig. 3 A).

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