Limits...
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.

Show MeSH

Related in: MedlinePlus

Cellular distribution of uPARs in preadherent U937 by immunofluorescence. Primed U937 cells were incubated in suspension  without (A and A′) or with 10 nM His-pro-uPAwt (B, B′, and D) or 10 nM His-pro-uPA138E/303E (C, C′, and E) for 1 h. After fixation, the  cells were incubated with anti-uPAR polyclonal antibodies, decorated with fluorescein-tagged antibodies, and mounted and examined  on a LSM 410 Zeiss confocal microscope. Single-focus images of representative fields (A, A′, B, B′, C, and C′) or sequential through- focus images of single cells (D and E) were captured.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2139889&req=5

Figure 8: Cellular distribution of uPARs in preadherent U937 by immunofluorescence. Primed U937 cells were incubated in suspension without (A and A′) or with 10 nM His-pro-uPAwt (B, B′, and D) or 10 nM His-pro-uPA138E/303E (C, C′, and E) for 1 h. After fixation, the cells were incubated with anti-uPAR polyclonal antibodies, decorated with fluorescein-tagged antibodies, and mounted and examined on a LSM 410 Zeiss confocal microscope. Single-focus images of representative fields (A, A′, B, B′, C, and C′) or sequential through- focus images of single cells (D and E) were captured.

Mentions: Like other GPI-anchored proteins, uPAR lateral mobility in the cell membrane allows its ligand-dependent migration toward specific regions of the cell (Myohanen et al., 1993). By the aid of immunofluorescence and confocal microscopy, we tested uPAR distribution in primed U937 cells, further treated with 10 nM His-pro-uPAwt or Hispro-uPA138E/303E for 1 h. Cells were fixed, incubated with affinity-purified anti-uPAR rabbit polyclonal antibody, and subsequently incubated with FITC-conjugated anti– rabbit IgG. Cells were kept in suspension throughout the entire procedure, so to exclude any effect of cell attachment on uPAR localization. First, the distribution of fluorescence in the 0.75-μm section at the cell equator was analyzed. Primed cells exhibited primarily a diffused staining pattern at the membrane level, with occasional concentration of fluorescence at one cell edge (Fig. 8, A and A′). After treatment with His-pro-uPAwt, uPAR staining became distinctly polarized in ∼70% of preadherent U937 cells (B and B′). On the contrary, treatment with the phosphorylation-like variant results in uPAR redistribution only in 20% of the cell population (C and C′). These values were calculated by taking sequential through-focus images of single cells, as fluorescent patches can be detected only by analyzing specific focal planes of preadherent cells (D). The data show that preadherent U937 cells undergo a ligand-dependent uPAR redistribution, which is severely impaired in cells treated with the phosphorylation-like, disubstituted pro-uPA variant.


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)

Cellular distribution of uPARs in preadherent U937 by immunofluorescence. Primed U937 cells were incubated in suspension  without (A and A′) or with 10 nM His-pro-uPAwt (B, B′, and D) or 10 nM His-pro-uPA138E/303E (C, C′, and E) for 1 h. After fixation, the  cells were incubated with anti-uPAR polyclonal antibodies, decorated with fluorescein-tagged antibodies, and mounted and examined  on a LSM 410 Zeiss confocal microscope. Single-focus images of representative fields (A, A′, B, B′, C, and C′) or sequential through- focus images of single cells (D and E) were captured.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 8: Cellular distribution of uPARs in preadherent U937 by immunofluorescence. Primed U937 cells were incubated in suspension without (A and A′) or with 10 nM His-pro-uPAwt (B, B′, and D) or 10 nM His-pro-uPA138E/303E (C, C′, and E) for 1 h. After fixation, the cells were incubated with anti-uPAR polyclonal antibodies, decorated with fluorescein-tagged antibodies, and mounted and examined on a LSM 410 Zeiss confocal microscope. Single-focus images of representative fields (A, A′, B, B′, C, and C′) or sequential through- focus images of single cells (D and E) were captured.
Mentions: Like other GPI-anchored proteins, uPAR lateral mobility in the cell membrane allows its ligand-dependent migration toward specific regions of the cell (Myohanen et al., 1993). By the aid of immunofluorescence and confocal microscopy, we tested uPAR distribution in primed U937 cells, further treated with 10 nM His-pro-uPAwt or Hispro-uPA138E/303E for 1 h. Cells were fixed, incubated with affinity-purified anti-uPAR rabbit polyclonal antibody, and subsequently incubated with FITC-conjugated anti– rabbit IgG. Cells were kept in suspension throughout the entire procedure, so to exclude any effect of cell attachment on uPAR localization. First, the distribution of fluorescence in the 0.75-μm section at the cell equator was analyzed. Primed cells exhibited primarily a diffused staining pattern at the membrane level, with occasional concentration of fluorescence at one cell edge (Fig. 8, A and A′). After treatment with His-pro-uPAwt, uPAR staining became distinctly polarized in ∼70% of preadherent U937 cells (B and B′). On the contrary, treatment with the phosphorylation-like variant results in uPAR redistribution only in 20% of the cell population (C and C′). These values were calculated by taking sequential through-focus images of single cells, as fluorescent patches can be detected only by analyzing specific focal planes of preadherent cells (D). The data show that preadherent U937 cells undergo a ligand-dependent uPAR redistribution, which is severely impaired in cells treated with the phosphorylation-like, disubstituted pro-uPA variant.

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