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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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Effect of phosphorylation on the ability of pro-uPA to  promote adhesion of differentiating U937 cells. (A) U937 cells  were grown to 0.8 × 106 cells/ml and then diluted 1:2 and treated  with TGF-β/vitamin D3 for 20 h. Then, they were incubated with  the following effectors at the concentration of 1 nM: His-pro-uPAwt,  His-pro-uPA138E, His-pro-uPA138E/303E, Ser-uPA (nonphosphorylated pro-uPA), Pser-uPA (phosphorylated pro-uPA), and DFPPser-uPA (DFP-inactivated Pser-uPA). Control samples included a combination of 5 nM His-pro-uPA138E/303E and 1 nM  His-pro-uPAwt (WT+138E/303E) and a preincubation of the  cells with 10 μg/ml anti-uPAR polyclonal antibody for 1 h before  the addition of 1 nM His-pro-uPAwt (WT+399Ab). The number  of adherent and non-adherent cells was counted 30 min later and  reported as a percentage of the maximal adherence observed  (37.5% of the total cell number, with 1 nM His-pro-uPAwt, over a  background of 12.6% due to the TGF-β/vitamin D3 addition).  The data represent the average of three experiments performed  in duplicate with standard deviations indicated by error bars. (B)  U937 cells were primed for the previous experiment and incubated with increasing concentrations of His-pro-uPAwt (□) or  His-pro-uPA138E/303E (○). The basal adherence of TGF-β/vitamin  D3-treated cells was 11.7 (SD = 2.12). The number of adherent  cells is reported as a percentage of the total cell number.
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Figure 5: Effect of phosphorylation on the ability of pro-uPA to promote adhesion of differentiating U937 cells. (A) U937 cells were grown to 0.8 × 106 cells/ml and then diluted 1:2 and treated with TGF-β/vitamin D3 for 20 h. Then, they were incubated with the following effectors at the concentration of 1 nM: His-pro-uPAwt, His-pro-uPA138E, His-pro-uPA138E/303E, Ser-uPA (nonphosphorylated pro-uPA), Pser-uPA (phosphorylated pro-uPA), and DFPPser-uPA (DFP-inactivated Pser-uPA). Control samples included a combination of 5 nM His-pro-uPA138E/303E and 1 nM His-pro-uPAwt (WT+138E/303E) and a preincubation of the cells with 10 μg/ml anti-uPAR polyclonal antibody for 1 h before the addition of 1 nM His-pro-uPAwt (WT+399Ab). The number of adherent and non-adherent cells was counted 30 min later and reported as a percentage of the maximal adherence observed (37.5% of the total cell number, with 1 nM His-pro-uPAwt, over a background of 12.6% due to the TGF-β/vitamin D3 addition). The data represent the average of three experiments performed in duplicate with standard deviations indicated by error bars. (B) U937 cells were primed for the previous experiment and incubated with increasing concentrations of His-pro-uPAwt (□) or His-pro-uPA138E/303E (○). The basal adherence of TGF-β/vitamin D3-treated cells was 11.7 (SD = 2.12). The number of adherent cells is reported as a percentage of the total cell number.

Mentions: As reported by others (Nusrat and Chapman, 1991), the proadhesive ability of uPA on differentiating myelomonocytic cell lines depends on receptor binding and is independent of uPA catalytic activity. However, the possibility that additional interactions involving uPA and matrix or membrane-associated proteins may concur to promote U937 cell adherence cannot be excluded. Cells were induced to differentiation with TGF-β/vitamin D3 for 20 h and then incubated in tissue culture multiwell plates for 30 min in the presence of 10% FBS, with or without 1 nM of various effectors. In the assay shown in Fig. 5 A, the adhesion promoted by 1 nM His-pro-uPAwt was taken as 100%. A single amino acid substitution at position 138 of histidine-tagged pro-uPA causes, at least, a 60% reduction of its proadhesive ability. It is noteworthy that His-prouPA138E/303E, in which the two amino acid substitutions are combined, fails to increase adhesion. Similar relative data were obtained using the same effectors at the concentration of 0.2 nM (not shown). The lack of stimulation is neither due to undesired mutations of the selected clones, nor to the COOH-terminal polyhistidine sequence, as it has been observed with untagged pro-uPA138E/303E purified from independent HeLa transfectants (not shown). Accordingly, A431 phosphorylated pro-uPA (Pser-uPA) shows a poor proadhesive ability with respect to the nonphosphorylated counterpart (Ser-uPA), at the same concentration (1 nM). In this case, the occurrence of nonphosphorylated molecules or molecules phosphorylated at single sites may likely account for the reduced, although still appreciable proadhesive effect of Pser-uPA, as compared to the disubstituted variant. Furthermore, the reduced proadhesive ability of Pser-uPA is not due to its inhibition-insensitive enzymatic activity, as it can be observed even after DFP treatment, which inactivates the residual two-chain activity. Consistently, the irreversible inactivation of two-chain uPA in the preparation of the phosphorylation-like variants did not alter the relative extent of the proadhesive effect. The proadhesive ability of pro-uPA is uPAR-dependent, as it can be prevented by pre-saturating the cells with anti-uPAR polyclonal antibody before the addition of the effectors. Also, 5 nM His-pro-uPA138E/303E can reverse the proadhesive effect of 1 nM pro-uPA, further supporting the possibility that binding to the receptor is required for the negative effect of the di-substituted variant. Fig. 5 B shows the percentage of differentiating U937 cells which have acquired adherence in the presence of increasing concentrations of His-pro-uPAwt or His-pro-uPA138E/303E. The results indicate that the di-substituted variant is indeed no longer capable to exert a proadhesive effect, even at concentrations 10-fold higher than those employed in the previous experiments. Further testing of the effects of phosphorylation on pro-uPA proadhesive ability employed THP-1 and HL-60 cell lines, all belonging to the myelomonocytic lineage. As shown in Table I, treatment of “primed” THP-1 and HL-60 cell lines with 0.2 nM His-pro-uPAwt under the conditions described for U937 cells causes 20– 25% of the cells to adhere to culture plates, in the presence of 10% FBS. A substantial reduction is observed with the monosubstituted variant His-pro-uPA138E, which approximately retains 20–25% of the wild-type proadhesive ability. As for U937 cell line, no proadhesive effect is exerted by the di-substituted 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)

Effect of phosphorylation on the ability of pro-uPA to  promote adhesion of differentiating U937 cells. (A) U937 cells  were grown to 0.8 × 106 cells/ml and then diluted 1:2 and treated  with TGF-β/vitamin D3 for 20 h. Then, they were incubated with  the following effectors at the concentration of 1 nM: His-pro-uPAwt,  His-pro-uPA138E, His-pro-uPA138E/303E, Ser-uPA (nonphosphorylated pro-uPA), Pser-uPA (phosphorylated pro-uPA), and DFPPser-uPA (DFP-inactivated Pser-uPA). Control samples included a combination of 5 nM His-pro-uPA138E/303E and 1 nM  His-pro-uPAwt (WT+138E/303E) and a preincubation of the  cells with 10 μg/ml anti-uPAR polyclonal antibody for 1 h before  the addition of 1 nM His-pro-uPAwt (WT+399Ab). The number  of adherent and non-adherent cells was counted 30 min later and  reported as a percentage of the maximal adherence observed  (37.5% of the total cell number, with 1 nM His-pro-uPAwt, over a  background of 12.6% due to the TGF-β/vitamin D3 addition).  The data represent the average of three experiments performed  in duplicate with standard deviations indicated by error bars. (B)  U937 cells were primed for the previous experiment and incubated with increasing concentrations of His-pro-uPAwt (□) or  His-pro-uPA138E/303E (○). The basal adherence of TGF-β/vitamin  D3-treated cells was 11.7 (SD = 2.12). The number of adherent  cells is reported as a percentage of the total cell number.
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Figure 5: Effect of phosphorylation on the ability of pro-uPA to promote adhesion of differentiating U937 cells. (A) U937 cells were grown to 0.8 × 106 cells/ml and then diluted 1:2 and treated with TGF-β/vitamin D3 for 20 h. Then, they were incubated with the following effectors at the concentration of 1 nM: His-pro-uPAwt, His-pro-uPA138E, His-pro-uPA138E/303E, Ser-uPA (nonphosphorylated pro-uPA), Pser-uPA (phosphorylated pro-uPA), and DFPPser-uPA (DFP-inactivated Pser-uPA). Control samples included a combination of 5 nM His-pro-uPA138E/303E and 1 nM His-pro-uPAwt (WT+138E/303E) and a preincubation of the cells with 10 μg/ml anti-uPAR polyclonal antibody for 1 h before the addition of 1 nM His-pro-uPAwt (WT+399Ab). The number of adherent and non-adherent cells was counted 30 min later and reported as a percentage of the maximal adherence observed (37.5% of the total cell number, with 1 nM His-pro-uPAwt, over a background of 12.6% due to the TGF-β/vitamin D3 addition). The data represent the average of three experiments performed in duplicate with standard deviations indicated by error bars. (B) U937 cells were primed for the previous experiment and incubated with increasing concentrations of His-pro-uPAwt (□) or His-pro-uPA138E/303E (○). The basal adherence of TGF-β/vitamin D3-treated cells was 11.7 (SD = 2.12). The number of adherent cells is reported as a percentage of the total cell number.
Mentions: As reported by others (Nusrat and Chapman, 1991), the proadhesive ability of uPA on differentiating myelomonocytic cell lines depends on receptor binding and is independent of uPA catalytic activity. However, the possibility that additional interactions involving uPA and matrix or membrane-associated proteins may concur to promote U937 cell adherence cannot be excluded. Cells were induced to differentiation with TGF-β/vitamin D3 for 20 h and then incubated in tissue culture multiwell plates for 30 min in the presence of 10% FBS, with or without 1 nM of various effectors. In the assay shown in Fig. 5 A, the adhesion promoted by 1 nM His-pro-uPAwt was taken as 100%. A single amino acid substitution at position 138 of histidine-tagged pro-uPA causes, at least, a 60% reduction of its proadhesive ability. It is noteworthy that His-prouPA138E/303E, in which the two amino acid substitutions are combined, fails to increase adhesion. Similar relative data were obtained using the same effectors at the concentration of 0.2 nM (not shown). The lack of stimulation is neither due to undesired mutations of the selected clones, nor to the COOH-terminal polyhistidine sequence, as it has been observed with untagged pro-uPA138E/303E purified from independent HeLa transfectants (not shown). Accordingly, A431 phosphorylated pro-uPA (Pser-uPA) shows a poor proadhesive ability with respect to the nonphosphorylated counterpart (Ser-uPA), at the same concentration (1 nM). In this case, the occurrence of nonphosphorylated molecules or molecules phosphorylated at single sites may likely account for the reduced, although still appreciable proadhesive effect of Pser-uPA, as compared to the disubstituted variant. Furthermore, the reduced proadhesive ability of Pser-uPA is not due to its inhibition-insensitive enzymatic activity, as it can be observed even after DFP treatment, which inactivates the residual two-chain activity. Consistently, the irreversible inactivation of two-chain uPA in the preparation of the phosphorylation-like variants did not alter the relative extent of the proadhesive effect. The proadhesive ability of pro-uPA is uPAR-dependent, as it can be prevented by pre-saturating the cells with anti-uPAR polyclonal antibody before the addition of the effectors. Also, 5 nM His-pro-uPA138E/303E can reverse the proadhesive effect of 1 nM pro-uPA, further supporting the possibility that binding to the receptor is required for the negative effect of the di-substituted variant. Fig. 5 B shows the percentage of differentiating U937 cells which have acquired adherence in the presence of increasing concentrations of His-pro-uPAwt or His-pro-uPA138E/303E. The results indicate that the di-substituted variant is indeed no longer capable to exert a proadhesive effect, even at concentrations 10-fold higher than those employed in the previous experiments. Further testing of the effects of phosphorylation on pro-uPA proadhesive ability employed THP-1 and HL-60 cell lines, all belonging to the myelomonocytic lineage. As shown in Table I, treatment of “primed” THP-1 and HL-60 cell lines with 0.2 nM His-pro-uPAwt under the conditions described for U937 cells causes 20– 25% of the cells to adhere to culture plates, in the presence of 10% FBS. A substantial reduction is observed with the monosubstituted variant His-pro-uPA138E, which approximately retains 20–25% of the wild-type proadhesive ability. As for U937 cell line, no proadhesive effect is exerted by the di-substituted 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