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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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Construction, expression, and selective purification of  polyhistidine-tagged pro-urokinase. (Upper part) In the BamHI– XhoI excised pcDNAneoI, a double strand oligonucleotide encoding the twelve COOH-terminal amino acids of pro-uPA followed by a “spacing-arm,” a stretch of six histidines and a stop  codon, was inserted. A large BamHI fragment containing the remainder of pro-uPA gene was subsequently inserted in the  unique BamHI site (see Materials and Methods). (Lower part)  The plasmids either encoding pro-uPA (pcDNAuPA) or Hispro-uPAwt (pcDNA6xHis-tagged uPA) were transiently transfected in HeLa cells: 48 h later, aliquots of the serum-free conditioned media from the transfectants were incubated with 5B4  anti-uPA (5B4) or with Ni-NTA (Ni2+) or with an irrelevant antibody (−), and the resulting samples were analyzed by SDSPAGE under reducing conditions.
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Figure 2: Construction, expression, and selective purification of polyhistidine-tagged pro-urokinase. (Upper part) In the BamHI– XhoI excised pcDNAneoI, a double strand oligonucleotide encoding the twelve COOH-terminal amino acids of pro-uPA followed by a “spacing-arm,” a stretch of six histidines and a stop codon, was inserted. A large BamHI fragment containing the remainder of pro-uPA gene was subsequently inserted in the unique BamHI site (see Materials and Methods). (Lower part) The plasmids either encoding pro-uPA (pcDNAuPA) or Hispro-uPAwt (pcDNA6xHis-tagged uPA) were transiently transfected in HeLa cells: 48 h later, aliquots of the serum-free conditioned media from the transfectants were incubated with 5B4 anti-uPA (5B4) or with Ni-NTA (Ni2+) or with an irrelevant antibody (−), and the resulting samples were analyzed by SDSPAGE under reducing conditions.

Mentions: Two complementary oligonucleotides (62 bases each) were synthesized which encode the carboxy-terminal sequence of pro-uPA followed by a “spacing-arm” (Gly-Ala-Gly) and six histidines before the stop codon, as diagrammed in Fig. 2. The two oligos were denatured at 95°C and slowly reassociated to allow the formation of a double strand oligo with BamHI– XhoI cohesive termini which has been inserted into the BamHI–XhoI excised pcDNA neoI vector, as depicted in the upper part of Fig. 2. For inframe fusion, a BamHI fragment containing most of the pro-uPA gene was introduced into the unique BamHI site to generate pcDNAneo-HisuPA plasmid encoding histidine-tagged pro-uPA (His-pro-uPAwt).


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)

Construction, expression, and selective purification of  polyhistidine-tagged pro-urokinase. (Upper part) In the BamHI– XhoI excised pcDNAneoI, a double strand oligonucleotide encoding the twelve COOH-terminal amino acids of pro-uPA followed by a “spacing-arm,” a stretch of six histidines and a stop  codon, was inserted. A large BamHI fragment containing the remainder of pro-uPA gene was subsequently inserted in the  unique BamHI site (see Materials and Methods). (Lower part)  The plasmids either encoding pro-uPA (pcDNAuPA) or Hispro-uPAwt (pcDNA6xHis-tagged uPA) were transiently transfected in HeLa cells: 48 h later, aliquots of the serum-free conditioned media from the transfectants were incubated with 5B4  anti-uPA (5B4) or with Ni-NTA (Ni2+) or with an irrelevant antibody (−), and the resulting samples were analyzed by SDSPAGE under reducing conditions.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Construction, expression, and selective purification of polyhistidine-tagged pro-urokinase. (Upper part) In the BamHI– XhoI excised pcDNAneoI, a double strand oligonucleotide encoding the twelve COOH-terminal amino acids of pro-uPA followed by a “spacing-arm,” a stretch of six histidines and a stop codon, was inserted. A large BamHI fragment containing the remainder of pro-uPA gene was subsequently inserted in the unique BamHI site (see Materials and Methods). (Lower part) The plasmids either encoding pro-uPA (pcDNAuPA) or Hispro-uPAwt (pcDNA6xHis-tagged uPA) were transiently transfected in HeLa cells: 48 h later, aliquots of the serum-free conditioned media from the transfectants were incubated with 5B4 anti-uPA (5B4) or with Ni-NTA (Ni2+) or with an irrelevant antibody (−), and the resulting samples were analyzed by SDSPAGE under reducing conditions.
Mentions: Two complementary oligonucleotides (62 bases each) were synthesized which encode the carboxy-terminal sequence of pro-uPA followed by a “spacing-arm” (Gly-Ala-Gly) and six histidines before the stop codon, as diagrammed in Fig. 2. The two oligos were denatured at 95°C and slowly reassociated to allow the formation of a double strand oligo with BamHI– XhoI cohesive termini which has been inserted into the BamHI–XhoI excised pcDNA neoI vector, as depicted in the upper part of Fig. 2. For inframe fusion, a BamHI fragment containing most of the pro-uPA gene was introduced into the unique BamHI site to generate pcDNAneo-HisuPA plasmid encoding histidine-tagged pro-uPA (His-pro-uPAwt).

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