* International Institute of Genetics and Biophysics, Consiglio Nazionale delle Ricerche, Via Marconi, 10, Napoli, Italy; Farmitalia Carlo Erba, Nerviano (Milano), Italy; and § Lepetit Spa Research Center, Gerenzano (Varese), Italy
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 prouPA 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 catalyticindependent ability of the mono- and di-substituted "phosphorylation-like" variants to bind to the GPIanchored 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.
Urokinase (uPA)1, one of the two plasminogen activators, is a secreted serine protease which converts the latent ubiquitous zymogen plasminogen
to plasmin. Plasmin is, in turn, capable of degrading a variety of intravascular proteins, such as fibrin and extracellular matrix components, including fibronectin, laminin, proteoglycans, as well as activating type IV pro-collagenase, thus indirectly hydrolyzing collagen (Dano et al., 1985 Urokinase catalyzes the rate-limiting step of this proteolytic cascade, thereby regulating a variety of events that
require extracellular proteolysis, such as cell migration,
tissue remodeling and involution, and tumor metastatization (Blasi et al., 1994 Materials
Expression vector pcDNAIneo as well as the MC1061-P3 bacterial strain
were from Invitrogen (San Diego, CA). The oligonucleotides were from
Primm (Milan, Italy). CNBr-activated Sepharose 4B-CL, protein A-Sepharose, and Fe3+ chelating Sepharose were obtained from Pharmacia (Uppsala,
Sweden). Ni-NTA resin was from Quiagen, GmbH (Hilden, Germany).
Chromogenic substrates for plasmin (H-D-Nle-HHT-Lys-pNA.2AcOH),
uPA Elisa kit, #399 anti-uPAR polyclonal antibody, and the amino-terminal fragment of uPA (aa 1-135) were from American Diagnostica (Greenwich, CT). FITC-conjugated polyclonal goat anti-rabbit IgG were from Jackson Immunoresearch Laboratories (West Grove, PA). Anti-human vitronectin polyclonal antibody was from Calbiochem (San Diego, CA).
Recombinant pro-uPA was a gift of Dr. P. Sarmientos, Farmitalia (Milan,
Italy). Plasminogen was purified from serum as previously described
(Franco et al., 1992 Subcloning of pro-uPA Gene and Site-directed
Mutagenesis of Its cDNA
A SacII-SspI fragment encompassing the pro-uPA gene, lacking the nontranslated exon I, was excised from pRSV-uPA (Riccio et al., 1985
Polyhistidine Tagging of pro-uPA
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).
Construction of the Mini-Gene138E
A FspI-FspI cDNA fragment carrying the S138E mutation was purified
and ligated to pcDNAneo-His-uPA digested with FspI. This subcloning
allows the formation of the mini-gene138E (unit encoding pro-uPA138E) retaining 6 out of 10 pro-uPA gene introns (B, C, D, E, I , J).
Construction of the Mini-Gene138E/303E
The FspI-EcoRI cDNA fragment carrying the S138E mutation, together
with the EcoRI-BamHI fragment containing the S303E mutation, were ligated to the FspI-BamHI excised pcDNAneo-His-uPA plasmid. As depicted in Fig. 1, the resulting mini-gene138E/303E (unit encoding prouPA138E/303E ) retains introns B, C, D, and E.
Cell Cultures and Transfections
A431 human epidermoid carcinoma, HeLa human cervix carcinoma, and
mouse LB6-19 expressing human uPAR cell lines were grown in DMEM
containing 5% FBS. HL-60 promyelocytic leukemia, U937 histiocytic lymphoma, and THP-1 monocytic leukemia cell lines were cultured in RPMI
containing 10% heat-inactivated FBS. All cell lines were grown in the
presence of 100 U/ml penicillin and 100 µg/ml streptomycin in a 5% CO2
atmosphere.
Transient transfections of 2 × 106 subconfluent HeLa cells were performed by electroporation at 250 V, 960 µFaraday in the presence of 30 µg
of plasmid DNA, in 0.8 ml of cell culture medium per sample. After the
transfection, the cells were diluted up to 3 ml and plated in a 6-cm dish.
The medium was replaced 24 h later and the cells assayed 48 h after the
transfection. Stable A431 transfectants were obtained by electroporating
107 subconfluent A431 cells at 360 V, 960 µFaraday with 80 µg of plasmid
DNA, in 0.9 ml of culture medium. The cells were then diluted to 10 ml
and seeded in 10-cm dishes. 24 h later, the medium was replaced. The neomycin analogue G418 was added at 0.8 mg/ml 48 h after the transfection
and the medium was changed twice a week. Drug resistant colonies appeared after ~2-3 weeks. The pro-uPA secreted by the resistant clones
was quantitated by enzymatic and ELISA assays from the serum-free conditioned media.
Metabolic Cell Labeling and Immuno-affinity
Purification of pro-uPA
To metabolically label A431 and HeLa cells, they were seeded at a density
of 3 × 106/10-cm dish in 10-ml DMEM with 5% FBS. After 24 h, the culture medium was substituted with 5 ml of either methionine-free plus 500 µCi of [35S]methionine or phosphate-free DMEM plus 1 mCi of [32P]orthophosphate. The 18-h labeling was performed in the presence of 5% dialyzed FBS, 1 mM NaI, 100 mM orthovanadate, 5 µg/ml aprotinin. 32P- and
35S-labeled pro-uPAs were purified by immuno-affinity chromatography of the conditioned media with the specific 5B4 anti-uPA antibody, as
previously described (Nolli et al., 1986 Purification of Polyhistidine-tagged pro-uPA
Histidine-tagged pro-uPAs were isolated from the serum-free conditioned
medium of 35S-labeled A431 transfectants or unlabeled HeLa transfectants, by Ni-NTA chromatography, according to the manufacturer's instructions, with minor modifications (see also Janknecht et al., 1991 Purification of Phosphorylated pro-uPA
Phosphorylated pro-uPA from conditioned medium of A431 cells was purified by Fe3+ chelated Sepharose chromatography, as previously described (Franco et al., 1992 Quantitation and DFP-Inactivation of uPA
Quantitative enzymatic assays were performed at 25°C with 0.3 mM of the
plasmin chromogenic substrate H-D-Nle-HHT-Lys-pNA.2AcOH, in the
presence of 1.0 µM human plasminogen, using a 50-mM Tris-HCl buffer,
pH 7.5. The reaction was monitored by measuring the absorbance at 400 nm.
Standard curves with recombinant pro-uPA were used as a reference.
Quantitation of pro-uPA and uPA was also performed by a commercial
enzyme immunoassay, following the manufacturer's instructions (see Materials and Methods). Nonreversible inactivation of two-chain uPA with
DFP (diisopropylfluorophosphate) was performed according to Cubellis
et al. (1989) Radioiodination, Receptor Binding Assays,
and Ligand Blotting
125I-labeling of ATF, His-pro-uPAwt, His-pro-uPA138E/303E, and vitronectin
was performed with Na125I using Iodo-Gen, as previously described (Del
Vecchio et al., 1993). The radiolabeled proteins were purified from unbound iodide by Sephadex G-25 chromatography and stabilized with 0.2 mg/ml BSA. The resulting specific activity of ATF and histidine-tagged pro-uPAs was 22 × 106 cpm/µg, whereas specific activity of vitronectin
was 2 × 106 cpm/µg. Binding competitions to the uPA receptor were carried out as described in Stoppelli et al. (1985) Blotting of vitronectin and subsequent probing with 125I-His-pro-uPAwt
and 125I-His-pro-uPA138E/303E was performed according to Moser et al. (1995) Adhesion Assays
For "priming," exponentially growing U937 cells were diluted to 0.4 × 106
cells/ml in RPMI-10% FCS and treated with 1 ng/ml TGF- Chemotaxis Assays
The assays were performed using Boyden chambers with 5 µm pore size
polycarbonate filters coated with collagen type I, according to Resnati et al.
(1996) Immunofluorescence and Confocal Laser
Scanning Microscopy
"Primed" U937 cells were incubated with the indicated effectors at 1.5 × 106 cells/ml, for 1 h at 37°C in plastic tubes under gentle agitation. Then,
they were washed with PBS, fixed with 3% paraformaldehyde for 10 min
on ice, and incubated with 10 µg/ml affinity purified anti-uPAR #399 polyclonal antibody and subsequently with 12 µg/ml affinity-purified FITCconjugated polyclonal goat anti-rabbit IgG. The cells were then washed
twice with PBS and mounted with PBS/Mowiol 4-88 (2:1). To observe labeled cells, we have used a Zeiss laser scanning microscope (LSM 410 invert) equipped with a plan apo oil (100×) immersion lens (NA = 1.3).
FITC emission was excited using the argon laser 488 nm line. The emission signals were filtered with a Zeiss 510-525-nm filter (fluorescein emission). The images were processed by a Zeiss CLSM instrument software.
Nonspecific fluorescence was assessed by incubating the cells with the secondary FITC-labeled anti-rabbit antibody and measuring the average intensity value. Optical serial sections were collected with a step size of 0.75 µm/section; regularly, noise levels were reduced by 16 lines averaging of
the scans.
High Level Expression of COOH-Terminal
Histidine-tagged Pro-Urokinase and Its Variants
Early work from our laboratory has shown that the in vivo
32P-labeled pro-urokinase (pro-uPA) from A431 human
carcinoma cell line is phosphorylated at least on two serine
residues, being located within the A and the B chain, respectively (Mastronicola et al., 1990 To selectively analyze the in vivo phosphorylation state
of pro-uPA specific variants, we had to take into account
the endogenous high level of pro-uPA in A431 cell line.
Therefore, a polyhistidine tagging was designed, as it does
not contain any phosphorylatable amino acid and, also,
should not interfere with protein secretion, if inserted at
the COOH-terminal end of the protein (Bush et al., 1991 Identification of A431 Pro-Urokinase
Phosphoseryl Residues
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).
To assess the in vivo phosphorylation state of pro-urokinase in which neither Ser138 nor Ser303 are available any
longer, the phosphorylation state of His-pro-uPA138E/303E
was analyzed and endogenous pro-uPA (pro-uPAwt) was
used as a reference. This approach allows the simultaneous analysis of both proteins, so excluding any effect
due to clonal variability. 1 10-cm dish of subconfluent
A431 transfectants was labeled with 200 µCi/ml of [32P]orthophosphate and one dish was incubated with 100 µCi/ml
of [35S]methionine, to ensure an internal control of prouPA synthesis. In both cases, 18 h later, the conditioned
medium was subjected to the Ni-NTA chromatography to
isolate His-pro-uPA138E/303E; the unbound proteins have
been further incubated with the 5B4 anti-uPA monoclonal
antibody to recover endogenous untagged pro-uPAwt. The
resulting samples have been loaded onto a 12.5% SDSPAGE under reducing conditions. Fig. 3 B shows that the
recovered untagged pro-uPAwt is labeled with 35S and 32P.
Conversely, His-pro-uPA138E/303E is exclusively labeled
with 35S and not with 32P. Quantitation of the enzymatic
activity of immunoprecipitates confirmed that the same
amount of pro-uPAwt and His-pro-uPA138E/303E is indeed
present in the last two samples shown in Fig. 3 B (not shown). The lack of His-pro-uPA138E/303E in vivo phosphorylation clearly establishes that pro-uPA phosphorylation by A431 cells is totally dependent on the availability of
Ser138 and Ser303.
Receptor Binding Ability of Phosphorylated Pro-uPA
and Its "Phosphorylation-like" Variants
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
Although the functional analysis of in vivo phosphorylated uPA could benefit from the Fe3+ Sepharose chromatography technique, this procedure does not allow purification of pro-uPAs homogeneously phosphorylated at one
or both relevant sites. So, to investigate the functional consequences of pro-uPA phosphorylation at single sites, we
analyzed the above described phosphorylation-like prouPA variants in which glutamic residues are expected to
mimic the presence of phosphate groups (Maciejewski et
al., 1995 Effect of Phosphorylation on Pro-Urokinase Ability to
Induce Adhesion of Differentiating Myeloid Cells
As reported by others (Nusrat and Chapman, 1991
Table I.
Effect of Phosphorylation on Pro-uPA-promoted
Adherence of TGF-).
). Increasing evidence shows that the
complex regulation of uPA activity and localization is accomplished through the integration of multiple modulators. These include specific inhibitors of the proteolytic activity, namely plasminogen activator inhibitor type 1 and 2 (PAI-1 and PAI-2) and protease nexin (PN), which directly interact with uPA catalytic moiety (Blasi et al., 1987
).
Cell surface-associated plasminogen activation is achieved
through the high affinity binding of uPA to the GPI-anchored
urokinase receptor (uPAR) (Vassalli et al., 1985
; Stoppelli
et al., 1985
, 1986. Roldan et al., 1990
). Secreted pro-urokinase is a zymogen that must be proteolytically cleaved in
two fragments (namely, A and B chain) to display its full
enzymatic activity, either in solution or on the cell surface: cell-associated plasmin cleaves receptor-bound pro-uPA
with an efficiency 50-fold higher than plasmin in solution
(Stephens et al., 1989
; Ellis et al., 1989
). Furthermore, the
activation of cell-associated plasminogen by receptorbound uPA is characterized by a 40-fold reduction in the
Km for plasminogen activation (Ellis et al., 1991
). The
membrane-bound plasmin activity generated by cellbound plasminogen is protected by inactivation from serum inhibitors, but it can be modulated by the plasminogen activator inhibitors 1 and 2 (Hall et al., 1991
; Cubellis
et al., 1990
). Receptor-bound uPA:PAI-1 complexes can
be cleared from the cell surface via the
2-macroglobulin
receptor-low density lipoprotein receptor-related protein
(Conese et al., 1994
). Many observations have suggested
that uPAR could direct uPA-dependent proteolysis to discrete regions of pericellular matrix, where the degrading activity is required by cells engaged in migratory processes
(Estreicher et al., 1990
). Considerable evidence has been
accumulated which indicates that receptor-bound uPA provides cells with a very efficient invasion machinery (Carriero et al., 1994
; reviewed in Dano et al., 1994
). However,
recent findings uncover additional roles for ligand-stimulated uPAR: in particular, the ability of pro-uPA to trigger
uPAR-dependent cell responses, such as cell migration, proliferation, chemotaxis, adherence, tyrosine phosphorylation, and transcription of specific genes, has received
considerable attention (Kirchheimer et al., 1989
; Nusrat and
Chapman, 1991
; Del Rosso et al., 1993; Dumler et al., 1993
;
Busso et al., 1994
; Gyetko et al., 1994
; Cao et al., 1995
). In
most cases, uPA proteolytic activity is not required, as the
same effect is exerted by catalytically inactivated uPA
(DFP-uPA) or its amino-terminal domain (ATF, aminoacids 1-135). In both cases, the occurrence of a COOH-terminal lysine (Lys135 or Lys158) is critical to the proadhesive effect
(Li et al., 1995
). Like other proteases of the fibrinolytic and
blood coagulation systems bearing large noncatalytic domains involved in the interaction with regulatory macromolecules, pro-uPA includes a 157-amino acid NH2-terminal moiety (A chain) which comprises the "growth factor-like" domain responsible for uPAR binding, the kringle domain, and the so called "mini-chain," which precedes the
Ile157-Lys158 activation site (Patthy, 1985
; Appella et al.,
1987
; Oswald et al., 1989
; Hansen et al., 1994
). The occurrence of ATF-dependent events leading to cell activation
raises an important question concerning the mechanisms
underlying the signaling ability of uPAR, as this receptor
lacks a transmembrane and a cytoplasmic region, which
could link it to the intracellular signal transduction pathways. Reversible association with CR3, the complement
receptor type III (CD11b/CD18) both in neutrophils and
in monocytes, has been reported (Kindzelskii et al., 1996
;
Sitrin et al., 1996
; Wei et al., 1996
). Furthermore,
2-integrins, Src kinases, and uPAR have been found in a single
complex from monocytes (Bohuslav et al., 1995
). Also,
functional evidence for the existence of a transmembrane
adaptor in THP-1 monocyte-like cells has been recently
provided (Resnati et al., 1996
). Although most of the uPAR-dependent cell responses are triggered by its ligand,
little attention has been given to the role of uPA in the early
steps of receptor activation. Cultured myelomonocytic cell
lines, like U937, HL-60, and THP-1, can be induced to differentiate with TGF-
/vitamin D3, and, if further treated
with nanomolar concentrations of uPA or ATF, they acquire an adherent phenotype within minutes (Waltz et al.,
1993
). Differentiation of U937 cells into cells displaying macrophage-like properties is accompanied by an increase
in the number of uPARs which parallels the acquisition of
an adherent phenotype (Stoppelli et al., 1985
; Picone at al.,
1989). Also, THP-1 motility is greatly stimulated by uPA
or ATF or proteolytically cleaved soluble uPAR (Resnati
et al., 1996
). To shed light on these ligand-dependent signaling processes, we analyzed the functional effects of pro-uPA
serine phosphorylation on uPAR-dependent chemotaxis and
adhesion. We have previously found that pro-urokinase synthesized by A431 human epidermoid carcinoma cells is
phosphorylated on seryl residues located within the A
chain and the B chain, respectively (Mastronicola et al.,
1990
). Tyrosine phosphorylation of uPA from human
urine and from HT-1080 cell line has also been reported (Barlati et al., 1991
). Phosphate groups linked to serine,
threonine, and tyrosine residues have been detected in
pro-uPA secreted from human melanoma cells (Barlati et
al., 1995
). Functional consequences of phosphorylation
have been described: we have shown that serine phosphorylated uPA from A431 cells is severely impaired in its interaction with PAI-1, although its catalytic activity is not affected (Mastronicola et al., 1992
; Franco et al., 1992
).
Moreover, an increased catalytic efficiency (kcat/Km) has
been reported for uPA phosphorylated by the Detroit 562 carcinomatous cells (Takahashi et al., 1992
). Early efforts
directed to the identification of pro-uPA phosphorylation
sites in A431 cell line, by tryptic phosphopeptide mapping,
have tentatively identified Ser138 and Ser303 as major phosphorylation sites (Welinder, K., M.P. Stoppelli, and F. Blasi, unpublished observations). In this paper, we describe the construction and the high level expression of engineered
COOH-terminal histidine-tagged pro-uPAs carrying single
amino-acid substitutions which alter the presumptive phosphorylation sites. These "phosphorylation-like" variants
have been employed for a final identification of the phosphorylation sites in vivo, and for a functional analysis of single phosphoserines at critical sites of pro-uPA. Evidence
is provided that the naturally occurring phosphate groups
on Ser138/303, as well as glutamic residues mimicking phosphoseryl residues at the same locations, strongly impair uPA
ability to induce uPAR-dependent myelomonocytic adherence and motility.
Materials and Methods
). 5B4 anti-uPA mAb has been previously described in
Nolli et al. (1986). Cell culture reagents were from GIBCO-BRL (Gaithersburg, MD). The Bradford protein assay method was from Biorad Labs.
(Hercules, CA). [35S]Methionine, [32P]orthophosphate, and prestained
molecular weight protein markers were obtained from Amersham (Amersham, UK). Restriction enzymes and human vitronectin were from
Promega (Madison, WI). Autoradiography was performed on X-omat,
films from Eastman Kodak Co. (Rochester, NY). Enlightning was from
New England Nuclear (Beverly, MA). Geneticin, TGF-
, and dihydroxyvitamin D3 were from Calbiochem-Novabiochem Corp. (La Jolla, CA).
; Nolli
et al., 1989
; see also Fig. 1) and subcloned in pcDNA neo I linearized with
EcoRV (pcDNAneo-uPA gene). pcUK176 from R. Miskin (Weizmann
Institute of Science, Rehovot, Israel) was the source of uPA cDNA (Axelrod et al., 1989
). Mutations were generated by PCR amplification with
pFC16 as template (described in Orsini et al., 1991
). The oligonucleotides
employed are n.1: 5
-TTC TTC CGG AGG TTC GGA GGG CTT TTT
TCC-3
, n.1/2: 5
-TCC TCC GGA AGA ATT AAA ATT TCA-3
, n.3: 5
CTG CTC CGG ATA GAG ATA GTC GGT TTC ATT CTC TTT TCC3
, n.5: GGA TCT GTG GGC ATG GTA (plasmid region downstream to
the pro-uPA cDNA), n.6: 5
-CCT GTT GAC AAT TAA TCA (pTrp promoter region). In particular, for the S138E mutation, two cDNA fragments were amplified using, respectively, primers n.6+1 and n.1/2+5.
Both the fragments were then restricted, respectively, with BglII+AccIII
and AccIII alone and subcloned into the pFC16 vector digested with the
same enzymes, therefore substituting the nonmutagenized fragments. For
the cloning procedure, in the primers n.1 and n.1/2, an AccIII site has been
introduced with no change in the protein sequence. For the S303E mutation, only one fragment was amplified using primers n.6+3 and subcloned
directly into the pFC16 vector, as described above. As a result, the codons encoding Ser138 (TCC) and Ser303 (TCT) were converted to GAA which
specifies for Glu (positions 2413 and 3794 of the uPA gene, according to Riccio et al., 1985
). Successful mutation was confirmed by DNA sequencing.
Fig. 1.
Construction of the
mini-genes encoding prouPA138E and pro-uPA138E/303E.
A map of the intron-exon organization of pro-uPA gene
showing the regions and the
restriction sites relevant to
the construction of the
cDNA/gene hybrids is shown
at the top of the figure. Exons are indicated by boxes
and roman numbers, introns
are indicated by solid lines and capital letters (B-J), broken lines correspond to absent introns. Specific restriction fragments of prouPA cDNA carrying the mutations were fused to genomic regions to generate new coding units or mini-genes, encoding full pro-urokinase variants. The figure depicts the mini-gene138E, carrying the introns B, C, D, E, I, and J and encoding pro-uPA138E and the minigene138E/303E, containing the introns B, C, D, E and encoding pro-uPA138E/303E.
[View Larger Version of this Image (10K GIF file)]
Fig. 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.
[View Larger Version of this Image (37K GIF file)]
; Stoppelli et al., 1986
). Receptorbound pro-uPA was extracted with a buffer containing 50 mM glycine-HCl,
0.1 M NaCl, pH 3, for 5 min and subjected to immunoaffinity purification,
as described (Stoppelli et al., 1986
). The resulting samples were analyzed
onto a 12.5% polyacrylamide gel electrophoresis under reducing conditions followed by autoradiography (Laemmli, 1970
). 32P-containing gels
were directly dried under a vacuum whereas 35S-containing gels were fixed
in 25% methanol, 10% acetic acid, embedded in Enlightning, and dried.
).
Briefly, 80 µl of Ni-NTA/Sepharose, equilibrated with 50 mM NaH2PO4,
pH 8.0, 300 mM NaCl, 20 mM Imidazole, were incubated with 1 ml of conditioned medium and 250 µl of 250 mM NaH2PO4, pH 8.0, 1.5 M NaCl,
100 mM Imidazole, 5 µg/ml aprotinin for 90 min at r.t. under gentle shaking. The sample was then centrifuged at 4°C, 2,000 rpm for 3 min in a microfuge and the supernatant saved as a source of nontagged uPA, whenever required. Then, the resin was washed three times with wash buffer
(50 mM NaH2PO4, pH 8.0, 300 mM NaCl, 20 mM Imidazole) and incubated with 500 µl of 50 mM NaH2PO4, pH 8.0, 300 mM NaCl, 250 mM Imidazole for 20 min at r.t. to elute histidine-tagged pro-uPA. The sample
was transferred to a clean tube and TCA-precipitated to remove salt. Negative controls were performed with glycine-blocked Sepharose CL-4B under the same conditions.
).
. Protein concentration was determined by the method of
Bradford (1976)
.
for U937 cells.
.
, 50 nM dihydroxyvitamin D3 in the presence of 10% FBS for 20 h in Petri dishes.
Then, 105 cells per sample were incubated in 24-multiwell plates with 0.2 nM
of the indicated effectors (unless otherwise specified) for 30 min at 37°C.
Non-adherent cells, harvested by pipetting and adherent cells, removed
with 0.05% trypsin, were counted in a hemocytometer. The number of adherent cells is expressed as a percentage of the total cell number and represents an average from three different experiments performed in duplicate.
. Briefly, 105 THP-1 were applied to the upper compartment in serum-free RPMI. A431 and LB6-19 cells were acid-washed before the assay
and resuspended in serum-free DMEM. Effectors were diluted in culture
medium at the indicated concentrations and added to the lower compartment. The chambers were then incubated at 37°C for 90 min, and afterwards, the filters were removed, fixed, and stained. The cells on the lower
side of the filter were counted and reported as a percentage of the basal
random migration in the absence of chemoattractant.
Results
). Further efforts directed to the localization of the phosphorylation sites by
tryptic phosphopeptide mapping of A431 pro-uPA tentatively identified Ser138 and/or Ser303 as major sites (Welinder, K., M.P. Stoppelli, and F. Blasi, unpublished observations). This paper addresses the localization of pro-uPA phosphoseryl residues by an independent approach. As
described in Materials and Methods, pro-uPA cDNA has
been subjected to site-directed mutagenesis to encode prouPA variants carrying a replacement of Ser138 and/or Ser303
with glutamic acid residues. However, transient transfection of these cDNAs driven by SV40 or CMV promoters
in HeLa cells resulted in a barely detectable level of prouPA, whereas a 10-20-fold greater amount of secreted prouPA was obtained from the genomic version under the
same conditions (not shown). The latter finding raises the
possibility that the presence of pro-uPA intronic regulatory sequences may stabilize its mRNA, therefore suggesting that a greater expression may arise from intron-bearing units encoding the full pro-uPA or its variants. On the
other hand, the requirement of, at least, one intron for optimal mRNA accumulation has been shown for many genes,
including those encoding
-globin, ribosomal protein L 32, tissue plasminogen activator, and purine nucleoside phosphorylase (Buchman and Berg, 1988
; Huang and Gorman,
1990
). Also, some genes require specific sequences for intron-independent gene expression (Liu and Mertz, 1995
).
In this case, although pcDNAneoI vector carries SV40 transcription termination and RNA processing signals to enhance mRNA stability, the expression of pro-uPA cDNA
was unsatisfying: therefore, a minimum of four introns, derived from pro-uPA gene, were inserted upstream to
the cDNA fragments carrying the mutations of interest,
following a strategy described in Materials and Methods
(Fig. 1). The protein encoded by these new intron-bearing
units or "mini-genes" is the 431 amino acids "pre-prourokinase," which is secreted concomitantly to the signal
peptide removal (Riccio et al., 1985
). Then, the expression level of these mini-genes, driven by CMV promoter in
pcDNAneoI vector, was tested in transient transfections
of HeLa cells: the results confirmed that fusion of genomic
sequences to the 5
region of pro-uPA cDNA enables accumulation of mRNA and protein to an intermediate level
as compared to the expression level of pro-uPA gene or
cDNA (not shown).
).
Also, it ensures rapid and efficient purification based on
its high affinity interaction with Ni2+Sepharose (Ni-NTA).
As described in Materials and Methods, a double strand
oligonucleotide encoding the COOH-terminal region of
pro-uPA, downstream to the BamHI site (exon XI), followed by an exahistidyl peptide and a stop codon, was
cloned into the BamHI-XhoI excised pcDNAneoI vector.
Afterwards, a BamHI fragment containing most of the prouPA gene or the mini-genes was inserted into the unique BamHI site. As shown in Fig. 2, upper part, a histidinetagged fusion protein with a slightly different carboxy-terminal sequence (His-pro-uPAwt) was generated. To test
the in vivo expression of this construct and the purification
procedure of histidine-tagged pro-uPA, a transient transfection of HeLa cells, displaying no detectable endogenous
pro-uPA, was performed. The cells were transfected as described in Materials and Methods and, 48 h later, labeled with 100 µCi/ml of [35S]methionine for 18 h. As shown by
the similar patterns displayed in Fig. 2 (lower part), incubation of the 35S-labeled HeLa transfectants' conditioned
medium with either 5B4-agarose or Ni2+Sepharose resulted in the purification of His-pro-uPAwt. In control experiments, untagged pro-uPAwt, encoded by pcDNAI
bearing uPA gene, could exclusively be purified with antiuPA antibody and not by Ni-NTA chromatography. In both cases, pro-uPA exhibits a mol wt of ~47 kD: the lower mol
wt bands correspond to the A and B chains of pro-uPA
which undergoes partial activation in cell culture. In other
experiments, a tag-dependent, slightly decreased electrophoretic mobility of B chain was detected (not shown).
Parallel ELISA and enzymatic assays of purified His-prouPAwt suggested no major effects of COOH-terminal tagging on its catalytic activity (not shown).
Fig. 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.
[View Larger Version of this Image (27K GIF file)]
). 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.
Fig. 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.
[View Larger Version of this Image (22K GIF file)]
). In this set of experiments, we took advantage of
the inability of HeLa cells to neither phosphorylate Ser138
nor Ser303, thus allowing individual analysis of the glutamic
substitutions (Iaccarino, C., P. Franco, and M.P. Stoppelli,
unpublished observations). For this reason, stably transfected HeLa cells overexpressing His-pro-uPAwt, His-prouPA138E, or His-pro-uPA138E/303E, at 1-2 µg uPA/106 cells
in 20 h, have been obtained. Histidine-tagged pro-uPA
variants have been purified on large scale Ni-NTA
Sepharose chromatography and quantitated by indirect
enzymatic assay, as phosphorylation of pro-uPA does not
affect its kinetic parameters for plasminogen activation
(Franco et al., 1992
). The results show that the receptor binding ability of unlabeled His-pro-uPAwt is indistinguishable from that of urinary uPA (Fig. 4 B). In addition,
the affinity of His-pro-uPA138E and His-pro-uPA138E/303E
for uPAR is similar to that of His-pro-uPAwt, as shown by
comparing the relative Kd s. In all cases, half-maximal
binding is attained ~0.4 nM, a value which is in the expected Kd range for pro-uPA binding to uPAR.
), 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-
/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.
Fig. 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-/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-
/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-
/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.
[View Larger Version of this Image (21K GIF file)]
/Vitamin D3 Primed THP-1 and HL-60
Cell Lines
Effect of Phosphorylation on Pro-Urokinase Enhancement of THP-1 Motility
It is known that pro-uPA or ATF can stimulate a chemotactic response of THP-1 monocyte-like cells: in this experiment, the extent of cell migration along a gradient formed
by His-pro-uPAwt or the relative phosphorylation-like variants was estimated. According to a procedure reported by
Resnati et al. (1996), THP-1 cells were allowed to migrate
in modified Boyden chambers. Directional migration was
assessed by counting the total cell number on the lower side
of each filter and reporting it as a percentage of basal cell
migration in the absence of chemoattractant. As shown in
Fig. 6, 0.2 nM His-pro-uPAwt does stimulate directional
migration, unlike His-pro-uPA138E/303E which fails to stimulate THP-1 cell motility at the same concentration (we
could detect even a slightly reduced migration with respect to random migration). Similar to the relative effects on adherence, His-pro-uPA138E retains ~30% of the chemotactic ability of His-pro-uPAwt. Consistently, Pser-uPA from
A431 cells is a weaker chemotactic agent than Ser-uPA.
Nonreversible inactivation by DFP does not alter the
chemotactic ability of any of the pro-uPAs, showing that
none of the observed effects is due to the trace amount of two-chain uPA in the preparation.
Interaction of His-pro-uPAwt and His-pro-uPA138E/303E with Vitronectin and uPAR
To gain some insight into the molecular mechanism underlying the impaired signaling ability of pro-uPA phosphorylated on Ser138/303, we investigated the direct interaction
of His-pro-uPAwt and His-pro-uPA138E/303E with denatured
vitronectin. Varying amounts of vitronectin were separated
by SDS-PAGE, transferred to a PVDF membrane, and
probed with 125I-His-pro-uPAwt or 125I-His-pro-uPA138E/303E,
according to a procedure published by Moser et al. (1995).
As shown in Fig. 7 A, both variants react to the same extent to 1.25 µg, 2.5 µg, and 5 µg of vitronectin whereas
they do not show an appreciable interaction with 5 µg of
fibronectin or collagen.
The possibility that phosphorylation of pro-uPA may interfere with uPAR binding to vitronectin was examined.
For this experiment, urea-denatured vitronectin, which
mimics the matrix-like form of vitronectin, was employed
(Wei et al., 1994). Primed U937 cells were either incubated
in RPMI without any effector or with 10 nM recombinant
pro-uPA, or His-pro-uPAwt or His-pro-uPA138E/303E, in the
presence of 125I-vitronectin. At the end of the incubation,
the cell-associated radioactivity was assessed. As shown in
Fig. 7 B, binding of 125I-vitronectin to the cells was similarly enhanced by all pro-uPAs used, ruling out the possibility that His-pro-uPA138E/303E may interfere with uPAR/
vitronectin interaction.
Chemotactic Ability of His-pro-uPAwt or His-pro-uPA138E/303E in Nonmyelomonocytic Cell Lines
Next, we investigated whether the lack of effect by the disubstituted phosphorylation-like variant can be exclusively observed in cells of the myelomonocytic lineage,
where uPAR interacts with the 2-integrin CD11b/CD18
or Mac-1 (Bohuslav et al., 1995
; Wei et al., 1996
). Therefore, the chemotactic ability of His-pro-uPAwt and Hispro-uPA138E/303E was tested in A431 human carcinoma and
mouse LB6-19, overexpressing human uPAR. As shown in
Table II, His-pro-uPAwt is a chemotactic factor, capable of
enhancing random cell migration of both cell lines. On the
contrary, the di-substituted variant fails to stimulate cell
motility in both cases. The data also show that migration
directed by His-pro-uPAwt is unaffected by 10 µg/ml antivitronectin antibodies, suggesting that uPAR binding to
vitronectin is not a prerequisite for uPA-dependent A431
cell migration. Taken together, these data suggest that the lack of His-pro-uPA138E/303E chemotactic ability is not dependent on tissue-specific, uPAR-associated factors and
raise the possibility that binding of phosphorylated prouPA may cause a general impairment of receptor function.
Table II. Chemotactic Ability of His-pro-uPAwt and His-prouPA138E/303E in Nonmyelomonocytic Cell Lines |
Cellular Distribution of uPA Receptors in Preadherent U937 Cells
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.
This study uncovers a novel regulatory mechanism of prourokinase proadhesive and chemotactic ability in myelomonocytic cells, which depends on phosphorylation at specific serine residues. First, we have identified Ser138/303 as the in vivo major phosphorylation sites of pro-uPA synthesized by A431 human carcinoma cells. Second, by analyzing histidine-tagged pro-uPA variants carrying Glu138 and/or Glu303 to mimic the occurrence of specific phosphoserines, we found that the di-substitution strongly impairs pro-uPA ability to promote myelomonocytic motility and adherence, although it does not alter the Kd of uPA for its receptor. Third, we found that the monosubstituted variant His-pro-uPA138E only retains 20-30% of the wild-type proadhesive and chemotactic ability. These results were confirmed by parallel controls with naturally occurring phosphorylated pro-uPA from A431 cells. Finally, the disubstituted variant does bind to vitronectin, but it does not stimulate uPAR polarization in preadherent U937 monocytes. The data reported here indicate that phosphorylation of pro-uPA may modulate uPAR signaling ability, possibly interfering with uPAR mobilization and dynamic association with other membrane partners.
The requirement for receptor binding is further confirmed by the finding that deletion of amino acids 10-135, which include the growth factor domain, renders pro-uPA
totally unable to interact with uPAR and to stimulate adherence (Chiaradonna, F., P. Franco, and M.P. Stoppelli,
unpublished observations). In addition, previous work has
shown that the amino-terminal fragment of uPA, namely ATF, completely retains pro-urokinase proadhesive ability (Nusrat and Chapman, 1991). However, these findings
do not exclude that other regions of pro-uPA may modulate its signaling mechanism. One of the two phosphorylation sites lies within a small region connecting A and B chain
of pro-uPA (135-158), or "mini-chain": The finding that a
negatively charged side chain at position 138 causes a 70-
80% reduction of both proadhesive and chemotactic ability of pro-uPA suggests that the surrounding region may
be responsible for critical interactions with membrane-
associated proteins, provided pro-uPA is receptor-bound.
Additional information on the relevance of uPA mini-chain
region to the uPA-enhanced adhesivness of U937 cells is
provided by Gurewich and coworkers which showed that
removal of Lys135 or Lys158 with carboxypeptidase A impairs uPA proadhesive effect, whereas it does not affect
uPAR binding (Li et al., 1995
). These data, taken together,
may lead to the conclusion that different local charges may
exert opposite effects on uPA proadhesive ability and
raise the possibility that the conformation of the mini-chain region may fulfill a regulatory function of uPA-dependent
signaling. However, we cannot rule out the possibility that
intramolecular charge interactions in phosphorylated uPA
may preclude proper formation of a functional contact region, thereby suppressing uPA-dependent signaling. In
our experiments, Glu303 seems to enhance the effects of
Glu138: this may lead to the interpretation that either the
full pro-uPA molecule bears two distant regions of interaction or that, in receptor-bound pro-uPA, Glu303 is spatially closer to Glu138, thereby enhancing the local negative
charge. On the other hand, NMR analysis of pro-urokinase has shown a substantial independent motion between
individual domains of the protein (Oswald et al., 1989
).
Functional alterations due to single phosphorylation sites or to negatively charged amino acids (Asp or Glu) replacing phosphoserines are not unique to this case: phosphorylation of Ser78 in the c-Jun transcriptional regulator enhances its DNA binding activity and so does the variant
carrying Asp78. The transcriptional regulator p53, if phosphorylated on Ser389, lacks its growth suppressor function;
regulation of isocitrate dehydrogenase by phosphorylation
of Ser113 leads to enzyme inactivation, which can be mimicked by substituting the critical Ser with Glu or Asp (Hoeffler et al., 1994
; Rolley and Milner, 1994
; Hurley et al.,
1990
).
A challenging question concerns the upstream molecular events of the uPAR-directed signaling, as this receptor,
lacking a transmembrane and a cytoplasmic region, is associated to the membrane via a glycosylphosphatidylinositol (GPI) anchor (Blasi et al., 1994). Very little is known
about the ligand-dependent activation and the early signaling steps of GPI-anchored proteins. However, their lateral mobility may be important for a dynamic coupling to
integral membrane proteins and therefore relevant to their transducing ability (Robinson, 1991
). It has recently been
reported that uPAR can interact with integrin receptors:
Work by Wei et al. (1996)
has shown complex formation
between uPAR and
1- or
2-integrins, which suggested
this as the link between uPAR and cytoskeleton, therefore
providing a mechanistic explanation for uPAR signaling.
In neutrophils, the interaction between uPAR and CD11b/
CD18 (Mac-1) is highly dynamic and depends upon cell
shape change from a spherical to a polarized morphology
during locomotion; interestingly, uPARs are concentrated
in lamellipodia-type structures (Kindzelskii et al., 1996
). It
is known that uPA binding to uPAR induces its relocalization to focal contact sites in human fibroblasts and rabdomyosarcoma cells (Myohanen et al., 1993
). Others have shown that uPAR undergoes ligand-dependent conformational changes (Plough et al., 1994
). This paper shows that
uPAR signaling ability may be modulated by single amino
acid substitutions in its ligand, in a nontissue-specific and
vitronectin-independent manner. We also show that wildtype tagged pro-uPA can induce uPAR redistribution in
preadherent U937 cells, and that this ligand-dependent
effect is prevented by phosphorylation of pro-uPA on
Ser138/303. The blockage of uPAR-dependent signaling is
further supported by the evidence that in cells treated with
the di-substituted variant, we could not observe cytoskeletal rearrangements, as in preadherent cells (Chiaradonna,
F., and M.P. Stoppelli, unpublished observations). Although the mechanistic role of pro-uPA as a trigger of motility and adhesion is presently unclear, different pieces of
evidence may converge on the possibility that ligand-activated uPAR has an increased lateral mobility which allows
its rapid redistribution to specific membrane regions and,
perhaps, a particular conformation leading to its dynamic
association with specific receptors. However, these aspects
deserve further investigation.
In any event, an interesting conclusion may be drawn
from the inability of phosphorylated pro-uPA to stimulate
both myelomonocytic adherence and motility, which suggests that these two processes share some common mediators in myelomonocytic cells. This finding is not surprising,
as the formation of adhesive cell-matrix contacts in cell
spreading and migration results from cooperation between the membrane-associated adhesive systems, the actin cytoskeleton, and the generation of force across regions of the
cell. Cell migration implies the occurrence of cytoskeletalmediated process extension (filopodia and lamellipodia)
and retraction, together with the formation and disruption
of adhesive contacts at the leading edge of the cell. In particular, focal adhesions comprise integrins as the major adhesion receptors and are thought to serve as sites for coordination between cell adhesion and motility (Gumbiner, 1996; Lauffenburger and Horwitz, 1996
). Despite the relevance of monocyte/macrophage motility and adhesion to
hemostasis, inflammation, and immunity, the molecular
mechanisms governing these processes have been studied
mostly in fibroblasts (Zachary and Rozengurt, 1992
). A
detailed molecular analysis of the effects triggered by prouPA may provide some clues to such mechanisms in myelomonocytic cells. In particular, the nonsignaling, di-substituted variant can be instrumental in additional studies
aimed at identifying the membrane partners of pro-uPA
and the molecular details of these critical interactions.
Also, the maximized expression level of the nonphosphorylatable variants encoded by the mini-genes will hopefully
give the opportunity to study both the regulation and the
tissue specificity of these sites.
In a previous paper, we have shown that phosphorylated pro-uPA is less sensitive to the inhibition by PAI-1; recent data suggest that it also render pro-uPA more susceptible to the activation by plasmin (Iaccarino, C., P. Franco, and M.P. Stoppelli, unpublished observations). Conversely, this work presents evidence that phosphorylation inhibits uPA-dependent control of myelomonocytic adherence and motility. In conclusion, phosphorylation of pro-uPA can be regarded as a remarkable mechanism which alters the properties of pro-uPA, indirectly enhancing its catalytic activity and reducing its signaling ability. It is tempting to speculate that in highly invasive cells phosphorylation of pro-uPA is a means to prevent pro-uPA-dependent control of cytoskeleton while favoring PAI-1-insensitive matrix degradation.
Received for publication 2 August 1996 and in revised form 20 December 1996.
1. Abbreviations used in this paper: DFP, diisopropylfluorophosphate; GPI, glycosylphosphatidylinositol; His-pro-uPAwt, histidine-tagged wildtype pro-uPA; PAI-1, plasminogen activator inhibitor type 1; Pro-uPA, pro-urokinase; Pro-uPAwt, wild-type pro-urokinase; Pser-uPA, serinephosphorylated uPA; Ser-uPA, nonphosphorylated uPA; uPA, urokinase.The authors thank Dr. P. Ragno for the generous gift of reagents; Dr. M. Carriero and S. Del Vecchio for useful discussions; and Dr. I. Buttino and A. Miralto for their help with the confocal microscope. The technical assistance of M. Terracciano is gratefully acknowledged.