BIOCHEMICAL

Vol. 182, No. 3, 1992 February 14, 1992

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1473-1481

ENZYMATICPROPERTIESOFTHEPHospHDRYLAl3ZDuRoKINRsE-TYPEPLASMINGQB ACI'IVATORISOLATEDFROMAHTBANCARCIXMAXXJSCEXLL~ Kei Takahashi, Hau C. Kwaan*, Enki Koh,** ardMasatakaTanabe*** Department of Physiology, Shimane Medical University, Izmo 693, Japan * Department of Medicine, Northwestern University Medical School, VA Lakeside Medical Center, Chicago, Illinois Division

of Research and Developnen& Nihon Pharmaceutical Co., LTD., **osaka 532, and Tokyo 724, Japan

Received January 8, 1992 Bqmatic properties of phosphorylated urokinase plasminogen activator (P-uPA) extracted fmn human carcinanatous cell line Detroit 562 cells were cunpared with those of non-phosphorylated uPA of urinary origin (nP-uPA). Using plasnincgen as a subsrate, the K,,, and Kmt of P-uPA were higher than that of nP-uPA while the Kcat/ was lower. By zymography, a greater degree of plasmincqen activation was Tiserv 0 Conomavalin A reacted to both the ed. enzymes. P-uPA had a lm affinity for the inhibitors of plasnimgen activator PAI-I and PAI-2, and was inhibited only by the excess amounts of inhibitors. For PAI-1, the KIs of P-uPA was greater and for PAI-2, KI was higher for PUPA. These alterations by phosphoqlation enable uPA to be more efficient in a focal proteolysis through plaminogen activation. 0 1992 Academic Press, Inc.

Urokinase (uPA) is a plasmincgen activator including

tumor cells

cells

rrajor

(lO,ll),

function

proteolysis the (9,15),

A-chain)

activity

(9).

It

is localized

and is phosphorylated by protein

of UPA is of interest

tor bound uPA may have physiological Cur previous

signaling

local or

receptors

suggesting that the recep-

functicm

562 line is phospohorylated

Its

fragment (ATF;

(17).

study demonstrated that the uPA associated

receptors of a Detroit

(8,12,13).

because it is not cnly bound to (16)

surface

facilitating

(14). The amino-terminal

lx& also stimulates “cell proliferation

cells

at adhesion sites

kinases

is believed to be that of an ectoenzyme,

at the adhesion sites

of

It birds to cell

is known to produce uPA (l-7).

receptors and has proteolytic of

present in a variety

(8).

with

surface

We are reporting

the

0006-291x/92$1.50 1473

Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol.

182, No. 3, 1992

enzymatic of

properties

and is

physiological

activate

uPA (I@-uPA) of urinary activating

not inhibited conditions.

amounts of inhibitors.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of this phosphorylated uPA (P-uPA) ccmparing with

non-phosphorylated

enzyamatic properties, cy,

BIOCHEMICAL

origin.

P-uPA has altered

plasminogen with greater

by plasminogen activators Canpetitive

inhibition

Thus, P-UPA in spite

those

PAI-

catalytic

efficien-

and PAI-

under

was observed only with large of cell

surface inhibitors

can

plasminogen leading to the focal degradation of extracellularmatrix

proteins. MAclTmALsAND-Materials. The chrancgenic substates S-2444 (L-Pyroglutamyl-L-glycyl-arginine-p-nitroanilide hydrcchlolide) and S-2251 (H-D-valyl-L-leucyl-L-lysyl-pnitroanilide hydrochrolide) were purchased from KaviVitrum (Stockholm, Sweden). Inhibitors for plasmincgen activators, PAI- and PAI- were purchased form TechncYClcne (GmbH, Vienna, Austria) and The Green Cross Corporation (Osaka, Japan), respectively. Cells. Cells used, C5, were one of the clones we obtained (18,19) from a cell line, Detroit 562 (AlCC CCL 138, Flow Laboratories, Inc., McLean, VA) (20). This clone was previously found to express uPA and receptor of uPA (18). Enzyme assay. Microtiter plates with 96-wells (Cell Wells, Corning Glass Works, Corning, NY) were used for the enzms assays. A portion of the purified proteins was incubated in 200 ,ul of a reaction mixture containing 50 nM TrisHCl, pH 8.8 or pH 7.5, 38 mMNaCl, and 0.1 to 0.5 mMS-2444. For plasmincgen activation, a similar reaction mixture containing 1 &I S-2251, 0.01% l'ween 20 and 0.1 to 0.7 p plasminogen (21) was incubated at 3PC for 20 min. Plate reader (SLT Labinstints, Austria) was used to determine the absorbace at 405 r-m for both the S-2444 and S-2251. To inhibit the enzymatic activity, inhibitors were supplemented in the reaction mixtures and incubated at 37oC. The inhibitors were employed in various units (IX) based on the calculation that cne unit of uPA is canpletely inhibited by cne unit of the inhibitor (22,23). The dependence of enzyme-catalyzed reaction or inhibition was determined by double reciprocal plot (24) of 1 /Vi versus l/[Sf, where vi is a substrate anverted per min and S is a substrate concentration in rrM. Purification of cell-associated UPA. The method for the purification of phosphoprylated uPA is described (8). SDS-polyacrvlamide gel electrophoresis and protein blotting. Proteins were separated an 10%SDS-polyacrylamide slab gel (26) and blotted onto a nitrocellulose paper (27). The antigenic proteins were examined by the method of Glass To detect mannose-containing sugar moiety of proteins, peroxi--et al (28). dase-conjugated Concanavalin A was used for the blots (29). Fibrin-autography To examine plasminqen activation by proteins, SDS-gel was overlayed on'a fibrin-agar indicator gel (30). The indicator gel was stained with 0.025% Amid0 Black in 10%acetic acid and 30%methanol and photoIntheabsenceofplasminogen,nolytic zoneswareobserved. SraMd. Protein assay. The concentration of proteins was determined by the method of Bradford (31) with bovine gamM globulin as the standard. FEw.LTs Catalytic

efficiency

of the phosphorvlated UpA.

UPA, purified

fran

human

urine on a benzamidine-Sepharose colusm, was found by imnunoblots, using anti1474

Vol.

182,

No.

3, 1992

BIOCHEMICAL

AND

BIOPHYSICAL

r

0

RESEARCH

COMMUNICATIONS

r

0.1

a2 0.3 s(mM)

0.4 O

‘O

l?“,

3o

4o

FIGURE1.

Snzym kinetics of urinary uPA and phosphorylated uPA. mzymatic was determinedby the incubationof urinary UPA or phosphorylated uPA with a chromogenicsubstrate, S-2444 fcx 20 tin at 37oC. Absorbanceat 405 nm was spectrophotomatrically determined. upper panels, phosphorylated UPA; 1me.r panels, urimry uPA. A, enzymekinetics-by various amountof S-2444; B, Double reciplocal plots of Lineweaver-Burk. V, velocity; S, concentration of the substrate in &I. read&m

body against phosphotyrosine, horylated lated

to ccmtain no phosphotyrceines.

uPA (nP-uPA) of urinary

uPA (P-uPA) derived fm

activities. the kinetic

Firstly,

origin was used to canpare with

clone 5 cells

for their

a chrcxmgenic substrate,

parameters (Fig.1

solutions

respective

S-2444, was used to

and Table 1).

enzymes ws determined in buffer

This non-phosp-

The optimal

@-I for

phcephoryenzymatic determine both the

of various pHs. The pH values that

TABLE1. Kinetic parameters of urinary uPA and phosphorylated uPA.OD;optical a chrancgenic subdensity at 405 m. Note that for plasninogen activation, strate S-2251 was used. UPA Sub6trates

Vmax

Km

Kcat

OD.mid

U

mine’

P-UPA Kcat / Km

Vmax

Km

ODmln

M

Kcat

Kc& I Km

mm-’

0.08

4.38x10-‘

0.45

1.0x103

0.04

297x1o-4

092

3.1x103

S-2444lpH75

0.12

6.08~10~

0.68

1. I x103

0.04

392xlo-4

086

2.2x103

PL3smhogm~75

0.21

243 xU7

2.30

95 x IO6

I 06

170x 1O-7 11.62

5-244L

IpHBB

1475

66x1@

Vol.

182, No. 3, 1992

BIOCHEMICAL

gave a mximun reaction

velocity

AND BIOPHYSICAL

(A405 rm/min) were fran 7.5 to 9.5 for the At pH 8.8, Vmax and. s

P-uPA and 8.8 to 9.5 for the nP-uPA. cme half of thoseof UPA.

the affinity

substrate

of the P-uPA were

nP-uPA and Kcat was about twice as much as that

Similar results

that

RESEARCH COMMUNICATIONS

were obtained at pH 7.5.

and the catalytic

weregreater

cm&ant

These experiments

of the P-uPA for

than those of thenP-uPA.

was used assubstrates,

the

the K,,, was seventy fold that of nP-uPA resulting

nP-

suggested synthetic

secondly, when plamimgen

of the P-uPA were five

theVmarxdKcat

of

in a Kcat/G

fold,

while

of the P-uPA 10

times lower than that of nP-uPA (Table 1). Inhibition

& --PAI-I

and PAI-2.

and PAI-

on both the P-uPA and nP-uPA.

nP-uPA was inhibited inhibit ticn~

We tested the inhibitmy under ccnditiom

by me unit of PAI-2,

only 29% (standard deviation; of P-uPA by both PAI-

where me

cme unit of PAI-I

6.5%) of nP-uPa.

and PAI-

effects

unit

of

was fourd

to

In contrast,

were almost negligible

of PAI-

under

inhibisimilar

conditicms (Table 2), being 0.8% (sd; 1) and 2.9% (sd; 3) respectively. that

ShOWS

PAI-I

P-uPA was less sensitive

and PAI-2. The inhibition

inhibitors that

was also studied.

the inhibition

than I-@-uPAto the inhibition

for

P-uPA and PAI-

7.8~10~~’

or PAI-

both

The Lineweaver-Burk plots in Figure 2 indicate

was ozfnpetitive.

were respectively

by

of P-UPA and nP-UPA by excess anmunts of these

The dissociation

lated fran these plots are shcxm in Table 3. PAI-

This

ad

The KI's

5.9~10-‘~

were much higher,

constants (KI)

cxlcu-

for nP-uPA and PAI-

(ml/l).

In contrast,

being 1.0x10-'

TABLE 2.

those

and 2.4~10~'

Inhibition of urinary UPA and phosphorylated uPA by plaaninogen inhibitors. One unit of uri.naryuPA andone unit of phos@oxylated UPA ware used for the inhibition by PAI(one unit) & pAI(me unit). Ensymeandinhibitco:were incubated at37oc for20 minand there&dual a&ivity was spectrophotonetrically determined at 405 nm. Chranogenic substrate,

ator

S-2444

was used.

% Inhibition Inhibitors PAI -1 PAI-

UPA

P- UPA

2 9.1 + 6.5

0.60’1.10

100.0 1476

2.90’ 260

or

Vol.

182, No. 3, 1992

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

FIGURE2. Lineweaver-Burk plots of enzyme reaction in the presence of plass-activator inhibitors. Upper panels; urinary uPA (A) or phosphorylated uPA (B) was incubated with PAI-1. Inhibitim by PAI- was observed at the level of 30%and 45%,respectively. Iower panels; urinary WA (C) or phosphorylated uPA (D) was incubated with PAI-2. Inhibiticm by PAI- was observed at the level of 50%and 32%, respectively.

respectively.

These findings

indicate

that P-uPA had less affinity

for

PAI-

and PAI-2. Characterization lectin

of the pbcxpboqlated

UPA.

F'urther analysis of proteins

blots of SDS-polyacrylamide gel sbcmedthat there was no difference

TABLE3. Dissociation constants of urinary UPA and phosphorylated uPA for plasni&en activator inhibitors. Vnder the experimental ccxditicns, PAI-1, using me ard twenty f-units, inhibited cmeunit of urinaly uPA and phoschprylated UPA by 30%ard 45%, respectively, while PAI-2, using 0.5 and forty units, inhibited me unit of urinary uPA and phosphorylated uPA by 50% and 32%, respectively. PAI -1

PA1-2

UPA

7.8 x 10-l’

5.9 x lo-‘O

P-d?4

1.0 x 1o-g

2.4 x lo-’

1477

by in

Vol.

182,

No.

3, 1992

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

FIGURE 3. The birding of Cohcanavalin A to UpAs. After the blotting of proteins, pemcidase cmjugated Ccccanavalin Awas incubated. M denotesnolecular weight markers; lane 1, urinary UPA; lane 2, UrinaryuPA; lane 3, phosphorylated UPA. BlotAwasstainedbyAmidoBlackandblotLwasstainadbypercxidase-conjugated Cmcanavalin A. Numbers denote the mclecular weight in kilodaltons.

the birding weight

of

Concanavalin

(LMV, 33 KD) species

A lath

to high

(HMV, 55 KD) and low molecular

of nP-uPA and P-uPA (Fig.

@-~oresed

gel was examined on fibrin-agar

dcme in

the presence

indicator

of plsminogen,

3).

A similar

gel.

electro-

When zymography

P-uPA shawed a greater

lysis

of

was fibrin

thannP-uPA. To

study

incubated

plasmincgen

with

plasminogen.

mercaptoethanol show

and

mannex with

P-uPA and

The activation

and electm@oresed

the generation

chain),

activation,

mixture

ware

gel.

one of 80 KD being

of 27 KD being a Bchain

(light

both P-UPA and nP-UPA. are able to activate

respectively

was then reduced

on SDS-polyacrylamide

of two polypeptides,

the other

nP-uPA

by

The

results

a A-chain

chain)

in

2-

(heavy identical

plasminogen

in a simi-

larmanner. DISCUSSION lhoqh

the substrate

PAI-

have been well

lated

fom

ampares urinary

specificity

characterized

of uPA and its (21-23,32).

(P-uPA) have been published.

P-uPA of tumor cell origin origin.

by PAI-

No studies on the

Thepresent

of

efficien-

When plaminogen was used

for P-uPAwas found tobetentims 1478

studies

uPA (nP-uPA)

With the chramgenic subsrate S-2444, the catalytic

theK,&I$,

and

phosphory-

eazymological

with non-phosphorylated

cy (Kcat/Ks,) of P-uPA was 2-3 folds that of nP-uPA. as a substrate,

inhibition

lower

than

Vol.

182,

that

No.

3, 1992

of nP-uPA,

raphy

due to a very

fibrinolysis

I

indicating

that

plasninqen minor

by

into

generated

units

tion,

since

ciency

the A-chain

so

that

surface,

receptor.

a highly

efficient

P-uPA could

catalyze

the

effects

condition

of PAI-

where

(22).

one unit

The residthan

that

Though the catalytic

effi-

for

under --in vivo

condi-

being attached

the lawer of

substrate

nP-uPA was inhibited

P-UPA, which

partial

inhibitions

were observed

are,

tions

(35).

creases

its

These

however,

These observations

to

affinity

of

plasmincgen.

much more

effi-

for

findings

are not due to changes

forms

of

since

remains

by one

by either

unit

of 30%, PAI-I

of PAI-2),

of 45% and 32%,

or only

respectively.

under physiological

the phospohorylation

condiof uPA de-

the inhibitors.

P-uPAandnP-uPA.

same way.

cell

that

under

at the level

and 40 units

than those

indicate

studied

inhibited

of PAI-I

of PAI-I

at the levels levels

were

was not inhibited

affinity

because thebinding

properties

(8),

(24 units

much higher

zyme,

plasminogen

by one unit

is of a two-chain inhibitors

These

and PAI-

of nP-uPA was ccmpletely

With excess

from

pep-

activa-

activator

of P-uPA and nP-uPA by PAI-I

PAI-2.

the

a

in the

available,

its

unidentified

was much less

of nP-uPA,

This can canpensate

P-uPA is still

of uPA proteolyse

(33,34).

of nP-uPA.

is readily

nP-uPA

than nP-uPA.

Inhibitory

lilt

formaticn

that

than that

of

KD represintirq

14

of P-uPA

By zymog-

that

additional

P-UPA is more efficient

plasminogen

Once bound to plasmincqen, ciently

An

that

of P-uPA is lower

plasminqen

P-uPA

fold

than

Path forms

of plasnin

by only 7 units

Kcat is five

cn the cell

surface

activator.

and the B-chain.

of nP-UPA suggesting

COMMUNICATIONS

of P-uPA for plasminogen.

was also produced. of approximate

(Kc.&.,,)

tions

RESEARCH

P-uPA was found to be greater

plasmincgen

its

BIOPHYSICAL

low affinity

in the complex process

ual unadivated 14

AND

P-uPA was an efficient

polypeptide

tide

by

BIOCHEMICAL

of ConcanavalinAwas Nor is it

related

both P-uPA and nP-uPA were The mechanism by which to be elucidated.

membrane proteins

in the mannose moiety

the

en-

similartobothHMW

and

LMW

to the activation

process

of

plasminogen

in

found to activate

phosphorylation ThephosphorylateduPA

and represents 1479

the receptor

of

alters

the was

bound uPA

enzyme

extracted. (8).

The

Vol.

182, No. 3, 1992

BIOCHEMICAL

above alterrations enabling in

in

efficient

AND BIOPHYSICAL

its enzymatic propeties

plasninagen activation

focal proteolysis

RESEARCH COMMUNICATIONS

is thus

of

significance

on the cell surface.

This

important in the process of cell migration

breakdcwn of extracellular

matrix

in

results

through

the

(14).

This research is supported in part by a Grant-in-Aid (63570038) for Scientific Research fran the Ministry of Education, Science, and Culture, Japan.

1. ~eurath,

2. Rijken,

H. (1984) Scienos 224, 350-357. D. C., and Grceneveld, E. (1991) Biochim. Biophys.Res. Carmun.

174, 432-438. 3. Ossowski, L., 4312-4320s 4. Wilson, E. L., (1980) Cancer 5. Vihko, K. K., 31, 383-389. 6. Takemura, R., 7. Beers, W. H.,

8. 9. 10. 11. 12. 13.

J. P., and Reich, E. (1974)

J. Biol.Chem. 249,

Becker, M. L., Hoal, E. G., and Dcwdle, E. B. Res. 40, 933-938. Suaninen, J. J. O., and Parvinen, M. (1984) Biol.

Reprod.

and Werb, 2. (1984) Amer. J. Physiol. 246, Cl-C5. Strickland, S., and Reich, E. (1975) Cell 6, 387-394. Takahashi, K., Kwaan, H.C., Ikeo, K., and Kch, E. (1992) Biochem. Biophys. Res. Commun.182,1466-1472. Vassalli, J.-D., Baccino, D., and Belin, D. (1985) J. Cell Biol. 100, 8692. Burridge, K., Fath, K., Kelly, T., Nucholls, G., and Turner, C. (1988) Am. Rev. Cell Biol. 4, 487-525. Mangel, W. F. (1990) Nature 344, 488-489. Mastroniwla, M. R., Stoppelli, M. P., Migliaccio, A., Auricchio, F., and Blasi, F. (1990) FEBS Lett. 266, 109-114. Barlati, S., Daracini, F., Belletti, D., and DePetro, G.(1991) FEE%3Lett. 281,

14.

aUigley,

137-140.

C!ken, W-T., Olden, K., Bernark, B. A., and Chu, F-F.

(1984)

J. Cell Biol.

98, 1546-1555. 15. Appella, E., Robinson, E. A.,

Ullrich, S. J., Stoppelli, M. P., Croti, A., and Blasi, F. (1987) J. Biol. Chem. 262, 4437-4440. 16. Rabbani, S. A., Desjardins, J., Bell, A. W., Banville, D., Mazar, A., Henkin, J., and Goltzman, D. (1990) Bicchem. Bio#ys. Res. Ccsrnun. 173,

1058-I 064. 17. Ploug, M., (1991) J. 18. Takahashi, 405-413. 19. Takahashi,

Ronne, E., Behrerdt, N., Jensen, A. L., Blasi, F., and Da&, K. Biol. Chum. 266, 1926-1933. K., Gojobori, T., and Tanifuji, M. (1991) Exp. Cell Res. 192,

K., Ikeo, K., Gojobori, T., and Tanifuji, M. (1990) Thranb. Res. Suppl. X, 55-61. 20. Peterson, Jr. W. D., Stulberg, C. S., and Simpson, W. F. (1971) Proc. Sot. Exp. Biol. Med. 136, 1187-1191. 21. Lijnen, H. R., Van Hoef, B., azl Collen, D. (1986) Biochim. Biophys. Acta 884, 402-408. 22. Kawano, T., Morimoto, K., and uenura, Y. (1970) J. Biochem. 67, 333-342. 23. Christensen, U., Holmberg, L., and Astedt. B. (1982) Thranb. Haermstas. 48, 24-26. 24. Lineweaver, H., and Burk, D. (1934) J. Am. Chem. Sot. 56, 658-666.

1480

Vol.

25.

182, No. 3, 1992

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Holmberg, L., Bladh, B., and ktedt,

B. (1976) Biochim. Biophys. Acta 445,

215-222.

Laenanli, U. K. (1970) Nature 227, 680-685. T&bin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. USA 76, 4350-4354. 28. Glass, W. F., II, Briggs, R. C., and Hnilica, L. S. (1981) Science 26. 27.

211.

70-72.

29. Kijimoto-Ochiai, 147, 30. 31. 32. 33. 34. 35.

S., Katagiri,

Y. U, and Ochiai,

H. (1985)

Anal.

222-229.

Granell-Pipemo, A., and Reich, E. (1978) J. Exp. Med. 148, Bradford, M. M. (1976) Anal. Biochern. 72, 248-254. Ellis, V., Wun, T-C., Behrendt, N., Ronne, E., and Dan@, K. (1990) J. Biol., Chem. 765, 9904-9908. Rouy, D., and Angles-&no, E. Biochem. J. (1990) 271, 51-57. Wiman, E., and Wallen, P. (1973) Eur. J. Biochem. 14, 25-31. Sprengers, E.D., and Kluft, C. (1987) Blood 69, 381-387.

1481

223-234.

Biochem.