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50 Structural and functional specificity of the domain of the human urokinase receptor in cell migration
ORAL COMMUNICATIONS III-3 Signal Transduction
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Signal Transduction and Biologic Effects Induced by Crosslinking of Receptor-Bound Urokinase in the Human Kidney Epithelial Tumor Cell Line TCL-598 Ruthner M, Koshelnick Yu and Binder BR Dept. Vase. Biol. & Thromb. Res.. Univ. Vienna, Vienna, Austria
S T R U C T U R A L AND F U N C T I O N A L SPECIFICITY O F THE DOMAIN OF THE HUMAN UROKINASE RECEPTOR IN CELL MIGRATION.
The tumor cell line TCL-598 expresses high levels of scu-PA and of its receptor uPAR. Clustering of receptor bound sco-PA by a specific monocloanl antibody (scuPA8) directed against the protease domain of scu-PA elicited a mitogenic/migratory response in TCL-598 in a wounding assay, while a control antibody directed against a different domain in the scu-PA molecule that did not induce clustering of the receptors had no effect. Parallel to the biologic response, receptor clustering also induced tyrosine phosphorylafion of several proteins (p30, p50-60, p75-90, p120-130 and p>200). Tyrosine phosphorylatiun occurred fast within three minutes and reached a maximum after 5 to 10 minutes. When caveolin containing low density Triton X-100 insoluble complexes were analyzed after receptor clustering, uPAR, seu-PA and the crosslinking antibody were found in the "cavenlae" together with the transmembrane protein gpl30 and the tyrosine kinases JAK1 and fyn and the JAKI substrate STATI. Ten minutes after receptor clustering tyrosine kinases were found to be increased but STAT1 was phospbotylated and became excluded from the "caveolae" and appeared in the cytosol in its dimeric form. Consistently, caveolae obtained after receptor clustering also exhibited in vitro kinase activity giving raise to specificallytyrosine phosphorylated proteins (p4050, p75-90, p125, p165 and p>200). Analysis of phospbotyrosine--containing proteins co-precipitated with the uPAR after receptor clustering revealed only one hand with a molecular weight of ~50kd, which upon Western blotting could be identified as fyn. After receptor clustering STATI was not only found in its dimeric form in the cytosol but 20 minutes alter receptor clustering STAT1 was excluded from the cytosul and appeared in the nuclear fraction. In the nuclear matrix receptor clustering induced among others the appearance of three new proteins (p32/4.85, p32/4.93 and I>45/5.6)which could be identified as related to cyclin D1, D2 and E, all belonging to the GI/S family of cyclins. These data suggest that in the TCL-598 cell line clustering of scu-PA bound to its receptor induces a signal via tyrosine kinases involving "caveolae", the JAK/STAT pathway and the GI/S family of cyclins. The uPA-R itself seems to be linked to the src kinase fyn in this cell line.
M. R e s n a l l , N. S l d e n / u s , L . R / I t t i n e n a n d F r a n e e s c o BIasi. DIBIT, San Raffaele Scientif~ Institute, Milan, and Department o f Genetics and Microbial Biology, University of Milan, Italy.
W e have previously shown that catalytically inactive uPA derivatives stimulates a chemotactic response in human monocytic THP-1 and in murine LB6 fibroblastic ceils expressing human uPAR. Transduction of the migratory signal takes place via uPAR, involves a time-dependent activation o f the tyrosine kinase p56/p59 hck and requires an as yet undefined transmembrane adaptor capable o f connecting the extracellular ligand-binding uPAR to intracellular transducer(s), such as p56/p59 ~ k (1). The existence of such an adaptor is strongly supported by the finding that a chymotrypsin-cleaved form o f soluble uPAR (suPAR) is a potent chemoattractant not only in THP-1 ceils, but also in cells lacking endogenous human uPAR, such as the parental untrasfected LB6 cells and macrophages obtained from uPAR-/- mice (1). LB6 cells expressing human uPAR deletion mutants containing only some of the receptor's domains (2) were analysed for thei ability to migrate towards a human ATF gradient. By this approach we were able to identify the uPAR's domain specifically required for signalling and thus possibly for the interaction with the transmembrane adaptor. To confirm these data and to define more finely the uPAR region responsible for promoting cell migration we have also constructed and purified different soluble uPAR sequences which have been used as chemoattractant in murine untrasfected LB6 cells. (1) Resnati et al., 1996, EMBO J., in press. (2) Riittinen et al. 1996, Febs Lea., in press.
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CYCLIC AMP-MEDIATED SIGNAL TRANSDUCTION IN UROKINASE-INDUCED MONOCYTE ADHESION Li C, Gurcwich V and Liu JN Vascular Research Laboratory, Institute for the Prevention of Cardiovascular Disease, Deaconess Hospital, Harvard Medical School, Boston, MA, USA.
ROLE O F p p b 0 Src IN u P A R - D E P E N D E N T MYEI~OMONOCYTIC A D H E R E N C E .
Urokinase (u-PA) is a well characterized, highly restricted serine protease that converts plasminogen to plasmin. In addition, u-PA or its amino-terminal fragment (ATF) has been shown to induce monocyte adhesion. The cell adhesion effect of u-PA is independent of the protease function of u-PA. Instead, it is related to occupation of a u-PA receptor (u-PAR) found on the surface of many cells. However, the signal transduction system triggered by u-PA receptor binding remains to be identified. In the present study, U937 monocyte adhesion to a plastic surface was used to investigate the signal transduction involved in u-PA induced monocyte adhesion. The u-PA promoted adhesion was found to be inhibited by cycloheximide (an inhibitor of protein synthesis) or actinomycin D (an inhibitor of RNA synthesis), implicating protein synthesis and gene expression in u-PA induced monocyte adhesion. Adhesion was also prevented by 2'-deoxyadenosine 3'-monophosphate, an inhibitor of adenylate cyclase, indicating that a cAMP-dependent pathway of signal transduction was involved. This concept was supported by the complementary finding that u-PA induced adhesion was greatly promoted by forskolin (a direct activator of adenylate cyclase), cholera toxin (an indirect activator of adenylate cyclase through modification o f G-protein) or 8-bromo-cAMP, which by themselves induced little adhesion. Furthermore, similar to many other cAMP-dependent activities, cGMP diminished u-PA induced adhesion. When uPA/ATF was treated with immobilized carboxypeptidase B, its proadhesive effect was abolished, implicating the involvement of carboxyl-terminal lysine residues (Lys 158on u-PA and Lys 135 on ATF). In conclusion, the present study indicates that u-PA/ATF induced monocyte adhesion involves cAMP mediated signal transduction, which is initiated by u-PAR binding with u-PA/ATF which has carboxyl-terminal lysine residue.
F. C h i a r a d o n n a , ('. l a v a r o n e , M. V. C a r r i e r o °, P. Franco, ('. laccarino, 5. Del Vecchio ° , M.V. Barone*, M.P. Stoppelli International Institute of Genetics and Biophysics, CNR, Naples, Italy; ' National Tumor Institute, Naples, Italy; * Differentiation Progran'm~e, EMBL, Heidelberg, Germany. D i f f e r e n t i a t i o n of m o n o c v t e - l i k e cells into a d h e r e n t m a c r o p h a g e s is a c c o m p a n i e d by an i n c r e a s e in the n u m b e r of u P A R s . We observed increased adherence of U937 m o n o c v t e - l i k e cell l i n e transiently overexpressing uPARs. Further increase m a d h e r e n c e is a c h i e v e d by treating d i f f e r e n t i a t i n g m v e l o m o n o c v t i c cells w i t h ATF. This p r o c e s s is not d e p e n d e n t on R N A or p r o t e i n s y n t h e s i s , r e q u i r e s metabolic activity a n d it is affected b v i n h i b i t o r s of tvrosine and serine phosphorylation, uPAd e p e n d e n t c v t o s k e l e t a l r e a r r a n g e m e n t s o c c u r r in mvelomonocvtic cells c o m m i t t e d to a d h e s i o n . F u r t h e r m o r e , t r a n s i e n t e x p r e s s i o n of a k i n a s e inactive ppt~0Src increased a d b e r e n c e of the c u l t u r e up to 80%. A c c o r d i n g l y , e x p r e s s i o n of active pp60Src and ppOOSrc Phe527 slightly r e d u c e d the basal level of a d h e r e n c e of t h e s e cells. T h e s e r e s u l t s u n c o v e r a novel s i g n a l l i n g p a t h w a y l e a d i n g to m a c r o p h a g e a d h e r e n c e a n d s u g g e s t t h a t m e d i a t o r s of t i s s u e i n f l a m m a t i o n , like uPA a n d c v t o k i n e s can t r i g g e r these effects by m o d u l a t i n g ppOOSrc activity.
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Signal Transduction and Biologic Effects Induced by Crosslinking of Receptor-Bound Urokinase in the Human Kidney Epithelial Tumor Cell Line TCL-598 Ruthner M, Koshelnick Yu and Binder BR Dept. Vase. Biol. & Thromb. Res.. Univ. Vienna, Vienna, Austria
S T R U C T U R A L AND F U N C T I O N A L SPECIFICITY O F THE DOMAIN OF THE HUMAN UROKINASE RECEPTOR IN CELL MIGRATION.
The tumor cell line TCL-598 expresses high levels of scu-PA and of its receptor uPAR. Clustering of receptor bound sco-PA by a specific monocloanl antibody (scuPA8) directed against the protease domain of scu-PA elicited a mitogenic/migratory response in TCL-598 in a wounding assay, while a control antibody directed against a different domain in the scu-PA molecule that did not induce clustering of the receptors had no effect. Parallel to the biologic response, receptor clustering also induced tyrosine phosphorylafion of several proteins (p30, p50-60, p75-90, p120-130 and p>200). Tyrosine phosphorylatiun occurred fast within three minutes and reached a maximum after 5 to 10 minutes. When caveolin containing low density Triton X-100 insoluble complexes were analyzed after receptor clustering, uPAR, seu-PA and the crosslinking antibody were found in the "cavenlae" together with the transmembrane protein gpl30 and the tyrosine kinases JAK1 and fyn and the JAKI substrate STATI. Ten minutes after receptor clustering tyrosine kinases were found to be increased but STAT1 was phospbotylated and became excluded from the "caveolae" and appeared in the cytosol in its dimeric form. Consistently, caveolae obtained after receptor clustering also exhibited in vitro kinase activity giving raise to specificallytyrosine phosphorylated proteins (p4050, p75-90, p125, p165 and p>200). Analysis of phospbotyrosine--containing proteins co-precipitated with the uPAR after receptor clustering revealed only one hand with a molecular weight of ~50kd, which upon Western blotting could be identified as fyn. After receptor clustering STATI was not only found in its dimeric form in the cytosol but 20 minutes alter receptor clustering STAT1 was excluded from the cytosul and appeared in the nuclear fraction. In the nuclear matrix receptor clustering induced among others the appearance of three new proteins (p32/4.85, p32/4.93 and I>45/5.6)which could be identified as related to cyclin D1, D2 and E, all belonging to the GI/S family of cyclins. These data suggest that in the TCL-598 cell line clustering of scu-PA bound to its receptor induces a signal via tyrosine kinases involving "caveolae", the JAK/STAT pathway and the GI/S family of cyclins. The uPA-R itself seems to be linked to the src kinase fyn in this cell line.
M. R e s n a l l , N. S l d e n / u s , L . R / I t t i n e n a n d F r a n e e s c o BIasi. DIBIT, San Raffaele Scientif~ Institute, Milan, and Department o f Genetics and Microbial Biology, University of Milan, Italy.
W e have previously shown that catalytically inactive uPA derivatives stimulates a chemotactic response in human monocytic THP-1 and in murine LB6 fibroblastic ceils expressing human uPAR. Transduction of the migratory signal takes place via uPAR, involves a time-dependent activation o f the tyrosine kinase p56/p59 hck and requires an as yet undefined transmembrane adaptor capable o f connecting the extracellular ligand-binding uPAR to intracellular transducer(s), such as p56/p59 ~ k (1). The existence of such an adaptor is strongly supported by the finding that a chymotrypsin-cleaved form o f soluble uPAR (suPAR) is a potent chemoattractant not only in THP-1 ceils, but also in cells lacking endogenous human uPAR, such as the parental untrasfected LB6 cells and macrophages obtained from uPAR-/- mice (1). LB6 cells expressing human uPAR deletion mutants containing only some of the receptor's domains (2) were analysed for thei ability to migrate towards a human ATF gradient. By this approach we were able to identify the uPAR's domain specifically required for signalling and thus possibly for the interaction with the transmembrane adaptor. To confirm these data and to define more finely the uPAR region responsible for promoting cell migration we have also constructed and purified different soluble uPAR sequences which have been used as chemoattractant in murine untrasfected LB6 cells. (1) Resnati et al., 1996, EMBO J., in press. (2) Riittinen et al. 1996, Febs Lea., in press.
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CYCLIC AMP-MEDIATED SIGNAL TRANSDUCTION IN UROKINASE-INDUCED MONOCYTE ADHESION Li C, Gurcwich V and Liu JN Vascular Research Laboratory, Institute for the Prevention of Cardiovascular Disease, Deaconess Hospital, Harvard Medical School, Boston, MA, USA.
ROLE O F p p b 0 Src IN u P A R - D E P E N D E N T MYEI~OMONOCYTIC A D H E R E N C E .
Urokinase (u-PA) is a well characterized, highly restricted serine protease that converts plasminogen to plasmin. In addition, u-PA or its amino-terminal fragment (ATF) has been shown to induce monocyte adhesion. The cell adhesion effect of u-PA is independent of the protease function of u-PA. Instead, it is related to occupation of a u-PA receptor (u-PAR) found on the surface of many cells. However, the signal transduction system triggered by u-PA receptor binding remains to be identified. In the present study, U937 monocyte adhesion to a plastic surface was used to investigate the signal transduction involved in u-PA induced monocyte adhesion. The u-PA promoted adhesion was found to be inhibited by cycloheximide (an inhibitor of protein synthesis) or actinomycin D (an inhibitor of RNA synthesis), implicating protein synthesis and gene expression in u-PA induced monocyte adhesion. Adhesion was also prevented by 2'-deoxyadenosine 3'-monophosphate, an inhibitor of adenylate cyclase, indicating that a cAMP-dependent pathway of signal transduction was involved. This concept was supported by the complementary finding that u-PA induced adhesion was greatly promoted by forskolin (a direct activator of adenylate cyclase), cholera toxin (an indirect activator of adenylate cyclase through modification o f G-protein) or 8-bromo-cAMP, which by themselves induced little adhesion. Furthermore, similar to many other cAMP-dependent activities, cGMP diminished u-PA induced adhesion. When uPA/ATF was treated with immobilized carboxypeptidase B, its proadhesive effect was abolished, implicating the involvement of carboxyl-terminal lysine residues (Lys 158on u-PA and Lys 135 on ATF). In conclusion, the present study indicates that u-PA/ATF induced monocyte adhesion involves cAMP mediated signal transduction, which is initiated by u-PAR binding with u-PA/ATF which has carboxyl-terminal lysine residue.
F. C h i a r a d o n n a , ('. l a v a r o n e , M. V. C a r r i e r o °, P. Franco, ('. laccarino, 5. Del Vecchio ° , M.V. Barone*, M.P. Stoppelli International Institute of Genetics and Biophysics, CNR, Naples, Italy; ' National Tumor Institute, Naples, Italy; * Differentiation Progran'm~e, EMBL, Heidelberg, Germany. D i f f e r e n t i a t i o n of m o n o c v t e - l i k e cells into a d h e r e n t m a c r o p h a g e s is a c c o m p a n i e d by an i n c r e a s e in the n u m b e r of u P A R s . We observed increased adherence of U937 m o n o c v t e - l i k e cell l i n e transiently overexpressing uPARs. Further increase m a d h e r e n c e is a c h i e v e d by treating d i f f e r e n t i a t i n g m v e l o m o n o c v t i c cells w i t h ATF. This p r o c e s s is not d e p e n d e n t on R N A or p r o t e i n s y n t h e s i s , r e q u i r e s metabolic activity a n d it is affected b v i n h i b i t o r s of tvrosine and serine phosphorylation, uPAd e p e n d e n t c v t o s k e l e t a l r e a r r a n g e m e n t s o c c u r r in mvelomonocvtic cells c o m m i t t e d to a d h e s i o n . F u r t h e r m o r e , t r a n s i e n t e x p r e s s i o n of a k i n a s e inactive ppt~0Src increased a d b e r e n c e of the c u l t u r e up to 80%. A c c o r d i n g l y , e x p r e s s i o n of active pp60Src and ppOOSrc Phe527 slightly r e d u c e d the basal level of a d h e r e n c e of t h e s e cells. T h e s e r e s u l t s u n c o v e r a novel s i g n a l l i n g p a t h w a y l e a d i n g to m a c r o p h a g e a d h e r e n c e a n d s u g g e s t t h a t m e d i a t o r s of t i s s u e i n f l a m m a t i o n , like uPA a n d c v t o k i n e s can t r i g g e r these effects by m o d u l a t i n g ppOOSrc activity.
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