Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1994 Jul 1;126(1):259–270. doi: 10.1083/jcb.126.1.259

Induction of cell migration by pro-urokinase binding to its receptor: possible mechanism for signal transduction in human epithelial cells

PMCID: PMC2120093  PMID: 7517943

Abstract

A human epithelial cell line, WISH, and a mouse cell line, LB6-uPAR, transfected with the human urokinase receptor (uPAR), both expressed high affinity uPAR but undetectable levels of urokinase (uPA). In two independent assays, binding of exogenous pro-uPA produced an up to threefold enhancement of migration. The migration was time and concentration dependent and did not involve extracellular proteolysis. This biologic response suggested that uPAR can trigger an intracellular signal. Since this receptor is a glycosyl-phosphatidylinositol-linked protein, we postulated that it must do so by interacting with other proteins, among which, by analogy to other systems, would be a kinase. To test this hypothesis, we carried out a solid phase capture of uPAR from WISH cell lysates using either antibodies against uPAR or pro-uPA adsorbed to plastic wells, followed by in vitro phosphorylation of the immobilized proteins. SDS-PAGE and autoradiography revealed two phosphorylated protein bands of 47 and 55 kD. Both proteins were phosphorylated on serine residues. Partial sequence of the two proteins showed a 100% homology to cytokeratin 18 (CK18) and 8 (CK8), respectively. A similar pattern of phosphorylation was obtained with lysates from A459 cells, a lung carcinoma, but not HL60, LB6-uPAR or HEp3 cell lysates, suggesting that the identified multiprotein uPAR- complex may be specific for simple epithelia. Moreover, immunocapture with antibody to another glycosyl-phosphatidylinositol-linked protein, CD55, which is highly expressed in WISH cells, was ineffective. The kinase was tentatively identified as protein kinase C, because it was inhibited by an analogue of staurosporine more specific for PKC and not by a PKA or tyrosine kinase inhibitors. The kinase was tentatively identified as PKC epsilon because of its resistance to PMA down- modulation, independence of Ca2+ for activity, and reaction with a specific anti-PKC epsilon antibody in Western blots. Cell fractionation into cytosolic and particulate fractions revealed that all four proteins, the kinase, uPAR, CK18, and CK8, were present in the particulate fraction. In vivo, CK8, and to a lesser degree CK18, were found to be phosphorylated on serine residues. Occupation of uPAR elicited a time-dependent increase in the phosphorylation intensity of CK8, a cell shape change and a redistribution of the cytokeratin filaments. These results strongly suggest that uPAR serves not only as an anchor for uPA but participates in a signal transduction pathway resulting in a pronounced biological response.

Full Text

The Full Text of this article is available as a PDF (3.9 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Appella E., Robinson E. A., Ullrich S. J., Stoppelli M. P., Corti A., Cassani G., Blasi F. The receptor-binding sequence of urokinase. A biological function for the growth-factor module of proteases. J Biol Chem. 1987 Apr 5;262(10):4437–4440. [PubMed] [Google Scholar]
  2. Baribault H., Blouin R., Bourgon L., Marceau N. Epidermal growth factor-induced selective phosphorylation of cultured rat hepatocyte 55-kD cytokeratin before filament reorganization and DNA synthesis. J Cell Biol. 1989 Oct;109(4 Pt 1):1665–1676. doi: 10.1083/jcb.109.4.1665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Baxter G., Oto E., Daniel-Issakani S., Strulovici B. Constitutive presence of a catalytic fragment of protein kinase C epsilon in a small cell lung carcinoma cell line. J Biol Chem. 1992 Jan 25;267(3):1910–1917. [PubMed] [Google Scholar]
  4. Blasi F. Urokinase and urokinase receptor: a paracrine/autocrine system regulating cell migration and invasiveness. Bioessays. 1993 Feb;15(2):105–111. doi: 10.1002/bies.950150206. [DOI] [PubMed] [Google Scholar]
  5. Brown D. A., Rose J. K. Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface. Cell. 1992 Feb 7;68(3):533–544. doi: 10.1016/0092-8674(92)90189-j. [DOI] [PubMed] [Google Scholar]
  6. Chou C. F., Omary M. B. Phorbol acetate enhances the phosphorylation of cytokeratins 8 and 18 in human colonic epithelial cells. FEBS Lett. 1991 Apr 22;282(1):200–204. doi: 10.1016/0014-5793(91)80477-k. [DOI] [PubMed] [Google Scholar]
  7. Chu Y. W., Runyan R. B., Oshima R. G., Hendrix M. J. Expression of complete keratin filaments in mouse L cells augments cell migration and invasion. Proc Natl Acad Sci U S A. 1993 May 1;90(9):4261–4265. doi: 10.1073/pnas.90.9.4261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cooper J. A., Sefton B. M., Hunter T. Detection and quantification of phosphotyrosine in proteins. Methods Enzymol. 1983;99:387–402. doi: 10.1016/0076-6879(83)99075-4. [DOI] [PubMed] [Google Scholar]
  9. Cubellis M. V., Nolli M. L., Cassani G., Blasi F. Binding of single-chain prourokinase to the urokinase receptor of human U937 cells. J Biol Chem. 1986 Dec 5;261(34):15819–15822. [PubMed] [Google Scholar]
  10. Danø K., Andreasen P. A., Grøndahl-Hansen J., Kristensen P., Nielsen L. S., Skriver L. Plasminogen activators, tissue degradation, and cancer. Adv Cancer Res. 1985;44:139–266. doi: 10.1016/s0065-230x(08)60028-7. [DOI] [PubMed] [Google Scholar]
  11. Del Rosso M., Anichini E., Pedersen N., Blasi F., Fibbi G., Pucci M., Ruggiero M. Urokinase-urokinase receptor interaction: non-mitogenic signal transduction in human epidermal cells. Biochem Biophys Res Commun. 1993 Jan 29;190(2):347–352. doi: 10.1006/bbrc.1993.1054. [DOI] [PubMed] [Google Scholar]
  12. Dumler I., Petri T., Schleuning W. D. Interaction of urokinase-type plasminogenactivator (u-PA) with its cellular receptor (u-PAR) induces phosphorylation on tyrosine of a 38 kDa protein. FEBS Lett. 1993 May 3;322(1):37–40. doi: 10.1016/0014-5793(93)81106-a. [DOI] [PubMed] [Google Scholar]
  13. Estreicher A., Wohlwend A., Belin D., Schleuning W. D., Vassalli J. D. Characterization of the cellular binding site for the urokinase-type plasminogen activator. J Biol Chem. 1989 Jan 15;264(2):1180–1189. [PubMed] [Google Scholar]
  14. Fernandez J., DeMott M., Atherton D., Mische S. M. Internal protein sequence analysis: enzymatic digestion for less than 10 micrograms of protein bound to polyvinylidene difluoride or nitrocellulose membranes. Anal Biochem. 1992 Mar;201(2):255–264. doi: 10.1016/0003-2697(92)90336-6. [DOI] [PubMed] [Google Scholar]
  15. Fibbi G., Ziche M., Morbidelli L., Magnelli L., Del Rosso M. Interaction of urokinase with specific receptors stimulates mobilization of bovine adrenal capillary endothelial cells. Exp Cell Res. 1988 Dec;179(2):385–395. doi: 10.1016/0014-4827(88)90277-7. [DOI] [PubMed] [Google Scholar]
  16. Gudewicz P. W., Gilboa N. Human urokinase-type plasminogen activator stimulates chemotaxis of human neutrophils. Biochem Biophys Res Commun. 1987 Sep 30;147(3):1176–1181. doi: 10.1016/s0006-291x(87)80193-6. [DOI] [PubMed] [Google Scholar]
  17. Hendrix M. J., Seftor E. A., Chu Y. W., Seftor R. E., Nagle R. B., McDaniel K. M., Leong S. P., Yohem K. H., Leibovitz A. M., Meyskens F. L., Jr Coexpression of vimentin and keratins by human melanoma tumor cells: correlation with invasive and metastatic potential. J Natl Cancer Inst. 1992 Feb 5;84(3):165–174. doi: 10.1093/jnci/84.3.165. [DOI] [PubMed] [Google Scholar]
  18. Hébert C. A., Baker J. B. Linkage of extracellular plasminogen activator to the fibroblast cytoskeleton: colocalization of cell surface urokinase with vinculin. J Cell Biol. 1988 Apr;106(4):1241–1247. doi: 10.1083/jcb.106.4.1241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Jallal B., Schlessinger J., Ullrich A. Tyrosine phosphatase inhibition permits analysis of signal transduction complexes in p185HER2/neu-overexpressing human tumor cells. J Biol Chem. 1992 Mar 5;267(7):4357–4363. [PubMed] [Google Scholar]
  20. Ledinko N., Costantino R. L. Modulation of p53 gene expression and cytokeratin 18 in retinoid-mediated invasion suppressed lung carcinoma cells. Anticancer Res. 1990 Sep-Oct;10(5A):1335–1339. [PubMed] [Google Scholar]
  21. McNeill H., Jensen P. J. A high-affinity receptor for urokinase plasminogen activator on human keratinocytes: characterization and potential modulation during migration. Cell Regul. 1990 Oct;1(11):843–852. doi: 10.1091/mbc.1.11.843. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Mignatti P., Rifkin D. B. Biology and biochemistry of proteinases in tumor invasion. Physiol Rev. 1993 Jan;73(1):161–195. doi: 10.1152/physrev.1993.73.1.161. [DOI] [PubMed] [Google Scholar]
  23. Mira-y-Lopez R., Ossowski L. Hormonal modulation of plasminogen activator: an approach to prediction of human breast tumor responsiveness. Cancer Res. 1987 Jul 1;47(13):3558–3564. [PubMed] [Google Scholar]
  24. Morioka S., Lazarus G. S., Baird J. L., Jensen P. J. Migrating keratinocytes express urokinase-type plasminogen activator. J Invest Dermatol. 1987 Apr;88(4):418–423. doi: 10.1111/1523-1747.ep12469754. [DOI] [PubMed] [Google Scholar]
  25. Munson P. J., Rodbard D. Ligand: a versatile computerized approach for characterization of ligand-binding systems. Anal Biochem. 1980 Sep 1;107(1):220–239. doi: 10.1016/0003-2697(80)90515-1. [DOI] [PubMed] [Google Scholar]
  26. Odekon L. E., Sato Y., Rifkin D. B. Urokinase-type plasminogen activator mediates basic fibroblast growth factor-induced bovine endothelial cell migration independent of its proteolytic activity. J Cell Physiol. 1992 Feb;150(2):258–263. doi: 10.1002/jcp.1041500206. [DOI] [PubMed] [Google Scholar]
  27. Omary M. B., Baxter G. T., Chou C. F., Riopel C. L., Lin W. Y., Strulovici B. PKC epsilon-related kinase associates with and phosphorylates cytokeratin 8 and 18. J Cell Biol. 1992 May;117(3):583–593. doi: 10.1083/jcb.117.3.583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Ossowski L., Clunie G., Masucci M. T., Blasi F. In vivo paracrine interaction between urokinase and its receptor: effect on tumor cell invasion. J Cell Biol. 1991 Nov;115(4):1107–1112. doi: 10.1083/jcb.115.4.1107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Ossowski L., Reich E. Experimental model for quantitative study of metastasis. Cancer Res. 1980 Jul;40(7):2300–2309. [PubMed] [Google Scholar]
  30. Pepper M. S., Vassalli J. D., Montesano R., Orci L. Urokinase-type plasminogen activator is induced in migrating capillary endothelial cells. J Cell Biol. 1987 Dec;105(6 Pt 1):2535–2541. doi: 10.1083/jcb.105.6.2535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Ploug M., Behrendt N., Løber D., Danø K. Protein structure and membrane anchorage of the cellular receptor for urokinase-type plasminogen activator. Semin Thromb Hemost. 1991 Jul;17(3):183–193. doi: 10.1055/s-2007-1002608. [DOI] [PubMed] [Google Scholar]
  32. Pöllänen J., Hedman K., Nielsen L. S., Danø K., Vaheri A. Ultrastructural localization of plasma membrane-associated urokinase-type plasminogen activator at focal contacts. J Cell Biol. 1988 Jan;106(1):87–95. doi: 10.1083/jcb.106.1.87. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Rabbani S. A., Mazar A. P., Bernier S. M., Haq M., Bolivar I., Henkin J., Goltzman D. Structural requirements for the growth factor activity of the amino-terminal domain of urokinase. J Biol Chem. 1992 Jul 15;267(20):14151–14156. [PubMed] [Google Scholar]
  34. Roldan A. L., Cubellis M. V., Masucci M. T., Behrendt N., Lund L. R., Danø K., Appella E., Blasi F. Cloning and expression of the receptor for human urokinase plasminogen activator, a central molecule in cell surface, plasmin dependent proteolysis. EMBO J. 1990 Feb;9(2):467–474. doi: 10.1002/j.1460-2075.1990.tb08132.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Rosen E. M., Meromsky L., Setter E., Vinter D. W., Goldberg I. D. Quantitation of cytokine-stimulated migration of endothelium and epithelium by a new assay using microcarrier beads. Exp Cell Res. 1990 Jan;186(1):22–31. doi: 10.1016/0014-4827(90)90205-o. [DOI] [PubMed] [Google Scholar]
  36. Schaap D., Parker P. J., Bristol A., Kriz R., Knopf J. Unique substrate specificity and regulatory properties of PKC-epsilon: a rationale for diversity. FEBS Lett. 1989 Jan 30;243(2):351–357. doi: 10.1016/0014-5793(89)80160-7. [DOI] [PubMed] [Google Scholar]
  37. Stefanová I., Horejsí V., Ansotegui I. J., Knapp W., Stockinger H. GPI-anchored cell-surface molecules complexed to protein tyrosine kinases. Science. 1991 Nov 15;254(5034):1016–1019. doi: 10.1126/science.1719635. [DOI] [PubMed] [Google Scholar]
  38. Steinert P. M., Liem R. K. Intermediate filament dynamics. Cell. 1990 Feb 23;60(4):521–523. doi: 10.1016/0092-8674(90)90651-t. [DOI] [PubMed] [Google Scholar]
  39. Stoppelli M. P., Tacchetti C., Cubellis M. V., Corti A., Hearing V. J., Cassani G., Appella E., Blasi F. Autocrine saturation of pro-urokinase receptors on human A431 cells. Cell. 1986 Jun 6;45(5):675–684. doi: 10.1016/0092-8674(86)90782-8. [DOI] [PubMed] [Google Scholar]
  40. Strulovici B., Daniel-Issakani S., Baxter G., Knopf J., Sultzman L., Cherwinski H., Nestor J., Jr, Webb D. R., Ransom J. Distinct mechanisms of regulation of protein kinase C epsilon by hormones and phorbol diesters. J Biol Chem. 1991 Jan 5;266(1):168–173. [PubMed] [Google Scholar]
  41. Thomas P. M., Samelson L. E. The glycophosphatidylinositol-anchored Thy-1 molecule interacts with the p60fyn protein tyrosine kinase in T cells. J Biol Chem. 1992 Jun 15;267(17):12317–12322. [PubMed] [Google Scholar]
  42. Toullec D., Pianetti P., Coste H., Bellevergue P., Grand-Perret T., Ajakane M., Baudet V., Boissin P., Boursier E., Loriolle F. The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C. J Biol Chem. 1991 Aug 25;266(24):15771–15781. [PubMed] [Google Scholar]
  43. Vassalli J. D., Baccino D., Belin D. A cellular binding site for the Mr 55,000 form of the human plasminogen activator, urokinase. J Cell Biol. 1985 Jan;100(1):86–92. doi: 10.1083/jcb.100.1.86. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Vassalli J. D., Sappino A. P., Belin D. The plasminogen activator/plasmin system. J Clin Invest. 1991 Oct;88(4):1067–1072. doi: 10.1172/JCI115405. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES