title: Induction of cell migration by pro-urokinase binding to its receptor: possible mechanism for signal transduction in human epithelial cells.
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.
authors.affiliation: Department of Medicine, Mount Sinai School of Medicine, New York 10029.
authors.forename: N
authors.initials: N
authors.lastname: Busso
authors.forename: S K
authors.initials: SK
authors.lastname: Masur
authors.forename: D
authors.initials: D
authors.lastname: Lazega
authors.forename: S
authors.initials: S
authors.lastname: Waxman
authors.forename: L
authors.initials: L
authors.lastname: Ossowski
mesh_terms: D000595:Amino Acid Sequence; D002460:Cell Line; D002465:Cell Movement / Q000502:physiology*; D048429:Cell Size / Q000502:physiology; D004792:Enzyme Precursors / Q000378:metabolism; D004847:Epithelial Cells; D004848:Epithelium / Q000201:enzymology; D006801:Humans; D007633:Keratins / Q000378:metabolism; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes; D010766:Phosphorylation; D010960:Plasminogen Activators / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism*; D055293:Receptors, Urokinase Plasminogen Activator; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism*
chemical_list: D004792:Enzyme Precursors; D046911:Macromolecular Substances; D046912:Multiprotein Complexes; C524898:PLAUR protein, human; C524899:Plaur protein, mouse; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D011994:Recombinant Proteins; D007633:Keratins; D011505:Protein-Tyrosine Kinases; D010960:Plasminogen Activators; D014568:Urokinase-Type Plasminogen Activator; C067551:saruplase