title: Receptor-mediated endocytosis of urokinase-type plasminogen activator is regulated by cAMP-dependent protein kinase.
abstract: Internalization of the urokinase-type plasminogen activator (uPA) requires two receptors, the uPA receptor (uPAR) and the low density lipoprotein receptor-related protein (LRP)/alpha2-macroglobulin (alpha2M) receptor. Here, we address whether protein kinases are involved in the internalization of uPA by human melanoma cells. Initially, we found that the internalization of uPA was significantly inhibited by the serine/threonine protein kinase inhibitors staurosporine, K-252a and H-89, but not by the tyrosine kinase inhibitors, genistein and lavendustin A. Internalization of uPA was also inhibited by a pseudosubstrate peptide for cAMP-dependent protein kinase (PKA), but not by a pseudosubstrate peptide for protein kinase C. We confirmed a requirement for PKA-activity and implicated a specific isoform by using an antisense oligonucleotide against the regulatory subunit RI alpha of PKA which suppresses PKA-I activity. Exposure of cells to this oligonucleotide led to a specific, dose-dependent decrease in RI alpha protein and to a significant inhibition in the rate of uPA internalization. We further demonstrate that treatment of melanoma cells with either H-89 or PKA RI alpha antisense oligonucleotides also resulted in a decreased internalization of two other ligands of LRP, activated alpha2M and lactoferrin, indicating that PKA activity is associated with LRP. Finally, we demonstrate that PKA activity is also required for the internalization of transferrin, but not for the internalization of the epidermal growth factor or adenovirus 2, suggesting that in melanoma cells, PKA activity is not generally required for clathrin-mediated endocytosis, but is rather associated with specific internalization receptors.
authors.affiliation: The Scripps Research Institute, Department of Immunology, La Jolla, CA 92037, USA.
authors.forename: L
authors.initials: L
authors.lastname: Goretzki
authors.forename: B M
authors.initials: BM
authors.lastname: Mueller
mesh_terms: D000260:Adenoviruses, Human / Q000378:metabolism; D002227:Carbazoles / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004705:Endocytosis / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000378:metabolism; D019833:Genistein; D006801:Humans; D026121:Indole Alkaloids; D007211:Indoles / Q000494:pharmacology; D007529:Isoflavones / Q000494:pharmacology; D007546:Isoquinolines / Q000494:pharmacology; D007781:Lactoferrin / Q000378:metabolism; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D008301:Maleimides / Q000494:pharmacology; D008545:Melanoma / Q000201:enzymology; D009281:Naphthalenes / Q000494:pharmacology; D010636:Phenols / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D011971:Receptors, Immunologic / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D019311:Staurosporine / Q000494:pharmacology; D013449:Sulfonamides; D014407:Tumor Cells, Cultured / Q000187:drug effects; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism*
chemical_list: D002227:Carbazoles; D004791:Enzyme Inhibitors; D026121:Indole Alkaloids; D007211:Indoles; D007529:Isoflavones; D007546:Isoquinolines; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D008301:Maleimides; D009281:Naphthalenes; D010636:Phenols; D011971:Receptors, Immunologic; D013449:Sulfonamides; C062898:lavendustin A; D004815:Epidermal Growth Factor; C049985:staurosporine aglycone; D019833:Genistein; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D007781:Lactoferrin; D014568:Urokinase-Type Plasminogen Activator; D019311:Staurosporine; C058819:calphostin C; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide; C088060:bisindolylmaleimide