Cell-surface cytokeratin 8 is the major plasminogen receptor on breast cancer cells and is required for the accelerated activation of cell-associated plasminogen by tissue-type plasminogen activator.
Cytokeratin 8 (CK 8) has been identified on the external surfaces of viable, unpermeabilized epithelial cells (Hembrough, T. A., Vasudevan, J., Allietta, M. M., Glass, W. F., and Gonias, S. L. (1995) J. Cell Sci. 108, 1071-1082). In this study, we demonstrated that CK 8 is the major plasminogen-binding protein in plasma membrane fractions isolated from three breast cancer cell lines, BT20, MCF-7, and MDA-MB-157. To assess the function of CK 8 as a plasminogen receptor, monoclonal antibody 1E8 was raised against the carboxyl-terminal 12 amino acids of CK 8. The 1E8 epitope was present on the external surfaces of breast cancer cells, as determined by immunofluorescence microscopy. 125I-1E8 bound to MCF-7 cells; the maximum binding capacity (1.5 x 10(6) sites per cell) was comparable with that determined for plasminogen. When MCF-7 cells were incubated with Fab fragments of 1E8, specific 125I-plasminogen binding was decreased up to 82%. Specific plasminogen binding was decreased up to 67%, even when the unbound 1E8 Fab was removed by washing the cells prior to adding 125I-plasminogen. Preincubation with 1E8 Fab decreased plasminogen binding to BT20 and MDA-MB-157 cells, although to a lesser extent than with MCF-7 cells. Plasminogen activation by tissue-type plasminogen activator was greatly accelerated, due to a large decrease in Km, when the plasminogen was bound to MCF-7 cells. Pretreatment with 1E8 Fab decreased the rate of plasminogen activation by up to 83%, implicating CK 8 in the MCF-7 cell-accelerated reaction. These studies identify cell-surface CK 8 as a major plasminogen receptor in breast cancer cells and as a required component for the rapid activation of cell-associated plasminogen by tissue-type plasminogen activator.
Hembrough
T A
TA
Department of Biochemistry, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908, USA.
Li
L
L
Gonias
S L
SL
D000911:Antibodies, Monoclonal / Q000494:pharmacology; D000918:Antibody Specificity; D000936:Antigen-Antibody Complex; D001943:Breast Neoplasms / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D005260:Female; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000494:pharmacology; D007633:Keratins / Q000737:chemistry / Q000502:physiology*; D007700:Kinetics; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D010958:Plasminogen / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000502:physiology*; D055293:Receptors, Urokinase Plasminogen Activator; D013347:Subcellular Fractions / Q000378:metabolism; D010959:Tissue Plasminogen Activator / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D007140:Immunoglobulin Fab Fragments; C524898:PLAUR protein, human; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D007633:Keratins; D010958:Plasminogen; D010959:Tissue Plasminogen Activator