Characterization of the binding sites for plasminogen and tissue-type plasminogen activator in cytokeratin 8 and cytokeratin 18

Cytokeratin 8 (CK8) is an intermediate filament protein that penetrates to the external surfaces of breast cancer cells and is released from cells in the form of soluble heteropolymers. CK8 binds plasminogen and tissue-type p lasminogen activator (t-PA) and accelerates plasminogen activation on cance r cell surfaces. The plasminogen-binding site is located at the C-terminus of CK8. Ln this study, we prepared GST-fusion proteins which contained eith er 174 amino acids from the C-terminus of CK8 (CK8f) or 134 amino acids fro m the C-terminus of CK18 (CK18f). A third GST-CK fusion protein was identic al to CK8f except that the C-terminal lysine was mutated to glutamine (CK8f (K483Q)). CK8f bound plasminogen; the K-D was 0.5 mu M. Binding was complet ely inhibited by epsilon ACA. CK8f(K483Q) also bound plasminogen, albeit wi th decreased affinity (K-D approximate to 1.5 mu M). CK18f did not bind pla sminogen at all. All three fusion proteins bound t-PA equivalently, providi ng the first evidence that CK18 may function as a t-PA receptor. t-PA and p lasminogen cross-competed for binding to CK8f. Thus, t-PA and plasminogen c annot bind to the same CK8f monomer simultaneously. Nevertheless, CK8f stil l promoted plasminogen activation, probably reflecting the fact that CK8f w as purified in dimeric or tetrameric form. These studies demonstrate that C K8 may promote plasminogen activation by t-PA only when present in an oligo merized state. CK18 may participate in the oligomer, together with CK8, bas ed on its ability to bind t-PA.