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 plasminogen activator (t-PA) and accelerates plasminogen activation on cancer cell surfaces. The plasminogen-binding site is located at the C-terminus of CK8. In this study, we prepared GST-fusion proteins which contained either 174 amino acids from the C-terminus of CK8 (CK8f) or 134 amino acids from the C-terminus of CK18 (CK18f). A third GST-CK fusion protein was identical to CK8fexcept that the C-terminal lysine was mutated to glutamine (CK8fK483Q). CK8f bound plasminogen; the K(D) was 0.5 microM. Binding was completely inhibited by epsilonACA. CK8fK483Q also bound plasminogen, albeit with decreased affinity (K(D) approximately 1.5 microM). CK18f did not bind plasminogen at all. All three fusion proteins bound t-PA equivalently, providing the first evidence that CK18 may function as a t-PA receptor, t-PA and plasminogen cross-competed for binding to CK8f. Thus, t-PA and plasminogen cannot bind to the same CK8f monomer simultaneously. Nevertheless, CK8f still promoted plasminogen activation, probably reflecting the fact that CK8f was purified in dimeric or tetrameric form. These studies demonstrate that CK8 may promote plasminogen activation by t-PA only when present in an oligomerized state. CK18 may participate in the oligomer, together with CK8, based on its ability to bind t-PA.
Kralovich
K R
KR
Department of Biochemistry and Molecular Genetics, University of Virginia Health Sciences Center, Charlottesville 22908, USA.
Li
L
L
Hembrough
T A
TA
Webb
D J
DJ
Karns
L R
LR
Gonias
S L
SL
D015119:Aminocaproic Acid / Q000378:metabolism / Q000494:pharmacology; D001665:Binding Sites; D001667:Binding, Competitive; D019281:Dimerization; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D007633:Keratins / Q000235:genetics / Q000378:metabolism*; D008239:Lysine; D009154:Mutation; D010958:Plasminogen / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012996:Solutions; D010959:Tissue Plasminogen Activator / Q000187:drug effects / Q000378:metabolism*