Differential binding of plasminogen, plasmin, and angiostatin4.5 to cell surface beta-actin: implications for cancer-mediated angiogenesis.
Angiostatin4.5 (AS4.5) is the product of plasmin autoproteolysis and consists of kringles 1 to 4 and approximately 85% of kringle 5. In culture, cancer cell surface globular beta-actin mediates plasmin autoproteolysis to AS4.5. We now show that plasminogen binds to prostate cancer cells and that the binding colocalizes with surface beta-actin, but AS4.5 does not bind to the cell surface. Plasminogen and plasmin bind to immobilized beta-actin similarly, with a Kd of approximately 140 nmol/L. The binding is inhibited by epsilon-aminocaproic acid (epsilonACA), indicating the requirement for a lysine-kringle domain interaction. Using a series of peptides derived from beta-actin in competitive binding studies, we show that the domain necessary for plasminogen binding is within amino acids 55 to 69 (GDEAQSKRGILTLKY). Substitution of Lys61 or Lys68 with arginine results in the loss of the ability of the peptide to block plasminogen binding, indicating that Lys61 and Lys68 are essential for plasminogen binding. Other actin peptides, including peptides with lysine, did not inhibit the plasminogen-actin interaction. AS4.5 did not bind actin at concentrations up to 40 micromol/L. Plasminogen, plasmin, and AS4.5 all contain kringles 1 to 4; however, kringle 5 is truncated in AS4.5. Isolated kringle 5 binds to actin, suggesting intact kringle 5 is necessary for plasminogen and plasmin to bind to cell surface beta-actin, and the truncated kringle 5 in AS4.5 results in its release from beta-actin. These data may explain the mechanism by which AS4.5 is formed locally on cancer cell surfaces and yet acts on distant sites.
Wang
Hao
H
Division of Hematology/Oncology and Division of Pulmonary and Critical Care Medicine, Department of Medicine and Robert H. Lurie Comprehensive Cancer Center, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.
Doll
Jennifer A
JA
Jiang
Keyi
K
Cundiff
Deborah L
DL
Czarnecki
Jarema S
JS
Wilson
Mindy
M
Ridge
Karen M
KM
Soff
Gerald A
GA
D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D043168:Angiostatins / Q000378:metabolism*; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005341:Fibrinolysin / Q000378:metabolism*; D006801:Humans; D008297:Male; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009389:Neovascularization, Pathologic / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D010958:Plasminogen / Q000378:metabolism*; D011471:Prostatic Neoplasms / Q000098:blood supply* / Q000378:metabolism*; D011485:Protein Binding
D000199:Actins; D010446:Peptide Fragments; C483085:angiostatin4.5, human; D043168:Angiostatins; D010958:Plasminogen; D005341:Fibrinolysin