Proteomic identification of lynchpin urokinase plasminogen activator receptor protein interactions associated with epithelial cancer malignancy.
Urokinase plasminogen activator (uPA) and its high affinity receptor (uPAR) play crucial proteolytic and non-proteolytic roles in cancer metastasis. In addition to promoting plasmin-mediated degradation of extracellular matrix barriers, cell surface engagement of uPA through uPAR binding results in the activation of a suite of diverse cellular signal transduction pathways. Because uPAR is bound to the plasma membrane through a glycosyl-phosphatidylinositol anchor, these signalling sequelae are thought to occur through the formation of multi-protein cell surface complexes involving uPAR. To further characterize uPAR-driven protein complexes, we co-immunoprecipitated uPAR from the human ovarian cancer cell line, OVCA 429, and employed sensitive proteomic methods to identify the uPAR-associated proteins. Using this strategy, we identified several known, as well as numerous novel, uPAR associating proteins, including the epithelial restricted integrin, alphavbeta6. Reverse immunoprecipitation using anti-beta6 integrin subunit monoclonal antibodies confirmed the co-purification of this protein with uPAR. Inhibition of uPAR and/or beta6 integrin subunit using neutralizing antibodies resulted in the inhibition of uPA-mediated ERK 1/2 phosphorylation and subsequent cell proliferation. These data suggest that the association of beta6 integrin (and possibly other lynchpin cancer regulatory proteins) with uPAR may be crucial in co-transmitting uPA signals that induce cell proliferation. Our findings support the notion that uPAR behaves as a lynchpin in promoting tumorigenesis by forming functionally active multiprotein complexes.
Saldanha
Rohit G
RG
Australian Proteome Analysis Facility Ltd and Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, 2109, NSW Australia.
Molloy
Mark P
MP
Bdeir
Khalil
K
Cines
Douglas B
DB
Song
Xiaomin
X
Uitto
Pauliina M
PM
Weinreb
Paul H
PH
Violette
Shelia M
SM
Baker
Mark S
MS
D000951:Antigens, Neoplasm / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D004848:Epithelium / Q000473:pathology; D005260:Female; D006801:Humans; D047468:Immunoprecipitation; D039641:Integrin beta Chains / Q000378:metabolism; D016023:Integrins / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D046912:Multiprotein Complexes / Q000378:metabolism; D010051:Ovarian Neoplasms / Q000209:etiology* / Q000473:pathology; D010766:Phosphorylation; D040901:Proteomics / Q000379:methods*; D011956:Receptors, Cell Surface / Q000378:metabolism*; D055293:Receptors, Urokinase Plasminogen Activator
D000951:Antigens, Neoplasm; D039641:Integrin beta Chains; D016023:Integrins; D046912:Multiprotein Complexes; C524898:PLAUR protein, human; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; C073878:integrin alphavbeta6; C072170:integrin beta6; D048052:Mitogen-Activated Protein Kinase 3