Hepatocyte growth factor induces ERK-dependent paxillin phosphorylation and regulates paxillin-focal adhesion kinase association.
Hepatocyte growth factor (HGF) modulates cell adhesion, migration, and branching morphogenesis in cultured epithelial cells, events that require regulation of cell-matrix interactions. Using mIMCD-3 epithelial cells, we studied the effect of HGF on the focal adhesion proteins, focal adhesion kinase (FAK) and paxillin and their association. HGF was found to increase the tyrosine phosphorylation of paxillin and to a lesser degree FAK. In addition, HGF induced association of paxillin and activated ERK, correlating with a gel retardation of paxillin that was prevented with the ERK inhibitor U0126. The ability of activated ERK to phosphorylate and induce gel retardation of paxillin was confirmed in vitro in both full-length and amino-terminal paxillin. Several potential ERK phosphorylation sites in paxillin flank the paxillin-FAK association domains, so the ability of HGF to regulate paxillin-FAK association was examined. HGF induced an increase in paxillin-FAK association that was inhibited by pretreatment with U0126 and reproduced by in vitro phosphorylation of paxillin with ERK. The prevention of the FAK-paxillin association with U0126 correlated with an inhibition of the HGF-mediated FAK tyrosine phosphorylation and inhibition of HGF-dependent cell spreading and adhesion. An examination of cellular localization of FAK and paxillin demonstrated that HGF caused a condensation of focal adhesion complexes at the leading edges of cell processes and FAK-paxillin co-localization in these large complexes. Thus, these data suggest that HGF can induce serine/threonine phosphorylation of paxillin most probably mediated directly by ERK, resulting in the recruitment and activation of FAK and subsequent enhancement of cell spreading and adhesion.
Liu
Zhen-Xiang
ZX
Section of Nephrology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
Yu
Cheng Fang
CF
Nickel
Christian
C
Thomas
Sheila
S
Cantley
Lloyd G
LG
D000818:Animals; D002070:Butadienes / Q000494:pharmacology; D002448:Cell Adhesion; D002460:Cell Line; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D005982:Glutathione Transferase / Q000378:metabolism; D017228:Hepatocyte Growth Factor / Q000378:metabolism*; D007684:Kidney Tubules / Q000166:cytology; D020935:MAP Kinase Signaling System; D051379:Mice; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D009570:Nitriles / Q000494:pharmacology; D051419:Paxillin; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D013912:Threonine / Q000378:metabolism; D013997:Time Factors; D014443:Tyrosine / Q000378:metabolism
D002070:Butadienes; D003598:Cytoskeletal Proteins; D004791:Enzyme Inhibitors; D009570:Nitriles; D051419:Paxillin; D010750:Phosphoproteins; C496062:Pxn protein, mouse; D011993:Recombinant Fusion Proteins; C113580:U 0126; D013912:Threonine; D014443:Tyrosine; D012694:Serine; D017228:Hepatocyte Growth Factor; D005982:Glutathione Transferase; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496051:Ptk2 protein, mouse; D020928:Mitogen-Activated Protein Kinases