Different modes and qualities of tyrosine phosphorylation of Fak and Pyk2 during epithelial-mesenchymal transdifferentiation and cell migration: analysis of specific phosphorylation events using site-directed antibodies.
Integrin signaling is activated during epithelial-mesenchymal transdifferentiation (EMT) and cell migration, processes serving as models for carcinogenesis. We have shown that paxillin and p130Cas become highly tyrosine phosphorylated during these processes in NMuMG cells. Here, we examined the regulation of Fak and Pyk2, kinases implicated in this phosphorylation. Pyk2 became phosphorylated at the major autophosphorylation site (Tyr-402) and the potential Grb2-binding site (Tyr-881) during EMT. In contrast, phosphorylation of Fak at the corresponding autophosphorylation site (Tyr-397) occurred even in sedentary epithelial cells, whereas phosphorylation at Tyr-407 and Tyr-861 was induced during EMT. During cell migration, these phosphorylation events, except Fak Tyr-397, were augmented further, and phosphorylation of Fak Tyr-577 and the corresponding Pyk2 Tyr-580, both within the kinase activation loops, was also induced. In all cases, phosphorylation of the putative Grb2-binding site in Fak (Tyr-925) was almost undetectable. Although Fak and Pyk2 have several phosphorylation sites in common, Tyr-407 and Tyr-861 are unique to Fak. Our results revealed that Fak and Pyk2 are non-equivalent in the tyrosine phosphorylation events and thereby likely to evoke different downstream signaling cascades during EMT and cell migration of NMuMG cells. We also show that Fak Tyr-397 phosphorylation occurs exclusively at the cytoplasm, but not at focal contacts, in the sedentary epithelial cells. In contrast, all other tyrosine phosphorylated forms of Fak and Pyk2 are predominantly localized to focal adhesions and the cell periphery in motile cells, all colocalized with paxillin and p130Cas.
Nakamura
K
K
Department of Molecular Biology, Osaka Bioscience Institute, Suita, Osaka 565-0874, Japan.
Yano
H
H
Schaefer
E
E
Sabe
H
H
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D001665:Binding Sites; D002448:Cell Adhesion / Q000502:physiology; D002454:Cell Differentiation / Q000502:physiology; D002465:Cell Movement / Q000502:physiology*; D050719:Crk-Associated Substrate Protein; D003593:Cytoplasm / Q000201:enzymology; D003598:Cytoskeletal Proteins / Q000378:metabolism; D004789:Enzyme Activation; D004847:Epithelial Cells / Q000166:cytology / Q000201:enzymology; D051417:Focal Adhesion Kinase 1; D051418:Focal Adhesion Kinase 2; D051416:Focal Adhesion Protein-Tyrosine Kinases; D051379:Mice; D051419:Paxillin; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000276:immunology / Q000378:metabolism*; D011506:Proteins; D050717:Retinoblastoma-Like Protein p130; D015398:Signal Transduction / Q000502:physiology; D016212:Transforming Growth Factor beta / Q000494:pharmacology; D053773:Transforming Growth Factor beta1; D014443:Tyrosine / Q000378:metabolism
D000911:Antibodies, Monoclonal; C493842:Bcar1 protein, mouse; D050719:Crk-Associated Substrate Protein; D003598:Cytoskeletal Proteins; D051419:Paxillin; D010750:Phosphoproteins; D011506:Proteins; C496062:Pxn protein, mouse; D050717:Retinoblastoma-Like Protein p130; C508961:Tgfb1 protein, mouse; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051418:Focal Adhesion Kinase 2; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496051:Ptk2 protein, mouse; C496053:Ptk2b protein, mouse