Paxillin null embryonic stem cells are impaired in cell spreading and tyrosine phosphorylation of focal adhesion kinase.
Paxillin is a focal-adhesion associated protein implicated in the regulation of integrin signaling and organization of the actin cytoskeleton. Paxillin associates with numerous signaling molecules including adaptor molecules (p130Cas, CRK), kinases (FAK, Pyk2, PAK and SRC), tyrosine phosphatases (PTP-PEST), ARF-GAP proteins (p95pkl, PAG3) and papillomavirus E6 oncoproteins. Although paxillin is tyrosine phosphorylated in cellular processes such as cell attachment and spreading, little direct evidence is available about paxillin's role in these events. Targeted gene disruption was used to generate paxillin null mouse embryonic stem (ES) cells and paxillin null differentiated cells. Paxillin null ES cells exhibit delayed spreading on integrin binding substrates fibronectin and laminin, and there is reduced tyrosine phosphorylation of Focal Adhesion Kinase (FAK). Both of these phenotypes are recovered in paxillin knockout cells upon exogenous re-expression of paxillin. The individual LD motifs of paxillin that are binding sites for FAK, vinculin and ARF-GAP proteins, as well as tyrosine residues that when phosphorylated create binding sites for CRK family members, are dispensable for FAK phosphorylation and early cell spreading. These results demonstrate that paxillin contributes to attachment-dependent tyrosine phosphorylation of FAK and early cell spreading in ES cells.
Wade
Ramon
R
Case Western Reserve University School of Medicine, Institute of Pathology, Cleveland, Ohio, OH 44106, USA.
Bohl
Joanna
J
Vande Pol
Scott
S
D020816:Amino Acid Motifs; D000818:Animals; D001665:Binding Sites; D002448:Cell Adhesion / Q000502:physiology*; D048429:Cell Size; D002999:Clone Cells / Q000166:cytology / Q000201:enzymology; D003470:Culture Media; D003598:Cytoskeletal Proteins / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D004622:Embryo, Mammalian / Q000166:cytology; D020224:Expressed Sequence Tags; D005353:Fibronectins; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D018390:Gene Targeting; D016023:Integrins / Q000502:physiology; D007797:Laminin; D051379:Mice; D009838:Oligodeoxyribonucleotides / Q000235:genetics / Q000494:pharmacology; D051419:Paxillin; D010750:Phosphoproteins / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000502:physiology; D013234:Stem Cells / Q000166:cytology* / Q000201:enzymology; D014162:Transfection
D003470:Culture Media; D003598:Cytoskeletal Proteins; D005353:Fibronectins; D016023:Integrins; D007797:Laminin; D009838:Oligodeoxyribonucleotides; D051419:Paxillin; D010750:Phosphoproteins; C496062:Pxn protein, mouse; D011993:Recombinant Fusion Proteins; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496051:Ptk2 protein, mouse