Cell density regulates tyrosine phosphorylation and localization of focal adhesion kinase.
We have investigated tyrosine phosphorylation of cellular proteins at different cell densities. A tyrosine-phosphorylated protein of 120 kDa was detected when cells were plated sparsely. The phosphorylation level of the protein gradually declined as the cells were plated at higher densities or when the sparsely plated cells approached confluence. This density-dependent phosphorylation was also associated with cell attachment since it disappeared when the cells were detached from plates or when the cells were cultured in suspension. Immunoblotting and immunoprecipitation analyses with specific antibodies revealed that the 120-kDa protein corresponded to the focal adhesion kinase (FAK) and the protein level of FAK was not altered at different cell densities. In vitro kinase assays demonstrated that the kinase activity of FAK decreased with increasing cell densities in parallel with its dephosphorylation. Cell density also affects localization of FAK associated with rearrangement of actin stress fibers. At low cell densities, FAK and actin stress fiber are distributed around the periphery of cells while they are dispersed over the ventral surface in high-density cells. Finally, the density-regulated tyrosine phosphorylation and localization of FAK appeared to be mediated by an insoluble factor produced by high-density cells.
Xu
F
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Division of Hematology/Oncology, Vanderbilt-Ingram Cancer Center, Nashville, Tennessee, 37232-6305, USA.
Zhao
Z J
ZJ
D016475:3T3 Cells; D000818:Animals; D002448:Cell Adhesion / Q000502:physiology; D002452:Cell Count; D003902:Detergents; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D006367:HeLa Cells; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism; D051379:Mice; D017830:Octoxynol; D010766:Phosphorylation; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D012995:Solubility; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000378:metabolism*; D014638:Vanadates / Q000494:pharmacology
D003902:Detergents; D004791:Enzyme Inhibitors; C076715:pervanadate; D014638:Vanadates; D014443:Tyrosine; D017830:Octoxynol; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; C496051:Ptk2 protein, mouse; D017027:Protein Tyrosine Phosphatases