Microinjection of protein tyrosine phosphatases into fibroblasts disrupts focal adhesions and stress fibers.
Microinjection and scrape-loading have been used to load cells in culture with soluble protein tyrosine phosphatases (PTPs). The introduction of protein tyrosine phosphatases into cells caused a rapid (within 5 minutes) decrease in tyrosine phosphorylation of major tyrosine phosphorylated substrates, including the focal adhesion kinase and paxillin. This decrease was detected both by blotting whole cell lysates with anti-phosphotyrosine antibodies and visualizing the phosphotyrosine in focal adhesions by immunofluorescence microscopy. After 30 minutes, many of the cells injected with tyrosine phosphatases revealed disruption of focal adhesions and stress fibers. To determine whether this disruption was due to the dephosphorylation of FAK and its substrates in focal adhesions, we have compared the effects of protein tyrosine phosphatase microinjection with the effects of displacing FAK from focal adhesions by microinjection of a dominant negative FAK construct. Although both procedures resulted in a marked decrease in the level of phosphotyrosine in focal adhesions, disruption of focal adhesions and stress fibers only occurred in cells loaded with exogenous protein tyrosine phosphatases. These results lead us to conclude that although tyrosine phosphorylation regulates focal adhesion and stress fiber stability, this does not involve FAK nor does it appear to involve tyrosine-phosphorylated proteins within focal adhesions. The critical tyrosine phosphorylation event is upstream of focal adhesions, a likely target being in the Rho pathway that regulates the formation of stress fibers and focal adhesions.
Department of Prosthodontics, School of Dentistry, University of North Carolina, Chapel Hill 27599, USA. schneidg.dentce@mhs.unc.edu
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Schneider
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Gilmore
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Lohse
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Romer
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Burridge
D000818:Animals; D002448:Cell Adhesion / Q000502:physiology; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000201:enzymology; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D019204:GTP-Binding Proteins / Q000378:metabolism; D008845:Microinjections; D051419:Paxillin; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D017027:Protein Tyrosine Phosphatases / Q000494:pharmacology*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D051381:Rats; D013314:Stress, Mechanical; D014443:Tyrosine / Q000378:metabolism*; D020741:rho GTP-Binding Proteins
D015815:Cell Adhesion Molecules; D003598:Cytoskeletal Proteins; D051419:Paxillin; D010750:Phosphoproteins; C496063:Pxn protein, rat; D019000:Phosphotyrosine; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496052:Ptk2 protein, rat; D017027:Protein Tyrosine Phosphatases; D019204:GTP-Binding Proteins; D020741:rho GTP-Binding Proteins