Microinjection of Protein Tyrosine Phosphatases into Fibroblasts Disrupts Focal Adhesions and Stress Fibers

Galen B. Schneider, Andrew P. Gilmore, Daniel L. Lohse, Lewis H. Romer, Keith Burridge

Research output: Contribution to journalArticlepeer-review

Abstract

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.

Original languageEnglish (US)
Pages (from-to)207-219
Number of pages13
JournalCell Communication and Adhesion
Volume5
Issue number3
StatePublished - 1998
Externally publishedYes

Keywords

  • FAK
  • Focal adhesions
  • Protein tyrosine phosphatases
  • Rho
  • Tyrosine phosphorylation

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Cell Biology

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