Mouse Swiss 3T3 fibroblasts maintained in serum-free medium lose their actin stress fibres and focal adhesions. Addition of serum or lysophosphatidic acid (LPA) to these cells induced the rapid reformation of these structures, and the increased tyrosine phosphorylation of ppl25Fak and paxillin. The formation of focal adhesions and actin stress fibres was shown to require tyrosine phosphorylation; it was blocked by the tyrosine kinase inhibitor genistein, and stimulated by the tyrosine phosphatase inhibitors sodium orthovandate and phenyl arsine oxide. Moreover, the activity of a tyrosine phosphatase directed against ppl25Fak and paxillin was elevated in serum-starved cells. Despite the absence of focal adhesions and actin stress fibres, the adhesion of serum- starved cells was shown to be mediated by integrins, as peptides corresponding to the cell-binding motif of fibronectin caused cell detachment from the extracellular matrix. In addition, the integrin-mediated formation of actin stress fibres and protein tyrosine phosphorylation was blocked by introducing C3 transferase into cells, a result which establishes a role for the small GTP-binding protein rho in signalling via integrins. PKC was also required to maintain adhesion in serum-starved cells as the PKC inhibitor calphostin C caused cell retraction, a process that was accompanied by the formation of actin stress fibres. In contrast, activation of PKC with phorbol esters suppressed the LPA-induced assembly of actin stress fibres and focal adhesions, and caused loss of these structures in cells grown in serum. The results suggest that PKC negatively regulates the formation of actin stress fibres. The proposed role of vinculin phosphorylation in the formation of focal adhesions was found to be inconsistent with the finding that the phosphorylated form of the protein is not associated with the Triton X-100 insoluble fraction in Swiss 3T3 cells. Vinculin was a poor substrate for PKC in vitro, although a cryptic C-terminal phosphorylation site was exposed by PIP2.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:674492 |
| Date | January 1996 |
| Creators | Barry, Simon Thomas |
| Publisher | University of Leicester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/2381/35218 |
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