Watton, Sandra Jayne; (2000) Cell adhesion and growth factor regulation of Akt. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Akt is a protein kinase that contains a C-terminal pleckstrin homology (PH) domain and an N-terminal kinase domain. The activity of Akt is regulated by PI3-kinases that generate 3 phosphoinositides. Akt activation requires the binding of 3 phosphoinositides to the pleckstrin homology (PH) domain and phosphorylation at residues Thr308 and Ser473. Phosphospecific antibodies that recognise the phosphorylated sites and green fluorescent protein (GFP) fusions with mutated or truncated forms of Akt were used to follow the activation of Akt. Confocal microscopy showed that membrane translocation is required for Akt phosphorylation and is dependent on the PH domain but not kinase activity. A GFP fusion with the PH domain of Akt, termed GFP-AH, was assessed and used as a probe for sites of PI3-kinase activity in cells. GFP- AH was used to locate 3 phosphoinositides in epithelial cells that were plated on collagen to investigate extracellular matrix induced cell survival. GFP-AH localised to sites of cell-cell and cell-matrix contact, distinct from focal adhesions. This suggests that the attachment induced, PI3-kinase mediated, survival signal in epithelial cells is generated not only by cell-matrix but also by cell-cell contact. PI3-kinase activity was monitored during the disruption and formation of cell-cell junctions. Disruption of the junctions by calcium removal decreased PI3-kinase activity, whereas the formation of junctions preceded PI3-kinase activity. Akt became phosphorylated 4 h after junction formation was initiated and correlated with the tyrosine phosphorylation of the cell-cell junctional proteins y-catenin and p120 catenin. As p120 catenin is a substrate of activated Src and Src family kinase inhibitors reduced the cell-cell adhesion induced phosphorylation of Akt, this suggests a role for Src family kinases in the regulation of cell-cell adhesion induced PI3-kinase activity.

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