Molecular mapping of tyrosine-phosphorylated proteins in focal adhesions using fluorescence resonance energy transfer.

Microscopy-based fluorescence resonance energy transfer (FRET) provides an opportunity to monitor molecular processes in the natural environment in live cells. Here we studied molecular interactions and tyrosine phosphorylation of paxillin, Crk-associated substrate (CAS), and focal adhesion kinase (FAK) in focal adhesions. For that purpose, these focal adhesion phosphoproteins, fused to cyan or yellow fluorescent proteins (CFP or YFP) were expressed in cultured fibroblasts.

Measurement of protein tyrosine phosphorylation in cell adhesion.

This chapter describes biochemical, immunochemical, and microscopic approaches to measure protein tyrosine phosphorylation after cell adhesion. We have outlined detailed procedures to biochemically examine the phosphotyrosine content of cellular proteins by Western blotting, which in some cases can be performed using phospho-specific antibodies. Furthermore, we have described in detail the examination of subcellular localization of phosphotyrosine-containing proteins in focal adhesions using immunofluorescence.

A conserved tyrosine in the neck of a fungal kinesin regulates the catalytic motor core.

The neck domain of fungal conventional kinesins displays characteristic properties which are reflected in a specific sequence pattern. The exchange of the strictly conserved Tyr 362, not present in animals, into Lys, Cys or Phe leads to a failure to dimerize. The destabilizing effect is confirmed by a lower coiled-coil propensity of mutant peptides.