Structural studies of the paxillin interaction network and its role in focal adhesion dynamics and signalling. (360G-Wellcome-079742_Z_06_Z)

We propose to extend our recent structural studies (Arold et al., 2002; Hoellerer et al., 2003) to generate a dynamic picture of the interactions of the focal adhesion (FA) proteins FAK, ILK and actopaxin with the adaptor protein paxillin. We will define the role these interactions play in recruitment and activation of proteins to FAs as well as their eventual exclusion during FA disassembly. This picture will be developed at resolutionsthat extend from the atomic to the cellular level. Atomic details will be provided by NMR and X-ray crystallographic imaging of individual functional domains in complex with different binding partners. Molecular interactions will be characterised by studying binding events by NMR, isothermal titration calorimetry and surface plasmon resonance, using different fragments of FAK, ILK, actopaxin, paxillin, and GRB2. In particular, we will define which interactions are mutually permissive or mutually exclusive, how they are affected by phosphorylation, and we will seek to relate this information to the dynamic process of FA turnover. Models of molecular interactions developedthrough these studies will be tested in a cellular context by expression of suitable constructs in cultured cells, followed by fluorescent microscopy and cell adhesion and migration studies.