Laboratory of Molecular Biophysics Laboratory Journal 2001 Dr. M. E. M. Noble

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Focal adhesion kinase.

(Collaborators: Prof. M. Ginsberg, Scripps Research Clinic, Dr. J. Ladbury, UCL, London, Dr. J. Werner, Oxford, and Prof I. Campbell, Oxford).

Figure 1. Interactions of focal adhesion kinase....more.

Focal adhesions are found at the cell membrane where the cytoskeleton interacts with proteins of the extracellular matrix. The clustering of integrins at these sites attracts a large complex of proteins and initiates intracellular regulatory processes, by which such events as cell migration and anchorage-dependent differentiation are controlled[1]. Focal adhesion kinase (FAK) is a protein tyrosine kinase which is recruited at an early stage to focal adhesions and which mediates many of the downstream responses. The kinase activity of FAK is activated by the Src non-receptor tyrosine kinase, which is recruited at an SH2 binding site generated on FAK by autophosphorylation. FAK subsequently interacts with a number of down-stream signalling proteins, including the adaptor protein Grb2 and the p85 _-subunit of phosphatidylinositol 3 kinase (PI3 kinase) (Figure 1).

Activation of Fyn tyrosine kinase.

Figure 2. Comparison of SH3-SH2 domain orientation...more.

The regulatory fragment of Src kinases, comprising Src Homology (SH) 3 and 2 domains, is responsible for controlled repression of kinase activity. We have used a multi-disciplinary approach, involving crystallography, NMR, and isothermal titration calorimetry, to study the regulatory fragment of Fyn (FynSH32) and its interaction with a physiological activator: a fragment of focal adhesion kinase (FAK) which contains both phosphotyrosine and polyproline motifs[2]. Although flexible, the preferred disposition of SH3 and SH2 domains in FynSH32 resembles the inactive forms of Hck and Src, differing significantly from LckSH32. This difference, which results from variation in the SH3-SH2 linker sequences, will affect the potential of the regulatory fragments to repress kinase activity. This surprising result implies that the mechanism of repression of Src- family members may vary, explaining functional distinctions between Fyn and Lck. The interaction between FynSH32 and FAK is restricted to the canonical SH3 and SH2 binding sites, and does not affect the dynamic independence of the two domains. Consequently, the interaction shows no enhancement by an avidity effect. Such an interaction may have evolved to gain specificity through an extended recognition site, while maintaining rapid dissociation after signalling.

The focal adhesion targetting domain.

Figure 3. Two structures of the focal adhesion tar...more.

The localisation of focal adhesion kinase (FAK) to sites of integrin clustering initiates downstream signalling. The C-terminal focal adhesion targeting (FAT) domain [3] causes this localisation by interacting with talin and paxillin. FAT also mediates signalling through Grb2 via phosphorylated Y925. We have solved two crystal structures of the FAT domain. Isolated FAT folds into a four- helical bundle, which has the capacity to form domain-exchanged dimers (Figure 3), in which the N-terminal helix is swapped between constituent monomers. Large rearrangements of the structure are indicated to allow phosphorylation of Y925 and subsequent interaction with Grb2. Sequence homology and structural compatibility suggest a FAT-like fold for the C-terminal domains of CAS, Efs/Sin and HEF1. A structure-based alignment including these proteins and the vinculin tail domain reveals a conserved region which could play a role in focal adhesion targeting. Previously postulated 'paxillin binding subdomains' may contribute to structural integrity rather than directly to paxillin binding.

The interaction between the SH3 domain of PI3-kinase and the C- terminus of focal adhesion kinase.

Src homolgy (SH) 3 domains are found in many proteins associated with receptor tyrosine signal transduction complexes. Their ligands share the same core-binding motif, which consists of a proline rich region with the consensus PXXP. However, the binding interface between an SH3 domain and a protein ligand appears to encompass more interactions than are represented by that involving the proline-rich motif. These additional interactions together with variations within the proline-rich region are thought to confer specificity to SH3-mediated protein-protein interactions. To test this hypothesis we have analysed the interaction between the SH3 domain of the regulatory p85 subunit of PI3-kinase and a proline rich region in the C-terminus of Focal adhesion kinase (FAK) [4]. We have found that p85-SH3 binds with a 50-times higher affinity to the complete C-terminal domain of FAK (residues 852 to 1052) than to a peptide ligand mimicking the proline- rich region RP2 (residues 871 to 880). This indicates that interactions outside the core-binding site might indeed stabilise the binding of the SH3 domain of p85 to FAK. We have also made progress towards crystallisation of a FAK/p85-SH3 complex.

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