Dissecting interactions within focal adhesions: studies on vinculin

Vinculin and paxillin are proteins that localise to focal adhesions. Focal adhesions are<br/>specialised sites of cell attachment between the cytoplasmic side of the cell membrane and<br/>the extracellular matrix. They consist of receptors that link extracellular matrix ligands to<br/>the actin cytoskeleton via various protein assemblies. They act as mechanical links and as<br/>sites of signal transduction to transduce signals for cell locomotion, cell attachment and<br/>detachment, apoptosis and gene expression. A range of cellular responses depend critically<br/>on the composition and regulation of focal adhesions.<br/><br/>Vinculin and paxillin will interact in vitro. The tail domain of vinculin interacts with a<br/>motif on paxillin called an LD motif. The interaction between LD motifs and their target<br/>proteins is important for regulation of focal adhesion signalling, yet little is known<br/>regarding recognition mechanisms between LD domains and interaction partners.<br/><br/>In this thesis, the molecular nature of the vinculin tail (Vt) and paxillin LD motif<br/>interaction has been studied using purified Vt, synthesised LD peptide mimics and<br/>recombinant paxillin His-LD1/LD2. The 1H-15N-HSQC spectrum of a Vt/I997S mutant<br/>has been assigned and the NH assignments transferred to a wild-type Vt spectrum.<br/>Chemical shift perturbation studies have subsequently been undertaken using wild-type Vt<br/>and paxillin.<br/><br/>The data presented here is consistent with specificity for LD motifs and points to an<br/>interaction between Vt and LD1 and LD2. In contrast there is little or no interaction with<br/>LD4. Two binding models are proposed; a single binding site on Vt face 3-4 where LD<br/>motifs bind in an extended conformation, or a two-site binding on Vt face 3-4 with two<br/>LD motifs bound as a-helices. Both models need further analysis. NMR data for the<br/>paxillin His-LD1/LD2 construct suggests a predominantly unstructured molecule in<br/>solution that can catalyse precipitation of Vt when Vt is added to excess.<br/><br/>It is clear that the mechanism of interaction between Vt and paxillin LD motifs is distinct<br/>from that of focal adhesion kinase interacting with paxillin. Further investigation is<br/>required to elucidate the precise mechanism of binding. A comparison of this data with<br/>other LD-protein interactions suggests there are little similarities between the target<br/>sequences that LD motifs recognise and that target proteins can be structurally different.

Name dc:type.qualificationname

Ph.D.

Level dc:type.qualificationlevel

doctoral

Grantor dc:publisher.institution

University of Southampton

Year dc:date.issued

2008

Author dc:creator

• Cox, Clare Louise

Advisor dc:contributor.advisor

• Werner, Jorn