Interaction of Talin with Proteins Containing LD-Motifs

Talin is a 250 kDa cytoplasmic protein that activates integrins and provides a link to cytoskeletal actin, thus producing the necessary force to stabilise adhesions. Talin is classically defined as an integrin-activator, but here we show that talin also couples adhesion assemblies to cortical microtubules via the Cortical Microtubule Stabilising Complex (CMSC) and plays a role in the mechano-regulation of the cell cycle. Cross talk between cortical microtubules and focal adhesions (FA) plays a critical role in cell polarity and migration. Microtubules regulate the turnover of adhesions and, equally, FAs help capture and stabilise microtubules in their vicinity. The molecular basis for this mechanism was unknown and remained a key question within the field. Here, I describe biochemical and biophysical evidence that the interaction between KANK and talin is the crucial link between the macromolecular assemblies FAs and CMSC. Fluorescence polarisation (FP) and Nuclear Magnetic Resonance (NMR) data show that the conserved KN domain in KANK1 binds to the talin rod domain R7 via a LD talin-binding motif. Through the design of point mutants in both the KANK1 KN domain and talin R7 domain this interaction could be perturbed. Our data show that the KANK1 KN domain binds to talin through a helix addition mechanism. Immunofluorescence work in HeLa cells corroborates our findings on the importance of this interaction, and that a single talin point mutation (G1404L) is enough to abrogate the association of FAs with the CMSC and, in turn, disrupts microtubule dynamics at the cell edge. The discovery of KANK1 as a binding partner of talin provides the missing link for how microtubules are targeted to FAs. The talin:KANK interaction gave a new insight on LD talin-binding motifs and allowed us to develop a novel pipeline for identifying talin-binding partners. After designing a LD talin-binding search motif, we identified cyclin dependent kinase1 (CDK1) as a talin-binding protein. CDK1 is the master regulator of the cell cycle, helping to drive cells from G2 phase into mitosis. Using similar biochemical and biophysical techniques I characterise the interaction between talin and CDK1. FP and NMR show that CDK1 binds to the talin R8 domain via helices 32 and 33 and based on structural modelling, we propose that this interaction also occurs through helix addition. Biochemical data combined with phosphoproteomics shows that the CDK1-cyclinA complex phosphorylates talin1 and talin2 isoforms at two unique sites (Ser1589 in talin1 and Ser1489 in talin2). Interestingly, in talin2 this novel phosphorylation site is in the talin actin-binding site (ABS2). We postulate that this could be a mechanism to regulate the coupling/uncoupling of actin to talin. Talin phosphorylation could perturb actin binding, thus reducing tension across the adhesions and leading to their disassembly. This interaction gives an insight into how adhesions and the cell cycle are entwined and poses many questions regarding how adhesion formation and disassembly regulates or occurs within the cell cycle. My thesis describes the discovery of two novel talin-binding partners KANK and CDK1 which reveals talin is a crucial player in both coupling FAs to microtubules and gives new evidence into how adhesions can be involved in the regulation of the cell cycle