Wagner, Simona M2013-11-082013-11-0820082008Source: Dissertation Abstracts International, Volume: 69-08, Section: B, page: 4522.http://hdl.handle.net/10393/29535http://dx.doi.org/10.20381/ruor-13005The Ste20-like kinase (SLK) is a conserved serine-threonine kinase that is involved in cytoskeletal reorganization and apoptosis (Sabourin and Rudnicki, 1999; Sabourin et al., 2000). Overexpression of SLK in various cell lines was shown to induce the rapid disassembly of actin stress fibres and cell death (Sabourin et al., 2000). To investigate the physiological role of SLK we used MEF-3T3 fibroblasts spreading and migrating on fibronectin coated substrates. Using immunofluorescence studies, we report that SLK is redistributed with adhesion components at podosome-like adhesion sites in fibronectin stimulated fibroblasts. Furthermore, we show that SLK is associated with the microtubule network and can be co-precipitated with alpha-tubulin. However, in vitro kinase assays demonstrate that its activity in not modulated following fibronectin stimulation. Using microinjection studies, we show that ectopic expression of activated SLK induces the disassembly of actin stress fibres, a process that can be inhibited by dominant negative Rac1. Furthermore, the overexpression of SLK by adenoviral infection inhibits cell spreading on fibronectin. As the signalling pathways activated during cell spreading also regulate cell motility we tested the possibility that SLK may play a role in cell migration (Mitra and Schlaepfer, 2006; Webb et al., 2002). We first investigated the localization of SLK and other cytoskeletal markers following scratch wounding of fibroblasts monolayers, and observed that SLK is recruited at the leading edge of migrating cells with paxillin, Rac1 and the microtubules. Using short interfering RNA and dominant negative approaches, we show that SLK is required for microtubule-dependent focal adhesion turnover and cell migration. Scratch wounding of confluent monolayers have been shown to induce polarization and migration (Etienne-Manneville and Hall, 2001). Therefore, we investigated SLK kinase activity following scratch wound induced migration and report that SLK is activated during migration. In addition, we report that SLK activation is dependent on a multitude of signalling pathways such as FAK/c-Src, MAPK, PI3K and RhoGTPases, whereas SLK recruitment to the leading edge is Src-dependent but FAK-independent. Overall, we have established a role for SLK in cell motility as a novel molecular link between the adhesion-destabilizing activity of the microtubule network and actin-based cytoskeletal remodelling events.125 p.enBiology, Cell.Thesis