Analysis of signal transduction pathways linking L-plastin Ser5 phosphorylation to breast cancer cell invasion

The organisation of the actin cytoskeleton is regulated by abundant actin-binding proteins. Functional alteration of these proteins contributes to pathologies such as cancer where structural and functional modifications of the actin cytoskeleton are linked to uncontrolled cell motility and signalling. The actin-bundling protein L-plastin has initially been detected in haematopoietic cells where it plays a role in the immune response. L-plastin is also ectopically expressed in several solid tumours and is often considered as a metastatic marker. L-plastin is known to be phosphorylated in vitro and in vivo with residue serine 5 (Ser5) being the major phosphorylation site. Ser5 phosphorylation increases the F-actin-binding and -bundling activity of L-plastin and regulates actin turn-over. Recent findings demonstrate that L-plastin Ser5 phosphorylation is crucial for invasion and metastasis formation. This research work has unravelled the signalling pathways leading to L-plastin Ser5 phosphorylation in breast cancer cells.

Previously, protein kinase A, protein kinase C and phosphoinositide-3-kinase have been reported to play a role in L-plastin Ser5 phosphorylation depending on the cell type and environment. This work however reveals that RSK kinases are the predominant kinases responsible for L-plastin Ser5 phosphorylation in breast cancer cells. In vitro kinase assays revealed that RSK1 and RSK2 are able to directly phosphorylate L-plastin on Ser5 and a whole genome microarray analysis pointed to an involvement of the ERK/MAPK pathway in this event. The involvement of this pathway was consolidated by activation and inhibition studies as well as by siRNA-mediated knockdowns. To our knowledge, this is the first evidence that L-plastin Ser5 phosphorylation occurs via the downstream ERK/MAPK pathway kinases, RSK1 and RSK2.

Moreover, a computational modelling approach enabled us to show that RSK is the most important activator of L-plastin in breast cancer cell lines compared to other previously identified kinases.

We performed migration and invasion assays which showed that RSK knockdown, besides reducing L-plastin Ser5 phosphorylation, also impaired breast cancer cell migration and invasion. The identification of a novel substrate of RSK kinases whose phosphorylation is important for cancer cell invasion underlines the importance of RSK in cancer progression and highlights RSK as a promising drug target in certain invasive carcinomas.