Dissecting the role and regulation of MRL function in Drosophila

The Mig10/RIAM/Lpd (MRL) family of adapter proteins have been recognised in transducing signals derived from growth factor receptors to alterations in cell motility and adhesion via changes in actin dynamics. Reduction in the levels of MRL proteins results in diminished cell division rates, growth retardation, an increase in monomeric (G): filamentous (F) actin ratios, loss of cell migration, and lethality. Conversely, overexpression of MRL proteins reduces the ratio of G:F actin, thereby promoting Serum Response Factor (SRF) signalling, lamellipodia formation, cellular invasion and coordinated cell growth and proliferation. Members of the MRL family all share common structural characteristics, most notably the presence of highly conserved central Ras-association (RA) and Pleckstrin homology (PH) domains in addition to an N-terminal Talin binding site and multiple C-terminal SH3 and proline rich motifs capable of interacting with Ena/VASP and Profilin. This report extends previous work carried out on Pico, the Drosophila MRL homologue, by demonstrating physical interactions with Chickadee (Profilin), Rhea (Talin), Ras, and Protein Phosphatase 1 (PP1). Many of these binding partners were found to co-localise with Pico within highly dynamic membrane ruffles during Drosophila cell spreading, while only Enabled (Ena/VASP) co-localised at the periphery of cells once they had reached a maximal size and spreading had ceased, pointing to the existence of distinct Pico-associated complexes. This work also details the presence of a highly conserved MAPK-binding site adjacent to the RA domain which had not been previously recorded in the literature. Site-directed mutagenesis revealed this MAPK-binding motif to be required for Pico's interactions with the Erk1/2 homologue Rolled, while conservation of MAPK binding ability was demonstrated in both human Lpd and RIAM orthologues. Further analysis showed that Rolled might phosphorylate Pico at a serine residue (Ser 819) previously identified by high-throughput phosphoproteomics, which may in turn promote Pico's interactions with PP1. Wing growth assays performed using site-directed Pico mutants indicated that PP1 plays a role in negative regulation of Pico-mediated growth, although the relevant targets of the phosphatase remain to be identified. Interestingly, reporter gene experiments confirmed that Pico induced SRF-dependent gene expression in ii Drosophila cells while ectopic SRF signalling has been found to increase expression of rolled, suggesting the potential existence of a Rolled/PP1 mediated negative feedback loop regulating Pico functionality