Investigating Post-Translational Modification of the Net Protein Superfamily

Abstract

The Net protein superfamily represents a group of actin binding proteins contain a novel actin binding domain at their N termini. The aim of this project was to investigate post-translational modification of these proteins in order to explore how the interaction of these proteins with actin is regulated. A bioinformatics-based approach was used to predict sites for multiple types of modification, with a focus on phosphorylation, that were conserved between members of the superfamily. It became clear that there was some level of conservation of predicted post-translational modification sites at the C-terminus in multiple Net families. Net4B was identified as having a high probability of being phosphorylated at the C-terminus and predicted to have its N- and C-termini proximal to one another in its tertiary structure, and following this mutant forms of the protein were created to investigate how its actin binding activity would be affected if one site, S509, was phosphorylated or dephosphorylated. The mutants were transiently expressed in Nicotania benthamiana and the appearance of the leaf cells assessed.

Whilst phosphomimicry of S509 resulted in no appreciable change in the appearance of the GFP-tagged protein, mutation to a residue imitating a non-phosphorylatable serine resulted in the formation of punctae, in some cases much like the ‘beads-on-a-string’ seen in other members of the Net superfamily. This finding may have implications for the regulation of actin binding in other Net proteins and for other proteins outside of the superfamily. Two models are presented in both of these contexts. This project may also provide groundwork for future experiments concerning phosphorylation and acylation, and may illuminate the mechanism by which Net proteins interact with actin and with the membranes with which they are respectively localised.