Wiezlak, MK; (2013) Regulation and function of the RPEL protein - Phactr1. Doctoral thesis (PhD), UCL (University College London).

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Abstract

Actin-binding proteins play well established roles in the regulation of actin dynamics and assembly of F-actin based structures involved in cell motility and adhesion. The Phosphatase and actin regulator (Phactr) family of proteins each contain four G-actin binding RPEL motifs and has been found to bind protein phosphatase 1 (PP1) via their C-terminal domain. Their function is not well established and it has been unclear whether G-actin can be their regulator. Members of the Phactr family are highly expressed in the nervous system and in some metastatic cancers. The RPEL domain was previously shown to confer Rho-regulated nuclear shuttling and activation of Serum Response Factor (SRF) coactivator myocardin – related transcription factor A (MRTF-A, also known as MAL/MKL1). MRTF-A is cytoplasmic in unstimulated cells and accumulates in the nucleus upon activation of Rho-actin signalling. In this thesis I show that activation of Rho-actin signalling by serum stimulation induces nuclear accumulation of Phactr1, but not other Phactr family members (Phactr2-4). Actin binding by the three Phactr1 C-terminal RPEL motifs is required for Phactr1 cytoplasmic localisation in resting cells. Phactr1 nuclear accumulation is Importin α−β-dependent. I also reveal that G-actin and Importin α−β bind competitively to nuclear import signals associated with the N- and C-terminal RPEL motifs in Phactr1. All four motifs are required for the inhibition of serum-induced Phactr1 nuclear accumulation by elevated G-actin. G-actin and PP1 bind competitively to the Phactr1 C-terminal region, and expression of Phactr1 C-terminal RPEL mutants that cannot bind G-actin induces actomyosin foci dependent on PP1 binding. In CHL-1 metastatic melanoma cells, Phactr1 exhibits actin-regulated subcellular localisation and is required for stress fibre assembly, motility, and invasiveness. These data support a role for Phactr1 in actomyosin assembly and suggest that Phactr1 G-actin sensing allows its coordination with F-actin availability.

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