Prechova, M; (2018) Functional Analysis of PP1 Regulator Phactr1. Doctoral thesis (Ph.D), UCL (University College London).

Abstract

The Phosphatase and actin regulator (Phactr) protein family has been identified as a group of four proteins (Phactr1,2,3,4), interacting with Protein Phosphatase 1 (PP1) and G-actin. G-actin binding to Phactr1 RPEL motifs has been shown to sterically inhibit Phactr1 interaction with PP1 and its nuclear import. Members of the Phactr family are highly expressed in the nervous system and have been implicated in the regulation of the actin cytoskeleton, cell migration and angiogenesis. The molecular mechanism of their signalling is however not well understood. Here I show that Phactr1 functions as a novel regulatory subunit of PP1. Phactr1 contains several binding motifs typical of PP1 regulatory subunits, including an RVxF motif, located in the C-terminal part of Phactr1, partially overlapping with the G-actin binding RPEL motif. Phactr1 binding leaves the PP1 active site accessible for substrates, blocks one of the substrate-binding grooves and creates a highly basic pocket along the PP1 hydrophobic substrate binding groove, thereby defining Phactr1-PP1 specific protein substrates for dephosphorylation. A phosphoproteomic analysis revealed that activation of Phactr1-PP1 complex formation leads to the dephosphorylation of several cytoskeletal proteins (such as IRSp53 and Afadin), leading to rearrangements of the actin cytoskeleton. Induction of Phactr1-PP1 interaction leads to the formation of aberrant actomyosin structures in fibroblasts. The converse phenotype could be induced by non-PP1-binding Phactr1 mutant expression, leading to the formation of long cytoplasmic extensions. In neurons, Phactr1 was enriched in dendritic spines and activation of Phactr1-PP1 complex formation led to changes in dendritic spine morphology. Moreover, I show that Phactr1 L519R mutation, that has been implicated in epilepsy, decreases Phactr1 affinity to G-actin, and thus indirectly activates Phactr1 interaction with PP1. All this suggests that Phactr1 targets PP1 to dephosphorylate specific cytoskeletal proteins, leading to changes in the actin cytoskeleton, which in neurons modulates dendritic spine morphology, leading to changes in neuronal signalling.

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