Novel perspectives of target-binding by the evolutionarily conserved PP4 phosphatase.

Zoltan Karman,Zsuzsanna Rethi-Nagy,Akos Csonka,Lilla Fabri-Ordogh,David M Glover,Michal Dadlez,Peter Vilmos,Janusz Debski,Edit Abraham,Zoltan Lipinszki

doi:10.1098/rsob.200343

Abstract

Protein phosphatase 4 (PP4) is an evolutionarily conserved and essential Ser/Thr phosphatase that regulates cell division, development and DNA repair in eukaryotes. The major form of PP4, present from yeast to human, is the PP4c-R2-R3 heterotrimeric complex. The R3 subunit is responsible for substrate-recognition via its EVH1 domain. In typical EVH1 domains, conserved phenylalanine, tyrosine and tryptophan residues form the specific recognition site for their target's proline-rich sequences. Here, we identify novel binding partners of the EVH1 domain of the Drosophila R3 subunit, Falafel, and demonstrate that instead of binding to proline-rich sequences this EVH1 variant specifically recognizes atypical ligands, namely the FxxP and MxPP short linear consensus motifs. This interaction is dependent on an exclusively conserved leucine that replaces the phenylalanine invariant of all canonical EVH1 domains. We propose that the EVH1 domain of PP4 represents a new class of the EVH1 family that can accommodate low proline content sequences, such as the FxxP motif. Finally, our data implicate the conserved Smk-1 domain of Falafel in target-binding. These findings greatly enhance our understanding of the substrate-recognition mechanisms and function of PP4.

Highlights

Protein phosphorylation serves as a molecular switch to regulate the activity, subcellular localization, interacting partners, structure and half-life of proteins
As transgenic Falafel forms a functional complex with R2 and PP4c [8], we have hypothesized that the interaction between the target protein and PP4c-R2-Falafel could be transient, with the substrate being released upon dephosphorylation
We previously demonstrated that the EVH1 domain of PP4R3Flfl could bind to the low proline content FKKP motif localized in the Falafel-Interacting Motif (FIM) region of CENP-C and that mutation of either the F (Phe) or P (Pro) residues abolished the interaction [8]

Summary

Introduction

Protein phosphorylation serves as a molecular switch to regulate the activity, subcellular localization, interacting partners, structure and half-life of proteins. It is mediated by protein kinases and reversed by protein phosphatases. To better understand phosphoregulation-mediated cell signalling, it is important to define the modular composition of the various PPP holoenzymes, and to identify the mode of substrate-binding for their different catalytic (c) and regulatory (R) subunits. PP4R3 orthologues (Psy in yeast [26,27], Falafel (Flfl) in Drosophila [8,28] and PP4R3α/SMEK1 and PP4R3β/SMEK2 in mammals [24]) are highly conserved, share similar domain architecture and are responsible for substrate recognition and subcellular localization of the holoenzyme [5,16,22,29,30,31,32]. The N-terminal domains are followed by Armadillo (ARM/ HEAT) repeats in the middle, and a low complexity tail region of various lengths in the C-terminal parts of the orthologues (figure 1a)

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