Phosphorylation of Def Regulates Nucleolar p53 Turnover and Cell Cycle Progression through Def Recruitment of Calpain3.

Yihong Guan,Li Jan Lo,Yingchun Wang,Zuyuan Liao,Jinrong Peng,Hui Shi,Ce Gao,Delai Huang,Feng Chen,Ting Tao,Shuyi Zhao,Jun Chen,Douglas R Green

doi:10.1371/journal.pbio.1002555

Yihong Guan, Li Jan Lo + Show 11 more

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https://doi.org/10.1371/journal.pbio.1002555

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Abstract

Digestive organ expansion factor (Def) is a nucleolar protein that plays dual functions: it serves as a component of the ribosomal small subunit processome for the biogenesis of ribosomes and also mediates p53 degradation through the cysteine proteinase calpain-3 (CAPN3). However, nothing is known about the exact relationship between Def and CAPN3 or the regulation of the Def function. In this report, we show that CAPN3 degrades p53 and its mutant proteins p53A138V, p53M237I, p53R248W, and p53R273P but not the p53R175H mutant protein. Importantly, we show that Def directly interacts with CAPN3 in the nucleoli and determines the nucleolar localisation of CAPN3, which is a prerequisite for the degradation of p53 in the nucleolus. Furthermore, we find that Def is modified by phosphorylation at five serine residues: S50, S58, S62, S87, and S92. We further show that simultaneous phosphorylations at S87 and S92 facilitate the nucleolar localisation of Capn3 that is not only essential for the degradation of p53 but is also important for regulating cell cycle progression. Hence, we propose that the Def-CAPN3 pathway serves as a nucleolar checkpoint for cell proliferation by selective inactivation of cell cycle-related substrates during organogenesis.

Highlights

The nucleolus is a subcellular organelle primarily known for the biogenesis of the ribosomal small and large subunits in eukaryotic cells [1,2]
The nucleolus is primarily known as the subcellular organelle for the biogenesis of the ribosomal small and large subunits in eukaryotic cells
We show that Digestive organ expansion factor (Def), a nucleolar protein, determines the nucleolar localisation of the cysteine proteinase CAPN3, and they form a PLOS Biology | DOI:10.1371/journal.pbio

Summary

Introduction

The nucleolus is a subcellular organelle primarily known for the biogenesis of the ribosomal small and large subunits in eukaryotic cells [1,2]. Bioinformatic analyses of nucleolar proteomic studies in cultured human HeLa cells [3,4], Arabidopsis [5], and human T cells [6] have shown that a mere 30% of nucleolar proteins (approximately 200 from Arabidopsis and approximately 700 from human cells) have functions directly related to the production of ribosomal subunits; the rest are involved in a variety of biochemical processes including cell cycle control, stress response, and the biogenesis of ribonucleoproteins. It is increasingly evident that many nucleolar proteins have dual functions. Nucleophosmin functions in ribosome biogenesis and in duplication of centrosomes [7] and stress response [8]. The exact biological functions of many nucleolar proteins remain elusive

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