Phosphoproteomics reveals new ERK MAP kinase targets and links ERK to nucleoporin-mediated nuclear transport.

Division of Cellular Proteomics (BML), Institute of Medical Science, The University of Tokyo, Tokyo, Japan.
Nature Structural & Molecular Biology (Impact Factor: 11.63). 09/2009; 16(10):1026-35. DOI: 10.1038/nsmb.1656
Source: PubMed

ABSTRACT Many extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase substrates have been identified, but the diversity of ERK-mediated processes suggests the existence of additional targets. Using a phosphoproteomic approach combining the steroid receptor fusion system, IMAC, 2D-DIGE and phosphomotif-specific antibodies, we detected 38 proteins showing reproducible phosphorylation changes between ERK-activated and ERK-inhibited samples, including 24 new candidate ERK targets. ERK directly phosphorylated at least 13 proteins in vitro. Of these, Nup50 was verified as a bona fide ERK substrate. Notably, ERK phosphorylation of the FG repeat region of Nup50 reduced its affinity for importin-beta family proteins, importin-beta and transportin. Other FG nucleoporins showed a similar functional change after ERK-mediated phosphorylation. Nuclear migration of importin-beta and transportin was impaired in ERK-activated, digitonin-permeabilized cells, as a result of ERK phosphorylation of Nup50. Thus, we propose that ERK phosphorylates various nucleoporins to regulate nucleocytoplasmic transport.

  • Journal of Biological Chemistry 11/2012; 287(46):38515-38522. DOI:10.1074/jbc.M112.378893 · 4.60 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Autophagy is an evolutionarily conserved cellular process which degrades intracellular contents. The Atg17-Atg31-Atg29 complex plays a key role in autophagy induction by various stimuli. In yeast, autophagy occurs with autophagosome formation at a special site near the vacuole named the pre-autophagosomal structure (PAS). The Atg17-Atg31-Atg29 complex forms a scaffold for PAS organization, and recruits other autophagy-related (Atg) proteins to the PAS. Here, we show that Atg31 is a phosphorylated protein. The phosphorylation sites on Atg31 were identified by mass spectrometry. Analysis of mutants in which the phosphorylated amino acids were replaced by alanine, either individually or in various combinations, identified S174 as the functional phosphorylation site. An S174A mutant showed a similar degree of autophagy impairment as an Atg31 deletion mutant. S174 phosphorylation is required for autophagy induced by various autophagy stimuli such as nitrogen starvation and rapamycin treatment. Mass spectrometry analysis showed that S174 is phosphorylated constitutively, and expression of a phosphorylation-mimic mutant (S174D) in the Atg31 deletion strain restores autophagy. In the S174A mutant, Atg9-positive vesicles accumulate at the PAS. Thus, S174 phosphorylation is required for formation of autophagosomes, possibly by facilitating the recycling of Atg9 from the PAS. Our data demonstrate the role of phosphorylation of Atg31 in autophagy.
    Protein & Cell 03/2015; 6(4). DOI:10.1007/s13238-015-0138-4 · 2.85 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Transport regulation by the Ran GTPase requires its nuclear localization and GTP loading by the chromatin-associated exchange factor, RCC1. These reactions generate Ran protein and Ran nucleotide gradients between the nucleus and cytoplasm. Cellular stress disrupts the Ran gradients, but the specific mechanisms underlying the disruption have not been elucidated. We used biochemical approaches to determine how oxidative stress disrupts the Ran system. RCC1 exchange activity was reduced by diamide-induced oxidative stress and restored with dithiothreitol. Using mass spectrometry we found that multiple solvent-exposed cysteines in RCC1 are oxidized in cells treated with diamide. Cysteines oxidized in RCC1 included Cys93, which is solvent-exposed and unique because it becomes buried upon contact with Ran. A Cys93Ser substitution dramatically reduced exchange activity through an effect on RCC1 binding to RanGDP. Diamide treatment reduced the size of the mobile fraction of RCC1-GFP in cells, and inhibited nuclear import in digitonin-permeabilized cell assays. The Ran protein gradient was also disrupted by UV-induced stress, but without affecting RCC1 exchange activity. Our data suggest that stress can disrupt the Ran gradients through RCC1-dependent and RCC1-independent mechanisms, possibly dependent on the particular stress condition. Copyright © 2014, American Society for Microbiology. All Rights Reserved.
    Molecular and Cellular Biology 12/2014; 35(3). DOI:10.1128/MCB.01133-14 · 5.04 Impact Factor


Available from
Oct 3, 2014