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: 13.31). 09/2009; 16(10):1026-35. DOI: 10.1038/nsmb.1656
Source: PubMed


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.

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    • "Several cellular stresses induce the modulation of nucleocytoplasmic transport. Such stress signaling is often mediated by the activation of the mitogen-activated protein kinases (MAPK), containing MEK, ERK, and p38, and also induces changes in the cellular distribution of transport factors and efficiency of nucleocytoplasmic transport (Czubryt et al., 2000; Kelley and Paschal, 2007; Kodiha et al., 2009; Kosako et al., 2009). This is similar to what has been demonstrated for Ran, although target molecules of these kinases are currently unknown. "
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    ABSTRACT: During heat shock stress, importin β family-mediated nucleocytoplasmic trafficking is downregulated, whereas nuclear import of the molecular chaperone Hsp70s is upregulated. Here, we identify a nuclear import pathway that operates during heat shock stress and is mediated by an evolutionarily conserved protein named "Hikeshi," which does not belong to the importin β family. Hikeshi binds to FG-Nups and translocates through nuclear pores on its own, showing characteristic features of nuclear transport carriers. In reconstituted transport, Hikeshi supports the nuclear import of the ATP form of Hsp70s, but not the ADP form, indicating the importance of the Hsp70 ATPase cycle in the import cycle. In living cells, depletion of Hikeshi inhibits heat shock-induced nuclear import of Hsp70s, reduces cell viability after heat shock stress, and significantly delays the attenuation and reversion of multiple heat shock-induced nuclear phenotypes. Nuclear Hsp70s rescue the effect of Hikeshi depletion at least in part. Thus, Hsp70s counteract heat shock-induced damage by acting inside of the nucleus.
    Cell 04/2012; 149(3):578-89. DOI:10.1016/j.cell.2012.02.058 · 32.24 Impact Factor
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    • "Phosphorylation of Nups by mitotic kinases, in­ cluding Cdk1 or NIMA­related kinases, has been proposed to mediate NPC disassembly during prophase (Macaulay et al., 1995; Glavy et al., 1997, 2007; Onischenko et al., 2005; Lusk et al., 2007; Laurell et al., 2011). Function of the NPC in nuclear trans­ port is also regulated via phosphorylation of FG­Nups by extra­ cellular signal–regulated kinase (Kosako et al., 2009). In higher eukaryotes, Nup96 is ubiquitylated and degraded by the protea­ some in a cell cycle–dependent manner (Chakraborty et al., 2008). "
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    ABSTRACT: Nuclear pore complexes (NPCs) correspond to large protein transport complexes responsible for selective nucleocytoplasmic exchange. Although research has revealed much about the molecular architecture and roles of the NPC subcomplexes, little is known about the regulation of NPC functions by posttranslational modifications. We used a systematic approach to show that more than half of NPC proteins were conjugated to ubiquitin. In particular, Nup159, a nucleoporin exclusively located on the cytoplasmic side of the NPC, was monoubiquitylated by the Cdc34/SCF (Skp1-Cdc53-F-box E3 ligase) enzymes. Preventing this modification had no consequences on nuclear transport or NPC organization but strongly affected the ability of Nup159 to target the dynein light chain to the NPC. This led to defects in nuclear segregation at the onset of mitosis. Thus, defining ubiquitylation of the yeast NPC highlights yet-unexplored functions of this essential organelle in cell division.
    The Journal of Cell Biology 01/2012; 196(1):19-27. DOI:10.1083/jcb.201108124 · 9.83 Impact Factor
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    • "Importantly, all of these kinases are activated and/or redistributed by ROS (Table 1). Moreover Nup50, Nup153, and Nup214 are established ERK targets [130], and their phosphorylation changed several interactions that are important for nuclear transport. Specifically, ERK-dependent modification of Nup50 interfered with its binding to importin-β and transportin, which are both carriers of the importin-β family. "
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    ABSTRACT: Imbalances in the formation and clearance of reactive oxygen species (ROS) can lead to oxidative stress and subsequent changes that affect all aspects of physiology. To limit and repair the damage generated by ROS, cells have developed a multitude of responses. A hallmark of these responses is the activation of signaling pathways that modulate the function of downstream targets in different cellular locations. To this end, critical steps of the stress response that occur in the nucleus and cytoplasm have to be coordinated, which makes the proper communication between both compartments mandatory. Here, we discuss the interdependence of ROS-mediated signaling and the transport of macromolecules across the nuclear envelope. We highlight examples of oxidant-dependent nuclear trafficking and describe the impact of oxidative stress on the transport apparatus. Our paper concludes by proposing a cellular circuit of ROS-induced signaling, nuclear transport and repair.
    01/2012; 2012(2090-1739):208650. DOI:10.1155/2012/208650
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