Article

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

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.

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    • "may alter the barrier function of NPCs. In mammals, nucleoporin Nup50 is phosphorylated by ERK kinase, which in turn changes the affinity of importin β to NPCs (Kosako et al., 2009). Thus, the barrier function of NPCs during meiosis in S. pombe may be regulated by phosphorylation or other post-translational modifications of nucleoporins. "
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    ABSTRACT: Ran, a small GTPase, is required for the spindle formation and nuclear envelope (NE) formation. After NE breakdown (NEBD) during mitosis in metazoan cells, the Ran-GTP gradient across the NE is lost and Ran-GTP becomes concentrated around chromatin, thus affecting the stability of microtubules and promoting the assembly of spindle microtubules and segregation of chromosomes. Mitosis in which chromosomes are segregated subsequent to NEBD is called “open mitosis.” In contrast, many fungi undergo a process termed “closed mitosis” in which chromosome segregation and spindle formation occur without NEBD. Although the fission yeast Schizosaccharomyces pombe undergoes a closed mitosis, it exhibits a short period during meiosis (anaphase of the second meiosis; called “anaphase II”) when nuclear and cytoplasmic proteins are mixed in the presence of intact NE and nuclear pore complexes (NPC). This “virtual” nuclear envelope breakdown (vNEBD) involves changes in the localization of RanGAP1, an activator of Ran-GTP hydrolysis. Recently, Nup132, a component of the structural core Nup107-160 subcomplex of the NPC, has been shown to be involved in the maintenance of the nuclear cytoplasmic barrier in yeast meiosis. In this review, we highlight the possible roles of RanGAP1 and Nup132 in vNEBD and discuss the biological significance of vNEBD in S. pombe meiosis.
    Preview · Article · Feb 2016 · Frontiers in Cell and Developmental Biology
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    • "Regarding experimental MAPK network discovery, ERK2 has been the most widely explored. For example, several different methods were utilized to identify ERK2 substrates by large-scale phosphoproteomics (Kosako et al, 2009; Carlson et al, 2011; Courcelles et al, 2013). Unfortunately, pairwise overlaps between the lists of substrates are low across studies (e.g., around ~10%), with not a single overlap between five different studies that aimed to find ERK2-phosphorylated substrates (Courcelles et al, 2013), suggesting great dependence on the experimental conditions used. "
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    ABSTRACT: Mitogen-activated protein kinases (MAPK) are broadly used regulators of cellular signaling. However, how these enzymes can be involved in such a broad spectrum of physiological functions is not understood. Systematic discovery of MAPK networks both experimentally and in silico has been hindered because MAPKs bind to other proteins with low affinity and mostly in less-characterized disordered regions. We used a structurally consistent model on kinase-docking motif interactions to facilitate the discovery of short functional sites in the structurally flexible and functionally under-explored part of the human proteome and applied experimental tools specifically tailored to detect low-affinity protein-protein interactions for their validation invitro and in cell-based assays. The combined computational and experimental approach enabled the identification of many novel MAPK-docking motifs that were elusive for other large-scale protein-protein interaction screens. The analysis produced an extensive list of independently evolved linear binding motifs from a functionally diverse set of proteins. These all target, with characteristic binding specificity, an ancient protein interaction surface on evolutionarily related but physiologically clearly distinct three MAPKs (JNK, ERK, and p38). This inventory of human protein kinase binding sites was compared with that of other organisms to examine how kinase-mediated partnerships evolved over time. The analysis suggests that most human MAPK-binding motifs are surprisingly new evolutionarily inventions and newly found links highlight (previously hidden) roles of MAPKs. We propose that short MAPK-binding stretches are created in disordered protein segments through a variety of ways and they represent a major resource for ancient signaling enzymes to acquire new regulatory roles.
    Preview · Article · Nov 2015 · Molecular Systems Biology
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    • "Thus, in vivo experiments should be performed to confirm the physiological meaning of the phosphorylated substrates identified in vitro. Many researchers have performed mass spectrometry-based studies to identify the in vivo protein phosphorylations downstream of cellular signaling-related kinases such as MAPKs, PKAs and AKTs23242526. In vivo studies of protein phosphorylation identify all of the phosphorylations downstream of the activation/inhibition of the target kinase. "
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    ABSTRACT: Protein phosphorylation is a major and essential post-translational modification in eukaryotic cells that plays a critical role in various cellular processes. Recent progresses in mass spectrometry techniques have enabled the effective identification and analysis of protein phosphorylation. Mass spectrometry-based approaches to investigating protein phosphorylation are very powerful and informative and can further improve our understanding of protein phosphorylation as a whole, but they cannot determine the upstream kinases involved. We introduce several studies that attempted to uncover the relationships between various kinases of interest and substrates, including two methods we developed: an in vitro approach termed the kinase-interacting substrate screening (KISS) method and an in vivo approach termed the phosphatase inhibitor and kinase inhibitor substrate screening (PIKISS) method. This article is part of a Special Issue entitled:Inhibitors of Protein Kinases. Copyright © 2015. Published by Elsevier B.V.
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