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

Nature Structural & Molecular Biology volume 16, pages 1026–1035 (2009)Cite this article

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-β family proteins, importin-β and transportin. Other FG nucleoporins showed a similar functional change after ERK-mediated phosphorylation. Nuclear migration of importin-β 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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Figure 1: Detection of ERK pathway components with a combination of the estrogen receptor fusion system, IMAC, 2D-DIGE and phosphomotif-specific antibodies.
Figure 2: Identification of ERK pathway components and confirmation by two-dimensional western blotting.
Figure 3: In vitro kinase assays with candidate substrates for ERK.
Figure 4: Validation of Nup50 as a novel ERK substrate.
Figure 5: Phosphorylation of Nup50 by ERK reduces its affinity for importin-β and transportin.
Figure 6: ERK phosphorylates Nup153 and Nup214 and regulates their interaction with importin-β.
Figure 7: Nuclear migration of importin-β and transportin is impaired in ERK-activated, digitonin-permeabilized cells.
Figure 8: Phosphorylation of Nup50 is involved in the ERK-mediated inhibition of nuclear translocation of importin-β.

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Acknowledgements

We thank J. Hirano, J. Inagawa, T. Noda, M. Tanaka, M. Kikkawa and L. Yamada for technical assistance with 2D-DIGE and LC-MS/MS analysis; M.-Y. Han, M. Kobayashi, M. Machida, K. Ueda, R. Ohtsuka, T. Nishimura, N. Iida and H. Suzuki for experimental assistance; K. Shirakabe for constructing the plasmid expressing GST-fused DYNC1LI1 and critical comments; S. Matsubara and M. Iwata for secretarial assistance; and Y. Okada for fruitful discussions and advice. We also thank Y. Gu and Y. Ihara (Doshisha University) for providing C4G anti–CRMP-2 mouse mAb and M. McMahon (University of California, San Francisco) for providing ΔB-Raf:ER cells. This work was supported in part by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (to H.K.) and Encouraging Development Strategic Research Centers Program, the Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Culture, Sports, Science, and Technology (to S.H.). This work was also supported by grants from the Nakajima Foundation (to H.K.) and the Novartis Foundation (Japan) for the Promotion of Science (to S.H.). This work was developed and coordinated under the framework of the program for the International Research and Educational Institute for Integrated Medical Sciences (IREIIMS).

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Authors and Affiliations

  1. Division of Cellular Proteomics (BML), Institute of Medical Science, The University of Tokyo, Tokyo, Japan

    Hidetaka Kosako, Nozomi Yamaguchi, Masato Ushiyama & Seisuke Hattori

  2. Division of Disease Proteomics, Institute for Enzyme Research, The University of Tokushima, Tokushima, Japan

    Hidetaka Kosako & Hisaaki Taniguchi

  3. RD Center, BML, Inc., Saitama, Japan

    Nozomi Yamaguchi

  4. Department of Biochemistry, School of Pharmaceutical Sciences, Kitasato University, Tokyo, Japan

    Chizuru Aranami & Seisuke Hattori

  5. GE Healthcare Bio-Sciences KK, Tokyo, Japan

    Masato Ushiyama

  6. Cellular Dynamics Laboratory, Advanced Science Institute, RIKEN, Saitama, Japan

    Shingo Kose & Naoko Imamoto

  7. Department of Cell and Developmental Biology, Graduate School of Biostudies, Kyoto University, Kyoto, Japan

    Eisuke Nishida

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Contributions

H.K. conceived the whole study, designed and performed experiments and wrote the manuscript; N.Y., C.A. and M.U. performed experiments; S.K., N.I., H.T. and E.N. provided reagents and ideas to this work; S.H. provided the initial strategy for phosphoproteomics and advice; all of the authors discussed the results and commented on the manuscript.

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Correspondence to Hidetaka Kosako.

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Kosako, H., Yamaguchi, N., Aranami, C. et al. Phosphoproteomics reveals new ERK MAP kinase targets and links ERK to nucleoporin-mediated nuclear transport. Nat Struct Mol Biol 16, 1026–1035 (2009). https://doi.org/10.1038/nsmb.1656

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