Phosphoproteomics reveals new ERK MAP kinase targets and links ERK to nucleoporin-mediated nuclear transport.
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.
Kosako
Hidetaka
H
Division of Cellular Proteomics (BML), Institute of Medical Science, The University of Tokyo, Tokyo, Japan. kosako@ier.tokushima-u.ac.jp
Yamaguchi
Nozomi
N
Aranami
Chizuru
C
Ushiyama
Masato
M
Kose
Shingo
S
Imamoto
Naoko
N
Taniguchi
Hisaaki
H
Nishida
Eisuke
E
Hattori
Seisuke
S
D021581:Active Transport, Cell Nucleus; D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D006801:Humans; D028884:Karyopherins / Q000378:metabolism; D051379:Mice; D041681:NIH 3T3 Cells; D028861:Nuclear Pore Complex Proteins / Q000737:chemistry*; D010750:Phosphoproteins / Q000737:chemistry / Q000502:physiology*; D010766:Phosphorylation; D040901:Proteomics / Q000379:methods*; D028961:beta Karyopherins / Q000378:metabolism
D028884:Karyopherins; D028861:Nuclear Pore Complex Proteins; D010750:Phosphoproteins; D028961:beta Karyopherins; D048049:Extracellular Signal-Regulated MAP Kinases