FRET-based detection of different conformations of MK2.
MAP kinase-activated protein kinase 2 (MK2 or MAPKAP K2) is a stress-activated enzyme downstream to p38 MAPK. By fusion of green fluorescent protein variants to the N- and C-terminus we analysed conformational changes in the kinase molecule in vitro and in vivo. Activation of MK2 is accompanied by a decrease in fluorescence resonance energy transfer, indicating a transition from an inactive/closed to an active/open conformation with an increase in the apparent distance between the fluorophores of approximately 9 A. The closed conformation exists exclusively in the nucleus. Upon stress, the open conformation of MK2 rapidly becomes detectable in the cytoplasm and accumulates in the nucleus only when Crm1-dependent nuclear export is blocked. Hence, in living cells activation of MK2 and its nuclear export are coupled by a phosphorylation-dependent conformational switch.
Neininger
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A
Innovationskolleg Zellspezialisierung, Martin-Luther-Universität Halle Wittenberg, 06120 Halle, Germany.
Thielemann
H
H
Gaestel
M
M
D021581:Active Transport, Cell Nucleus / Q000502:physiology; D000841:Anisomycin / Q000494:pharmacology; D002460:Cell Line; D004735:Energy Transfer; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006801:Humans; D007202:Indicators and Reagents / Q000737:chemistry / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins / Q000737:chemistry / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000737:chemistry* / Q000378:metabolism*; D010766:Phosphorylation; D011487:Protein Conformation; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D000099190:MAP-Kinase-Activated Kinase 2
D000841:Anisomycin; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins; D020928:Mitogen-Activated Protein Kinases; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D011500:Protein Synthesis Inhibitors; D011993:Recombinant Fusion Proteins; D000099190:MAP-Kinase-Activated Kinase 2