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 ∼9 Å. 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.

[1]  A. Omelchenko,et al.  Inhibition of the Drosophila Na+/Ca2+ Exchanger, CALX1.1, by KB‐R7943 , 2002, Annals of the New York Academy of Sciences.

[2]  J. Tavaré,et al.  Rapid caspase‐3 activation during apoptosis revealed using fluorescence‐resonance energy transfer , 2000, EMBO reports.

[3]  Zygmunt Gryczynski,et al.  A FRET-Based Sensor Reveals Large ATP Hydrolysis–Induced Conformational Changes and Three Distinct States of the Molecular Motor Myosin , 2000, Cell.

[4]  U. Rapp,et al.  Serine/Threonine Kinases 3pK and MAPK-activated Protein Kinase 2 Interact with the Basic Helix-Loop-Helix Transcription Factor E47 and Repress Its Transcriptional Activity* , 2000, The Journal of Biological Chemistry.

[5]  O. Werz,et al.  5-lipoxygenase is phosphorylated by p38 kinase-dependent MAPKAP kinases. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[6]  Masatoshi Hagiwara,et al.  A fluorescent indicator for visualizing cAMP-induced phosphorylation in vivo , 2000, Nature Biotechnology.

[7]  E. Nishida,et al.  A conserved docking motif in MAP kinases common to substrates, activators and regulators , 2000, Nature Cell Biology.

[8]  E. Nishida,et al.  Two co‐existing mechanisms for nuclear import of MAP kinase: passive diffusion of a monomer and active transport of a dimer , 1999, The EMBO journal.

[9]  M. Gaestel,et al.  MAPKAP kinase 2 is essential for LPS-induced TNF-α biosynthesis , 1999, Nature Cell Biology.

[10]  R. Kraft,et al.  MAPKAP Kinase 2 Phosphorylates Serum Response Factor in Vitro and in Vivo* , 1999, The Journal of Biological Chemistry.

[11]  K. Sutoh,et al.  Swing of the lever arm of a myosin motor at the isomerization and phosphate-release steps , 1998, Nature.

[12]  C. Marshall,et al.  Nuclear export of the stress-activated protein kinase p38 mediated by its substrate MAPKAP kinase-2 , 1998, Current Biology.

[13]  M. Gaestel,et al.  Leptomycin B‐sensitive nuclear export of MAPKAP kinase 2 is regulated by phosphorylation , 1998, The EMBO journal.

[14]  E. Goldsmith,et al.  Phosphorylation of the MAP Kinase ERK2 Promotes Its Homodimerization and Nuclear Translocation , 1998, Cell.

[15]  L. Mahadevan,et al.  Anisomycin Selectively Desensitizes Signalling Components Involved in Stress Kinase Activation and fos andjun Induction , 1998, Molecular and Cellular Biology.

[16]  P. Cohen,et al.  A stress-activated kinase cascade can mediate the activation of tyrosine hydroxylase in chromaffin cells. , 1997, Biochemical Society transactions.

[17]  R. Tsien,et al.  Fluorescent indicators for Ca2+based on green fluorescent proteins and calmodulin , 1997, Nature.

[18]  P. Cohen,et al.  Participation of a stress-activated protein kinase cascade in the activation of tyrosine hydroxylase in chromaffin cells. , 1997, European journal of biochemistry.

[19]  A. Persechini,et al.  Detection in Living Cells of Ca2+-dependent Changes in the Fluorescence Emission of an Indicator Composed of Two Green Fluorescent Protein Variants Linked by a Calmodulin-binding Sequence , 1997, The Journal of Biological Chemistry.

[20]  J. Jongstra,et al.  LSP1 Is the Major Substrate for Mitogen-activated Protein Kinase-activated Protein Kinase 2 in Human Neutrophils* , 1997, The Journal of Biological Chemistry.

[21]  R. Davis,et al.  MKK3- and MKK6-regulated gene expression is mediated by the p38 mitogen-activated protein kinase signal transduction pathway , 1996, Molecular and cellular biology.

[22]  R. Tsien,et al.  Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer , 1996, Current Biology.

[23]  M. Gaestel,et al.  Constitutive Activation of Mitogen-activated Protein Kinase-activated Protein Kinase 2 by Mutation of Phosphorylation Sites and an A-helix Motif (*) , 1995, The Journal of Biological Chemistry.

[24]  M. Chalfie.  GREEN FLUORESCENT PROTEIN , 1995, Photochemistry and photobiology.

[25]  Chi‐Kuang Huang,et al.  Characterization of an Autoinhibitory Domain in Human Mitogen-activated Protein Kinase-activated Protein Kinase 2 (*) , 1995, The Journal of Biological Chemistry.

[26]  M. Gaestel,et al.  Characterization of the proline-rich region of mouse MAPKAP kinase 2: influence on catalytic properties and binding to the c-abl SH3 domain in vitro. , 1994, Biochemical and biophysical research communications.

[27]  S. Taylor,et al.  A conserved helix motif complements the protein kinase core. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[28]  David Stokoe,et al.  Identification of MAPKAP kinase 2 as a major enzyme responsible for the phosphorylation of the small mammalian heat shock proteins , 1992, FEBS letters.

[29]  J. Zheng,et al.  Crystal structure of the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase. , 1991, Science.

[30]  Fluorescence resonance energy transfer between blue-emitting and redshifted excitation derivatives of the green fluorescent protein * , 2003 .

[31]  Jiahuai Han,et al.  The p38 signal transduction pathway: activation and function. , 2000, Cellular signalling.

[32]  Susan S. Taylor,et al.  A genetically encoded, fluorescent indicator for cyclic AMP in living cells , 1999, Nature Cell Biology.

[33]  Y. Wang,et al.  Leptomycin B is an inhibitor of nuclear export: inhibition of nucleo-cytoplasmic translocation of the human immunodeficiency virus type 1 (HIV-1) Rev protein and Rev-dependent mRNA. , 1997, Chemistry & biology.

[34]  R. Mitra,et al.  Fluorescence resonance energy transfer between blue-emitting and red-shifted excitation derivatives of the green fluorescent protein. , 1996, Gene.

[35]  R. Clegg.  Fluorescence resonance energy transfer and nucleic acids. , 1992, Methods in enzymology.

[36]  Th. Förster.  Fluoreszenz organischer Verbindungen , 1951 .