Leptomycin B‐sensitive nuclear export of MAPKAP kinase 2 is regulated by phosphorylation

To study the intracellular localization of MAPKAP kinase 2 (MK2), which carries a putative bipartite nuclear localization signal (NLS), we constructed a green fluorescent protein–MAPKAP kinase 2 fusion protein (GFP–MK2). In transfected cells, this protein is located predominantly in the nucleus; unexpectedly, upon stress, it rapidly translocates to the cytoplasm. This translocation can be blocked by the p38 MAP kinase inhibitor SB203580, indicating its regulation by phosphorylation. Molecular mimicry of MK2 phosphorylation at T317 in GFP–MK2 led to a mutant which is located almost exclusively in the cytoplasm of the cell, whereas the mutant T317A shows no stress‐induced redistribution. Since leptomycin B, which inhibits the interaction of exportin 1 with the Rev‐type leucine‐rich nuclear export signal (NES), blocks stress‐dependent translocation of GFP–MK2, it is supposed that phosphorylation‐induced export of the protein causes the translocation. We have identified the region responsible for nuclear export in MK2 which is partially overlapping with and C‐terminal to the autoinhibitory motif. This region contains a cluster of hydrophobic amino acids in the characteristic spacing of a leucine‐rich Rev‐type NES which is necessary to direct GFP–MK2 to the cytoplasm. However, unlike the Rev‐type NES, this region alone is not sufficient for nuclear export. The data obtained indicate that MK2 contains a constitutively active NLS and a stress‐regulated signal for nuclear export.

[1]  Philip R. Cohen,et al.  A comparison of the substrate specificity of MAPKAP kinase‐2 and MAPKAP kinase‐3 and their activation by cytokines and cellular stress , 1996, FEBS letters.

[2]  A. Brunet,et al.  Growth factors induce nuclear translocation of MAP kinases (p42mapk and p44mapk) but not of their activator MAP kinase kinase (p45mapkk) in fibroblasts , 1993, The Journal of cell biology.

[3]  J. Blenis,et al.  Nuclear localization and regulation of erk- and rsk-encoded protein kinases , 1992, Molecular and cellular biology.

[4]  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.

[5]  Philip R. Cohen,et al.  Identification of anisomycin-activated kinases p45 and p55 in murine cells as MAPKAP kinase-2. , 1996, Oncogene.

[6]  T. Collins,et al.  Tumor Necrosis Factor α-Induced E-selectin Expression Is Activated by the Nuclear Factor-κB and c-JUN N-terminal Kinase/p38 Mitogen-activated Protein Kinase Pathways* , 1997, The Journal of Biological Chemistry.

[7]  Philip R. Cohen,et al.  SB 203580 is a specific inhibitor of a MAP kinase homologue which is stimulated by cellular stresses and interleukin‐1 , 1995, FEBS letters.

[8]  Philip R. Cohen,et al.  The substrate specificity and structure of mitogen-activated protein (MAP) kinase-activated protein kinase-2. , 1993, The Biochemical journal.

[9]  C. Dargemont,et al.  Evidence for a role of CRM1 in signal-mediated nuclear protein export. , 1997, Science.

[10]  Philip R. Cohen,et al.  Identification of novel phosphorylation sites required for activation of MAPKAP kinase‐2. , 1995, The EMBO journal.

[11]  Philip R. Cohen,et al.  FGF and stress regulate CREB and ATF‐1 via a pathway involving p38 MAP kinase and MAPKAP kinase‐2. , 1996, The EMBO journal.

[12]  M. Powers,et al.  Nuclear Export Receptors: From Importin to Exportin , 1997, Cell.

[13]  B. Cullen,et al.  Identification of the activation domain of equine infectious anemia virus rev , 1993, Journal of virology.

[14]  T. Hunter,et al.  Transcriptional control by protein phosphorylation: signal transmission from the cell surface to the nucleus , 1995, Current Biology.

[15]  T. Hope,et al.  Posttranscriptional effector domains in the Rev proteins of feline immunodeficiency virus and equine infectious anemia virus , 1994, Journal of virology.

[16]  P. Cohen,et al.  Phosphorylation and activation of human tyrosine hydroxylase in vitro by mitogen-activated protein (MAP) kinase and MAP-kinase-activated kinases 1 and 2. , 1993, European journal of biochemistry.

[17]  A. Brunet,et al.  Identification of MAP Kinase Domains by Redirecting Stress Signals into Growth Factor Responses , 1996, Science.

[18]  E. Nishida,et al.  A Novel Regulatory Mechanism in the Mitogen-activated Protein (MAP) Kinase Cascade , 1997, The Journal of Biological Chemistry.

[19]  E. Winter,et al.  An osmosensing signal transduction pathway in yeast. , 1993, Science.

[20]  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.

[21]  H. Rubinfeld,et al.  Nuclear translocation of mitogen-activated protein kinase kinase (MEK1) in response to mitogenic stimulation. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[22]  John D. Scott,et al.  Molecular Glue: Kinase Anchoring and Scaffold Proteins , 1996, Cell.

[23]  M. Gaestel,et al.  PMA‐induced activation of the p42/44ERK‐ and p38RK‐MAP kinase cascades in HL‐60 cells is PKC dependent but not essential for differentiation to the macrophage‐like phenotype , 1997, Journal of cellular physiology.

[24]  M E Greenberg,et al.  A cytoplasmic inhibitor of the JNK signal transduction pathway. , 1997, Science.

[25]  M. Gaestel,et al.  The MAP kinase‐activated protein kinase 2 contains a proline‐rich SH3‐binding domain , 1993, FEBS letters.

[26]  U. Kutay,et al.  The asymmetric distribution of the constituents of the Ran system is essential for transport into and out of the nucleus , 1997, The EMBO journal.

[27]  P. Barnes,et al.  Transcriptional Down-regulation of m2 Muscarinic Receptor Gene Expression in Human Embryonic Lung (HEL 299) Cells by Protein Kinase C (*) , 1995, The Journal of Biological Chemistry.

[28]  Erich A. Nigg,et al.  Nucleocytoplasmic transport: signals, mechanisms and regulation , 1997, Nature.

[29]  P. Cohen,et al.  On target with a new mechanism for the regulation of protein phosphorylation. , 1993, Trends in biochemical sciences.

[30]  E. Nishida,et al.  Interaction of MAP kinase with MAP kinase kinase: its possible role in the control of nucleocytoplasmic transport of MAP kinase , 1997, The EMBO journal.

[31]  J. Minna,et al.  3pK, a new mitogen-activated protein kinase-activated protein kinase located in the small cell lung cancer tumor suppressor gene region , 1996, Molecular and cellular biology.

[32]  E. Nishida,et al.  Cytoplasmic Localization of Mitogen-activated Protein Kinase Kinase Directed by Its NH2-terminal, Leucine-rich Short Amino Acid Sequence, Which Acts as a Nuclear Export Signal* , 1996, The Journal of Biological Chemistry.

[33]  Minoru Yoshida,et al.  CRM1 Is an Export Receptor for Leucine-Rich Nuclear Export Signals , 1997, Cell.

[34]  J. Hsuan,et al.  Interleukin-1 activates a novel protein kinase cascade that results in the phosphorylation of hsp27 , 1994, Cell.

[35]  T. Hunter,et al.  The protein kinases of budding yeast: six score and more. , 1997, Trends in biochemical sciences.

[36]  Jiahuai Han,et al.  Pro-inflammatory Cytokines and Environmental Stress Cause p38 Mitogen-activated Protein Kinase Activation by Dual Phosphorylation on Tyrosine and Threonine (*) , 1995, The Journal of Biological Chemistry.

[37]  B. Cullen,et al.  Protein sequence requirements for function of the human T-cell leukemia virus type 1 Rex nuclear export signal delineated by a novel in vivo randomization-selection assay , 1996, Molecular and cellular biology.

[38]  M. Gaestel,et al.  3pK, a novel mitogen-activated protein (MAP) kinase-activated protein kinase, is targeted by three MAP kinase pathways , 1996, Molecular and cellular biology.

[39]  Marc W. Kirschner,et al.  How Proteolysis Drives the Cell Cycle , 1996, Science.

[40]  R. Laskey,et al.  Nuclear shuttling: The default pathway for nuclear proteins? , 1993, Cell.

[41]  Karsten Weis,et al.  Exportin 1 (Crm1p) Is an Essential Nuclear Export Factor , 1997, Cell.

[42]  John C. Lee,et al.  Identification of Mitogen-activated Protein (MAP) Kinase-activated Protein Kinase-3, a Novel Substrate of CSBP p38 MAP Kinase (*) , 1996, The Journal of Biological Chemistry.

[43]  P. Cohen,et al.  MAPKAP kinase‐2; a novel protein kinase activated by mitogen‐activated protein kinase. , 1992, The EMBO journal.

[44]  Roger Y Tsien,et al.  Identification of a signal for rapid export of proteins from the nucleus , 1995, Cell.

[45]  Minoru Yoshida,et al.  CRM1 is responsible for intracellular transport mediated by the nuclear export signal , 1997, Nature.

[46]  Jerry L. Adams,et al.  A protein kinase involved in the regulation of inflammatory cytokine biosynthesis , 1994, Nature.

[47]  S. Wente,et al.  An RNA-export mediator with an essential nuclear export signal , 1996, Nature.

[48]  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.

[49]  Michel Morange,et al.  A novel kinase cascade triggered by stress and heat shock that stimulates MAPKAP kinase-2 and phosphorylation of the small heat shock proteins , 1994, Cell.