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ERK Nuclear Translocation Is Dimerization-independent but Controlled by the Rate of Phosphorylation*

  • Diane S. Lidke
    Footnotes
    Affiliations
    From the Laboratory of Cellular Dynamics, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany,
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  • Fang Huang
    Footnotes
    Affiliations
    the Department of Physics and Astronomy, University of New Mexico, Albuquerque, New Mexico 87131,
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  • Janine N. Post
    Footnotes
    Affiliations
    From the Laboratory of Cellular Dynamics, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany,
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  • Bernd Rieger
    Footnotes
    Affiliations
    From the Laboratory of Cellular Dynamics, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany,
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  • Julie Wilsbacher
    Footnotes
    Affiliations
    the Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75235, and
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  • James L. Thomas
    Footnotes
    Affiliations
    the Department of Physics and Astronomy, University of New Mexico, Albuquerque, New Mexico 87131,
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  • Jacques Pouysségur
    Affiliations
    the Institute of Developmental Biology and Cancer, CNRS UMR6543, Université de Nice, Centre A. Lacassagne, 06189 Nice, France
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  • Thomas M. Jovin
    Affiliations
    From the Laboratory of Cellular Dynamics, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany,
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  • Philippe Lenormand
    Correspondence
    To whom correspondence should be addressed: Centre A. Lacassagne, 06189 Nice, France. Tel.: 492-031227; Fax: 492-031225;
    Affiliations
    the Institute of Developmental Biology and Cancer, CNRS UMR6543, Université de Nice, Centre A. Lacassagne, 06189 Nice, France
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  • Author Footnotes
    * This work was supported by European Union FP5 Project QLRT-2000-02278 (MAP Kinase) (to T. M. J. and J. P.), the CNRS, Centre A. Lacassagne, Association pour la Recherche sur le Cancer Contract 3338, and American Cancer Society Grant IRG 192 (to D. S. L.). This work was supported in part by National Institutes of Health Grant DK34128 (to J. W.).
    ♦ This article was selected as a Paper of the Week.
    The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables S1 and S2, Figs. S1–S3, and Movies 1 and 2.
    1 Present address: Dept. of Pathology and Cancer Research and Treatment Center, University of New Mexico, Albuquerque, NM 87131.
    2 Supported by the Army Research Office Grant W911NF0510464.
    3 Present address: Molecular Cell Biology Faculty of Science and Technology, University of Twente, 7500AE Enschede, The Netherlands.
    4 Present address: Quantitative Imaging Group, Dept. of Imaging Science and Technology, Delft University of Technology, 2628CJ Delft, The Netherlands.
    5 Supported by a predoctoral fellowship from the Howard Hughes Medical Institute. Present address: Cancer Research, Global Pharmaceutical Research and Development, Abbott Laboratories, 100 Abbott Park Rd., Abbott Park, IL 60064.
Open AccessPublished:November 17, 2009DOI:https://doi.org/10.1074/jbc.M109.064972
      Upon activation, ERKs translocate from the cytoplasm to the nucleus. This process is required for the induction of many cellular responses, yet the molecular mechanisms that regulate ERK nuclear translocation are not fully understood. We have used a mouse embryo fibroblast ERK1-knock-out cell line expressing green fluorescent protein (GFP)-tagged ERK1 to probe the spatio-temporal regulation of ERK1. Real time fluorescence microscopy and fluorescence correlation spectroscopy revealed that ERK1 nuclear accumulation increased upon serum stimulation, but the mobility of the protein in the nucleus and cytoplasm remained unchanged. Dimerization of ERK has been proposed as a requirement for nuclear translocation. However, ERK1-Δ4, the mutant shown consistently to be dimerization-deficient in vitro, accumulated in the nucleus to the same level as wild type (WT), indicating that dimerization of ERK1 is not required for nuclear entry and retention. Consistent with this finding, energy migration Förster resonance energy transfer and fluorescence correlation spectroscopy measurements in living cells did not detect dimerization of GFP-ERK1-WT upon activation. In contrast, the kinetics of nuclear accumulation and phosphorylation of GFP-ERK1-Δ4 were slower than that of GFP-ERK1-WT. These results indicate that the differential shuttling behavior of the mutant is a consequence of delayed phosphorylation of ERK by MEK rather than dimerization. Our data demonstrate for the first time that a delay in cytoplasmic activation of ERK is directly translated into a delay in nuclear translocation.

      Introduction

      Stimulation of numerous cell surface receptors leads to activation of the Raf/MEK
      The abbreviations used are: MEK
      mitogen-activated protein kinase/ERK kinase
      FCS
      fluorescence correlation spectroscopy
      emFRET
      energy migration Förster resonance energy transfer
      FRAP
      fluorescence recovery after photobleaching
      F/N
      fluorescence intensity per mobile object
      ERK
      extracellular signal-regulated kinase
      GFP
      green fluorescent protein
      WT
      wild type
      ERK-Δ4
      human ERK1 deleted from Pro193–Asp196
      MEF
      mouse embryo fibroblasts
      LMB
      leptomycin B
      DMEM
      Dulbecco's modified Eagle's medium.
      /ERK signaling pathway. In this kinase cascade, Raf phosphorylates only MEK, and MEK phosphorylates only ERK, whereas ERK is able to phosphorylate many substrates in nearly all cell compartments (
      • Yoon S.
      • Seger R.
      ). Noncatalytic activation of a few partners by c-Raf is well documented, but the biological outcomes of the ERK pathway are predominantly driven by the kinase activity, as evidenced, for example, by chemical inhibition (reviewed in Ref.
      • Kohno M.
      • Pouyssegur J.
      ). ERK is primarily located in the cytoplasm of resting cells, although overexpression results in cytoplasmic and nuclear localization (
      • Lenormand P.
      • Sardet C.
      • Pagès G.
      • L’Allemain G.
      • Brunet A.
      • Pouysségur J.
      ). It has long been recognized that in the course of physiological signal transduction, ERK accumulates in the nucleus after acute stimulation of the cell (
      • Lenormand P.
      • Sardet C.
      • Pagès G.
      • L’Allemain G.
      • Brunet A.
      • Pouysségur J.
      ,
      • Chen R.H.
      • Sarnecki C.
      • Blenis J.
      ). Nuclear translocation of ERK is required for cell cycle entry. Thus, retention of ERK in the cytoplasm alters neither ERK kinase activity nor phosphorylation of cytoplasmic substrates, whereas ERK-dependent transcription and cell proliferation are blocked (
      • Brunet A.
      • Roux D.
      • Lenormand P.
      • Dowd S.
      • Keyse S.
      • Pouysségur J.
      ). It has been demonstrated that ERK phosphorylates the Phe-Gly nucleoporins Nup50, Nup153, and Nup154, reducing importin-β-mediated nucleocytoplasmic transport (
      • Kosako H.
      • Yamaguchi N.
      • Aranami C.
      • Ushiyama M.
      • Kose S.
      • Imamoto N.
      • Taniguchi H.
      • Nishida E.
      • Hattori S.
      ). This observation would expand the role of ERK nuclear entry to include the regulation of nucleocytoplasmic transport of certain classes of proteins while crossing the nucleopore.
      MEK functions as the cytoplasmic anchor for ERK such that MEK co-overexpression maintains the cytoplasmic localization of overexpressed ERK, whereas saturating levels of the ERK-binding site of MEK abrogates ERK export from the nucleus (
      • Adachi M.
      • Fukuda M.
      • Nishida E.
      ). MEK is sequestered in the cytoplasm as a consequence of active export out of the nucleus mediated by its nuclear export sequence. MEK binds to inactive ERK in resting cells (
      • Fukuda M.
      • Gotoh Y.
      • Nishida E.
      ). The natural regulation of ERK translocation has also been demonstrated by differential expression of cytoplasmic anchors such as PEA15 (
      • Formstecher E.
      • Ramos J.W.
      • Fauquet M.
      • Calderwood D.A.
      • Hsieh J.C.
      • Canton B.
      • Nguyen X.T.
      • Barnier J.V.
      • Camonis J.
      • Ginsberg M.H.
      • Chneiweiss H.
      ,
      • Gervais M.
      • Dugourd C.
      • Muller L.
      • Ardidie C.
      • Canton B.
      • Loviconi L.
      • Corvol P.
      • Chneiweiss H.
      • Monnot C.
      ) or Sef (
      • Torii S.
      • Kusakabe M.
      • Yamamoto T.
      • Maekawa M.
      • Nishida E.
      ) or expression of nuclear anchors such as DUSP5 (
      • Mandl M.
      • Slack D.N.
      • Keyse S.M.
      ) or Vanishing (
      • Sur R.
      • Ramos J.W.
      ). It has been shown that the stimulation-induced nuclear accumulation of ERK requires the synthesis of short lived nuclear anchors (
      • Volmat V.
      • Camps M.
      • Arkinstall S.
      • Pouysségur J.
      • Lenormand P.
      ). Clearly, regulation of ERK nuclear translocation is an essential feature of the Raf/MEK/ERK signaling cascade.
      The precise mechanism of ERK transport across the nuclear pore is not fully understood. ERK lacks a nuclear localization sequence, leading to the suggestion that ERK may enter by a piggyback mechanism via binding to nuclear localization sequence-containing proteins (
      • Brunet A.
      • Roux D.
      • Lenormand P.
      • Dowd S.
      • Keyse S.
      • Pouysségur J.
      ). Nuclear localization sequence-dependent mechanisms require energy for Ran-dependent cycling of importins (
      • Terry L.J.
      • Shows E.B.
      • Wente S.R.
      ). However, reconstituted import assays have shown that ERK can bind directly to FXFG sequences of nucleoporin in the lumen of the nuclear pore complex, indicating that it may enter the nucleus in the absence of energy sources or cytosolic factors (
      • Matsubayashi Y.
      • Fukuda M.
      • Nishida E.
      ,
      • Whitehurst A.W.
      • Wilsbacher J.L.
      • You Y.
      • Luby-Phelps K.
      • Moore M.S.
      • Cobb M.H.
      ). Point mutations of ERK revealed that inactive and active ERK interact with nucleoporins via different domains; thus, both active and inactive ERK can be transported across the nuclear pore in an energy-independent fashion (
      • Yazicioglu M.N.
      • Goad D.L.
      • Ranganathan A.
      • Whitehurst A.W.
      • Goldsmith E.J.
      • Cobb M.H.
      ). However, it has also been proposed that active transport of ERK may also occur and that it requires dimerization of the protein driven by phosphorylation of the TEY activation loop (
      • Khokhlatchev A.V.
      • Canagarajah B.
      • Wilsbacher J.
      • Robinson M.
      • Atkinson M.
      • Goldsmith E.
      • Cobb M.H.
      ). Indeed, in a reconstituted import assay, thiophosphorylated ERK2 import increased in the presence of energy (
      • Ranganathan A.
      • Yazicioglu M.N.
      • Cobb M.H.
      ). Overall, these observations suggest a role for both an energy-dependent (presumably via dimerization) and an energy-independent mechanism (via direct binding to nucleoporins) in ERK cytoplasmic-nuclear translocation.
      Surprisingly, the role of ERK dimerization in nucleocytoplasmic shuttling has proven to be controversial. Two distinct “dimerization mutants” have been used in several studies as follows: the mutation of histidine 176 to glutamic acid plus four leucines to alanines (H176E,L333A,L336A,L341A,L344A or H176E L4A mutant) or the removal of histidine 176 and three adjacent amino acids (deletion 174–177 or Δ4 mutant). Initially, both mutants were shown to not dimerize in vitro and to not accumulate in the nucleus (
      • Khokhlatchev A.V.
      • Canagarajah B.
      • Wilsbacher J.
      • Robinson M.
      • Atkinson M.
      • Goldsmith E.
      • Cobb M.H.
      ), and later it was demonstrated that they remained monomeric in vitro at physiological salt concentrations (
      • Wilsbacher J.L.
      • Juang Y.C.
      • Khokhlatchev A.V.
      • Gallagher E.
      • Binns D.
      • Goldsmith E.J.
      • Cobb M.H.
      ). However, further studies using the H176E L4A mutant indicated its translocation to be normal (unless fused to β-galactosidase) (
      • Adachi M.
      • Fukuda M.
      • Nishida E.
      ,
      • Burack W.R.
      • Shaw A.S.
      ), although others found another replacement mutant, H176A L4A, to accumulate even in starved cells (
      • Wolf I.
      • Rubinfeld H.
      • Yoon S.
      • Marmor G.
      • Hanoch T.
      • Seger R.
      ); this mutant without the glutamic charge was not tested in vitro for its capacity to dimerize. Moreover, dimerization could not be detected in live cells through FRET measurements between co-expressed yellow fluorescent protein-ERK and cyan fluorescent protein-ERK (
      • Burack W.R.
      • Shaw A.S.
      ). Recently, it has been suggested that ERK dimerization plays a role in the activation of cytoplasmic substrates but not nuclear substrates (
      • Casar B.
      • Pinto A.
      • Crespo P.
      ). In parallel investigations focused on the nature and functional role(s) of ERK interactions with mitochondria, it was determined that dimerization of human ERK1 was favored in the mitochondria, also occurred in the nuclei, but was hardly detectable in the cytosol of HeLa cells (
      • Galli S.
      • Jahn O.
      • Hitt R.
      • Hesse D.
      • Opitz L.
      • Plessmann U.
      • Urlaub H.
      • Poderoso J.J.
      • Jares-Erijman E.A.
      • Jovin T.M.
      ). In view of these and other somewhat disparate assessments of ERK dimerization, we examined in detail the dimerization mutant ERK1-Δ4 because several studies have reported that the related mutation in ERK2 leads to abnormal nuclear translocation (
      • Khokhlatchev A.V.
      • Canagarajah B.
      • Wilsbacher J.
      • Robinson M.
      • Atkinson M.
      • Goldsmith E.
      • Cobb M.H.
      ,
      • Adachi M.
      • Fukuda M.
      • Nishida E.
      ,
      • Horgan A.M.
      • Stork P.J.
      ).
      In attempts to retain a normal cytoplasmic localization of ERK transfected to high levels of expression, several studies have resorted to co-expression of MEK (
      • Adachi M.
      • Fukuda M.
      • Nishida E.
      ,
      • Burack W.R.
      • Shaw A.S.
      ,
      • Horgan A.M.
      • Stork P.J.
      ), although others have imaged cells expressing very low levels of ERK (
      • Costa M.
      • Marchi M.
      • Cardarelli F.
      • Roy A.
      • Beltram F.
      • Maffei L.
      • Ratto G.M.
      ). ERK co-expressed with MEK generally displays an abnormally short persistence in the nucleus following stimulation, ranging from 10 to 40 min (
      • Burack W.R.
      • Shaw A.S.
      ,
      • Horgan A.M.
      • Stork P.J.
      ,
      • Furuno T.
      • Hirashima N.
      • Onizawa S.
      • Sagiya N.
      • Nakanishi M.
      ) instead of several hours in the case of endogenous ERK. In our studies, we used a mouse embryo fibroblast ERK1-knock-out cell line (MEFERK1−/− (
      • Pagès G.
      • Guérin S.
      • Grall D.
      • Bonino F.
      • Smith A.
      • Anjuere F.
      • Auberger P.
      • Pouysségur J.
      )). The lack of endogenous ERK1 allowed us to transfect MEFERK1−/− cells with GFP-ERK1 while maintaining the MEK-ERK balance and ensuring that every ERK1 in the cell is GFP-tagged.
      Using this system, we examined ERK1 localization, cytoplasmic-nuclear translocation, and dimerization in live cells using fluorescence microscopy techniques. Our results demonstrate that the mutant displays delayed kinetics of nuclear entry/shuttling but no differences in overall nuclear accumulation. These real time measurements in ERK1 knock-out cells help to reconcile previous discrepancies between studies with ERK “dimerization mutants,” by emphasizing that the differences in mutant and WT ERK are subtle, and the ability to resolve these differences is dependent on the time scale of the measurements. We also found that mutation of the dimerization motif delayed ERK phosphorylation, suggesting that the mutant is less efficiently phosphorylated by MEK. Our study reveals that dimerization of ERK is not required for nuclear entry but rather that the efficient activation of ERK by MEK is the key determinant of rapid nuclear translocation.

      DISCUSSION

      The outcomes of signaling via the mitogen-activated protein kinase pathway are determined by the regulation of ERK nuclear translocation (
      • Brunet A.
      • Roux D.
      • Lenormand P.
      • Dowd S.
      • Keyse S.
      • Pouysségur J.
      ,
      • Gervais M.
      • Dugourd C.
      • Muller L.
      • Ardidie C.
      • Canton B.
      • Loviconi L.
      • Corvol P.
      • Chneiweiss H.
      • Monnot C.
      ,
      • Robinson M.J.
      • Stippec S.A.
      • Goldsmith E.
      • White M.A.
      • Cobb M.H.
      ), which is required for activation of many transcription factors. The duration and extent of translocation is dependent on the type of stimulus, and ERK nuclear translocation is required to induce specific responses in the form of gene expression (
      • Whitehurst A.
      • Cobb M.H.
      • White M.A.
      ). In some cases stimulus-dependent regulation of nuclear translocation may be linked to distinct elevation of calcium concentration triggered by agonists (
      • Chuderland D.
      • Marmor G.
      • Shainskaya A.
      • Seger R.
      ). Proteins that regulate ERK nuclear translocation have been identified, including PEA15, which anchors ERK in the cytosol (
      • Formstecher E.
      • Ramos J.W.
      • Fauquet M.
      • Calderwood D.A.
      • Hsieh J.C.
      • Canton B.
      • Nguyen X.T.
      • Barnier J.V.
      • Camonis J.
      • Ginsberg M.H.
      • Chneiweiss H.
      ) in part by inhibiting the capacity of ERK to bind to nucleoporins (
      • Whitehurst A.W.
      • Robinson F.L.
      • Moore M.S.
      • Cobb M.H.
      ), and Sef, which blocks the dissociation of MEK·ERK complexes (
      • Torii S.
      • Kusakabe M.
      • Yamamoto T.
      • Maekawa M.
      • Nishida E.
      ).
      Because of the rapid nature of ERK nuclear translocation, we fluorescently tagged ERK to study its shuttling dynamics in living cells by quantitative microscopy. To ensure that expression of GFP-ERK1 did not overwhelm the system, we expressed GFP-ERK1 in MEKERK1−/− cells, thus avoiding saturation of ERK1/2 partners. The average concentration of GFP-ERK1 in our experiments was on the order of 1 μm, a value similar to the endogenous levels of total ERK reported recently by Fujioka et al. (
      • Fujioka A.
      • Terai K.
      • Itoh R.E.
      • Aoki K.
      • Nakamura T.
      • Kuroda S.
      • Nishida E.
      • Matsuda M.
      ) for HeLa and Cos cells (0.96 and 0.81 μm, respectively.) These authors reviewed the results of other studies, two reporting low concentrations (0.26 and 0.36 μm) and four concentrations ranging from 1 to 2.1 μm. In further confirmation of the physiological relevance of our system, we observed rapid nuclear translocation of GFP-ERK1 within minutes of serum addition. As shown previously for endogenous ERK (
      • Volmat V.
      • Camps M.
      • Arkinstall S.
      • Pouysségur J.
      • Lenormand P.
      ), nuclear localization of GFP-ERK1 lasted for several hours, and it was retained in the nucleus after dephosphorylation. This observation confirms that inactive and active ERK can be sequestered in the nucleus, presumably via binding to nuclear anchoring proteins (
      • Volmat V.
      • Camps M.
      • Arkinstall S.
      • Pouysségur J.
      • Lenormand P.
      ).
      Nuclear anchoring, however, is not accompanied by immobilization. GFP-ERK1 was found to be highly mobile, and its mobility did not depend on activation state, indicating that ERK binding partners are also mobile or that interactions are transient. FCS measurements of diffusion constants (Table 1) and FRAP studies showed that GFP-ERK1 redistributed within seconds throughout the nucleus (Fig. 5). The protein mobility was similar for unstimulated and stimulated cells (15 min to 2 h post-stimulation). Free GFP diffusion (Table 1) and nuclear redistribution (Fig. 5) were much faster than for GFP-ERK1, presumably reflecting the smaller size and lack of binding partners. The FCS and emFRET measurements did not detect changes in the aggregation state of ERK1 upon activation in either compartment (Fig. 6 and Table 1), indicating that ERK does not homodimerize before or during nuclear translocation.
      By blocking CRM1-dependent nuclear export, accumulation of unstimulated ERK was observed (see Refs.
      • Adachi M.
      • Fukuda M.
      • Nishida E.
      ,
      • Volmat V.
      • Camps M.
      • Arkinstall S.
      • Pouysségur J.
      • Lenormand P.
      and our data not shown), indicating that the localization of ERK and ERK complexes is actively regulated. Constant shuttling of ERK was further demonstrated by FRAP experiments in resting cells, in which the nuclear pool of GFP-ERK1 recovered after photobleaching the entire nucleus (Fig. 4). Stimulation of the cells led to an increase in nuclear recovery of GFP-ERK1, consistent with previous observations (
      • Costa M.
      • Marchi M.
      • Cardarelli F.
      • Roy A.
      • Beltram F.
      • Maffei L.
      • Ratto G.M.
      ) and in support of previous models in which ERK is released from a cytoplasmic anchor upon activation, allowing rapid nuclear accumulation (
      • Adachi M.
      • Fukuda M.
      • Nishida E.
      ). Together with data demonstrating that ERK nuclear entry does not require energy (
      • Matsubayashi Y.
      • Fukuda M.
      • Nishida E.
      ,
      • Whitehurst A.W.
      • Wilsbacher J.L.
      • You Y.
      • Luby-Phelps K.
      • Moore M.S.
      • Cobb M.H.
      ), our results suggest that upon release from cytoplasmic anchoring after activation, ERK diffuses to the nucleus while being highly mobile in both compartments at all times.
      To gain more insight into the mechanism of ERK nuclear translocation, we examined the behavior of the only ERK mutant shown to display consistently altered nuclear translocation, ERK1-Δ4. To our surprise, ERK1-Δ4 accumulated in the nucleus to the same level as ERK1-WT. This result was observed when nontagged ERK1-Δ4 was expressed in cells lacking endogenous ERK1, and it became obvious following statistical measurements of time-lapse studies of GFP-ERK1-Δ4 serum-induced nuclear translocation. In unstimulated cells, ERK1-Δ4 diffusion in both compartments was similar to that of WT, consistent with the observation that the unphosphorylated form of the other dimerization mutant, ERK2-H176E L4A, translocated normally across the nucleopore in reconstituted assays comparing active and inactive transport (
      • Yazicioglu M.N.
      • Goad D.L.
      • Ranganathan A.
      • Whitehurst A.W.
      • Goldsmith E.J.
      • Cobb M.H.
      ). FRAP of the nucleus also revealed that GFP-ERK1-Δ4 shuttling was accelerated after stimulation but was not as rapid as GFP-ERK1-WT. Abnormal folding could not explain this phenomenon because the kinase activity of ERK2-Δ4 has been found to be in the range of ERK2 (
      • Wilsbacher J.L.
      • Juang Y.C.
      • Khokhlatchev A.V.
      • Gallagher E.
      • Binns D.
      • Goldsmith E.J.
      • Cobb M.H.
      ).
      The dynamics and extent of nuclear accumulation of ERK may reflect import or export rates or both. Indeed, nuclear accumulation can be driven by changes in export alone as demonstrated by treatment of resting cells with LMB. The observation that ERK-Δ4 eventually accumulates to the same extent as ERK-WT implies that, at steady state, the ratio of import to export rates is unaffected by the mutation, provided the mechanisms of import and export are unchanged. The slower accumulation of the mutant could arise from slowed kinetics (import and export) or reflect a delay in the conversion of ERK to an “import-eligible” form, i.e. a delayed phosphorylation. Nuclear exchange measurements indicate a contribution from the former, whereas the sigmoidal shape of the accumulation curves strongly implies the existence of an activation delay as well.
      The fact that the mobilities of WT and mutant ERK1 were similar in the resting state, despite a delay in nuclear accumulation subsequent to stimulation, led us to hypothesize that the defect in mutant behavior should be upstream of translocation. In accordance with this supposition, we found that ERK1-Δ4 was phosphorylated more slowly than ERK1-WT and evaluated by double phosphorylation on the TEY activating loop (Fig. 7A). This delay was missed in previous experiments that suggested normal activation of the ERK2 mutant (H176A L4A) in an end point immunoblot assay (5 min of 12-O-tetradecanoylphorbol-13-acetate treatment) (
      • Wolf I.
      • Rubinfeld H.
      • Yoon S.
      • Marmor G.
      • Hanoch T.
      • Seger R.
      ). The difference between ERK1-Δ4 and ERK1-WT was specific to activation because upon chemical inhibition of MEK, ERK1-Δ4 was dephosphorylated as rapidly as WT (Fig. 7B). This normal dephosphorylation is consistent with the observation that the ERK2 mutant H176A L4A bound normally to the phosphatase MKP3-CS (determined via immunolocalization studies) (
      • Wolf I.
      • Rubinfeld H.
      • Yoon S.
      • Marmor G.
      • Hanoch T.
      • Seger R.
      ). Abnormal activation is also consistent with failure of ERK2-H176A L4A to co-immunoprecipitate normally with MEK1 (
      • Wolf I.
      • Rubinfeld H.
      • Yoon S.
      • Marmor G.
      • Hanoch T.
      • Seger R.
      ). Our data suggest a close correlation between activation of ERK and the rate of nuclear accumulation. We conclude that the activation (double phosphorylation) of ERK constitutes the trigger of rapid nuclear translocation.
      It has been demonstrated that at the onset of stimulation ERK is released from MEK (
      • Fukuda M.
      • Gotoh Y.
      • Nishida E.
      ) and one can expect that a delay in activation leads to a delayed release of ERK. This delayed release would then lead to slower nuclear accumulation because MEK and inactive ERK complexes are retained in the cytosol via the nuclear export sequence of MEK (
      • Fukuda M.
      • Gotoh I.
      • Gotoh Y.
      • Nishida E.
      ). We interpret the delay of shuttling during stimulation as a consequence of the slower re-activation of inactive ERK in complexes with MEK. Indeed, the turnover of ERK activation is very fast, as demonstrated by the total extinction of phospho-ERK within 3 min after blocking MEK activation (Fig. 7B). Furthermore, it has been demonstrated that at peak stimulation only 5% of MEK molecules are active, whereas up to 60% of ERK molecules are active (
      • Fujioka A.
      • Terai K.
      • Itoh R.E.
      • Aoki K.
      • Nakamura T.
      • Kuroda S.
      • Nishida E.
      • Matsuda M.
      ). Hence, rapidly inactivated ERK molecules are trapped instantly by the large pool of inactive MEK inside the cytoplasm, and inefficient activation of ERK1-Δ4 would then retard further release from MEK. Our data demonstrate for the first time that a delay in cytoplasmic activation of ERK is immediately translated into a delay of nuclear translocation, highlighting that the constant shuttling of ERK is linked to a rapid turnover of activation. This constant exchange of ERK, regulated by interactions with binding partners, allows nuclear ERK responses to act as immediate sensors of signal strength.
      Recently, wild type ERK has been shown to migrate as an ∼80-kDa complex, whereas the ERK dimer mutant H176E L4A did not form an 80-kDa complex or co-precipitate with MEK (
      • Casar B.
      • Pinto A.
      • Crespo P.
      ,
      • Philipova R.
      • Whitaker M.
      ). Surprisingly, the mutant also prevented endogenous ERK from forming the complex. These results were interpreted as demonstrating the formation of homodimers. It is important to note that the presence of cytoplasmic extracts was required to form the 80-kDa complex and that it was observed only after addition of the reducing agent β-mercaptoethanol to reduce higher molecular weight complexes (
      • Philipova R.
      • Whitaker M.
      ). Therefore, although it is clear that the dimerization mutant is impeded in its ability to form high molecular weight complexes, it is unclear whether the 80-kDa complexes represent ERK homodimers. Indeed, prior to our present work, the existence of ERK dimers in vivo was already questioned due to the lack of hetero-FRET between ERKs fused to different fluorescent proteins (
      • Burack W.R.
      • Shaw A.S.
      ). Interestingly, dimerization in vitro was demonstrated to occur best at an osmolarity higher than that of living cells, likely because of the hydrophobic nature of the interface (
      • Wilsbacher J.L.
      • Juang Y.C.
      • Khokhlatchev A.V.
      • Gallagher E.
      • Binns D.
      • Goldsmith E.J.
      • Cobb M.H.
      ). We have not detected any differences in aggregation state between ERK1-WT and ERK1-Δ4, using both FCS and emFRET measurements in live cells. Our experimental conditions were optimal because all ERK1 molecules expressed were tagged with GFP, and the concentration of GFP-ERKs was in the recently determined range of endogenous ERK expression (
      • Fujioka A.
      • Terai K.
      • Itoh R.E.
      • Aoki K.
      • Nakamura T.
      • Kuroda S.
      • Nishida E.
      • Matsuda M.
      ). Our results demonstrate that ERK1-Δ4 activation is less efficient than that of WT ERK, possibly due to abnormal formation of the scaffold complex (
      • Wolf I.
      • Rubinfeld H.
      • Yoon S.
      • Marmor G.
      • Hanoch T.
      • Seger R.
      ,
      • Casar B.
      • Pinto A.
      • Crespo P.
      ). The ability of low levels of ERK dimerization mutants to displace endogenous ERK from scaffolding proteins such as KSR1 (
      • Casar B.
      • Pinto A.
      • Crespo P.
      ) may indicate a greater affinity of the mutants leading to a decrease in the efficiency of activation by MEK.
      It was reported recently that phosphorylation of the SPS motif of the kinase insert of ERK is necessary for ERK nuclear translocation (
      • Chuderland D.
      • Konson A.
      • Seger R.
      ). Phosphorylation of this SPS motif was shown to be uncoupled from the activating phosphorylation of the TEY motif, precluding an involvement of the SPS in the phenomenon described in this study. However, both mutants share problems with activation as follows: mutation of SPS reduces ERK activation markedly, whereas the Δ4 mutation delays ERK activation. Furthermore, SPS phosphorylation was shown to accelerate movement across the nucleopore (
      • Chuderland D.
      • Konson A.
      • Seger R.
      ), whereas ERK dimerization mutants translocate across the nucleopore normally (
      • Yazicioglu M.N.
      • Goad D.L.
      • Ranganathan A.
      • Whitehurst A.W.
      • Goldsmith E.J.
      • Cobb M.H.
      ).
      Our study demonstrates that although ERK is localized to cellular compartments, it is not immobilized. We conclude that the localization of ERK is dictated by the abundance of and affinity for anchoring proteins to which ERK binds. Furthermore, although ERK constantly shuttles between the cytoplasm and nucleus, nuclear exchange increases upon activation. The efficient activation of ERK leads to its release from cytoplasmic anchoring proteins and to rapid nuclear accumulation. Such a regulation of MEK-ERK coupling controlling signal transmission to the nucleus would potentiate a function of nuclear ERK activity in the discrimination of external signal strength.

      Acknowledgments

      The University of New Mexico Cancer Center Fluorescence Microscopy Facility received support from National Institutes of Health Grants S10 RR14668, S10 RR19287, S10 RR016918, P20 RR11830, and P30 CA118100 and from National Science Foundation Grant MCB9982161. We thank Sheli Ryan for cell culture assistance, Mary Raymond-Stintz for electron microscopy, Marta Vuckovic and Michael Schmidt for assistance with transfection and imaging, and Rainer Heintzmann for helpful discussion. We thank Deborah Rousseau and Jean-Claude Chambard for invaluable help with cell sorting.

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