Intracellular mobility and nuclear trafficking of the stress-activated kinase JNK1 are impeded by hyperosmotic stress

BBAMCR-17104; No. of pages: 12; 4C: 3, 4, 5, 6, 7, 8, 10 Biochimica et Biophysica Acta xxx (2013) xxx–xxx

Contents lists available at ScienceDirect

Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbamcr

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Mariya Misheva a, Gurpreet Kaur b,c, Kevin R.W. Ngoei a, Yvonne Y. Yeap a, Ivan H.W. Ng a,b, Kylie M. Wagstaff b, Dominic C.H. Ng a, David A. Jans b, Marie A. Bogoyevitch a,⁎ a b c

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Intracellular mobility and nuclear trafficking of the stress-activated kinase JNK1 are impeded by hyperosmotic stress

Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Victoria 3010, Australia Department of Biochemistry and Molecular Biology, Monash University, Victoria 3800, Australia European Molecular Biology Laboratory (EMBL) Australia, Australian Regenerative Medicine Institute, Monash University, Clayton, Victoria 3800, Australia

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Article history: Received 14 August 2013 Received in revised form 15 October 2013 Accepted 21 October 2013 Available online xxxx

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The c-Jun N-terminal kinases (JNKs) are a group of stress-activated protein kinases that regulate gene expression changes through specific phosphorylation of nuclear transcription factor substrates. To address the mechanisms underlying JNK nuclear entry, we employed a semi-intact cell system to demonstrate for the first time that JNK1 nuclear entry is dependent on the importin α2/β1 heterodimer and independent of importins α3, α4, β2, β3, 7 and 13. However, quantitative image analysis of JNK1 localization following exposure of cells to either arsenite or hyperosmotic stress did not indicate its nuclear accumulation. Extending our analyses to define the dynamics of nuclear trafficking of JNK1, we combined live cell imaging analyses with fluorescence recovery after photobleaching (FRAP) protocols. Subnuclear and subcytoplasmic bleaching protocols revealed the slowed movement of JNK1 in both regions in response to hyperosmotic stress. Strikingly, while movement into the nucleus of green fluorescent protein (GFP) or transport of a GFP-T-antigen fusion protein as estimated by initial rates and time to reach half-maximal recovery (t1/2) measures remained unaltered, hyperosmotic stress slowed the nuclear entry of GFP-JNK1. In contrast, arsenite exposure which did not alter the initial rates of nuclear accumulation of GFP, GFP-T-antigen or GFP-JNK1, decreased the t1/2 for nuclear accumulation of both GFP and GFPJNK1. Thus, our results challenge the paradigm of increased nuclear localization of JNK broadly in response to all forms of stress-activation and are consistent with enhanced interactions of stress-activated JNK1 with scaffold and substrate proteins throughout the nucleus and the cytosol under conditions of hyperosmotic stress. © 2013 Published by Elsevier B.V.

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Keywords: Nuclear import Sorbitol Live-imaging Fluorescence recovery after photobleaching

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1. Introduction

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The mitogen-activated protein kinases (MAPKs), a well-conserved family of proline-directed serine/threonine kinases, act within defined protein kinase cascades to direct eukaryotic cell responses to the changes in their environment [1]. In mammals, thirteen MAPKs are divided in three major subfamilies — the extracellular signal-regulated protein kinases (ERK1, ERK2, ERK3, ERK4 and ERK5) [2], the p38 MAPKs (p38α,

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Abbreviations: MAPK, mitogen-activated protein kinase; ERK, extracellular signalregulated kinase; JNK, c-Jun N-terminal kinase; Imp, importin; NLS, nuclear localization sequence; NTS, nuclear translocation signal; FRAP, fluorescence recovery after photobleaching; GFP, green fluorescent protein; GST, glutathione S-transferase; MKK, MAPK kinase; IAF, 5-iodoacetamidofluorescein; HTC cells, rat hepatoma tissue culture cells; CLSM, confocal laser scanning microscopy; TR70, Texas Red-labeled dextran; MEFs, murine embryonic fibroblasts; p45, nuclear factor erythroid 2 (NF-E2); TAg, Simian Virus SV40 large tumor antigen; Fn, nuclear fluorescence; Fc, cytoplasmic fluorescence; Fn/c, the nuclear to cytoplasmic ratio; t1/2, time to reach half-maximal recovery; SEM, standard error of the mean ⁎ Corresponding author at: Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, 30 Flemington Road, Parkville, Victoria 3010, Australia. Tel.: +61 3 8344 2289; fax: +61 3 9348 1421. E-mail address: [email protected] (M.A. Bogoyevitch).

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p38β, p38γ, and p38δ) [3] and the c-Jun N-terminal kinases (JNK1, JNK2 and JNK3) [4]. Each of these MAPK subfamilies can target the phosphorylation of specific substrate proteins, and the MAPK-mediated phosphorylation of transcription factors notably drives altered gene expression patterns in response to a diverse range of environmental and important developmental cues [1]. Access to the nucleus and nuclear resident transcription factors is typically dependent on members of the importin (Imp) superfamily of carrier proteins. Through their recognition of a nuclear localization sequence (NLS) within the cargo protein, Imps mediate movement of the NLS-containing cargo across the nuclear envelope via the nuclear pore complex [5–7]. This canonical pathway for nuclear entry has been demonstrated for ERK5, with two canonical NLSs defined within its C-terminal non-kinase domain [8]. In contrast, ERK1/2 can enter the nucleus by both carrier-dependent and -independent mechanisms [9]. In addition, ERK1/2 can bind and phosphorylate a number of nucleoporins that make up the nuclear pore complex, thus indicating again the possibility of carrier-independent ERK1/2 nuclear translocation as well as a more direct role for ERK signaling in the regulation of the nucleo-cytoplasmic transport machinery [10,11]. Intriguingly, ERK1/2 can also be translocated into the nucleus following phosphorylation

0167-4889/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.bbamcr.2013.10.017

Please cite this article as: M. Misheva, et al., Intracellular mobility and nuclear trafficking of the stress-activated kinase JNK1 are impeded by hyperosmotic stress, Biochim. Biophys. Acta (2013), http://dx.doi.org/10.1016/j.bbamcr.2013.10.017

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2.1. In vitro nuclear import assay

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The preparation and purification of active full-length JNK1α1 as a glutathione S-transferase (GST) fusion protein using the Sf9 cell/baculovirus system co-expressing active forms of the upstream MAPK kinases (MKKs), MKK4 and MKK7, have been described [20]. GST was removed by cleavage with PreScission Protease (GE Healthcare) prior to further use. Purified proteins were labeled with 5-iodoacetamidofluorescein (IAF; Molecular Probes) as previously [21,22]. Briefly, following buffer exchange of purified JNK1 (50 mM Tris (pH 7.0), 150 mM NaCl, 1 mM EDTA), this protein was mixed (9:1) with 4 mg/ml IAF in 250 mM Bicine (pH = 7.8), the pH readjusted to 7.8, and the mixture incubated (90 min, 25 °C, in the dark). The reaction was stopped by the addition of 2-mercaptoethanol, unreacted IAF was removed from the protein solution via size exclusion chromatography (PD-10 column, GE Healthcare), and the final IAF-JNK1 concentrated using an Amicon-30 filtration device. Mechanically perforated rat hepatoma tissue culture (HTC) cells were used to assess the nuclear import of IAF-JNK1 by confocal laser scanning microscopy (CLSM), as previously [22–24]. Incubations (5 μl total volume) included 1 μM IAF-JNK1, 70 kDa Texas Red-labeled dextran (TR70; Molecular Probes, Invitrogen) as a control to assess nuclear integrity, cytosol (untreated reticulocyte lysate, Promega) or bovine serum albumin (45 mg/ml), and an ATP regenerating system (0.125 mg/ml creatinine kinase, 30 mM creatinine phosphate, 2 mM ATP). Where indicated, antibodies specific to different Imps [Imps α2, α3, α4, and β1 (Abcam), β2 (transportin; BD Biosciences), β3 (Santa Cruz), 7 (Imgenex) and 13 (T. Tao, Xiamen University)] were used for inhibition experiments. In certain experiments, CHAPS (0.025%) which permeabilizes the nuclear envelope, was included [23,25], while IAF-labeled nuclear factor erythroid 2 (NF-E2 or p45) was included as a control for the impact of Imp7 inhibition and GFP-TAg was included as a control for the impact of inhibition of Imps α2, α3, α4, and β1. Cells were imaged by CLSM (Bio Rad MRC-500) using a 60× oil objective and image analysis of digitized confocal images performed using the ImageJ 1.38 public domain software (National Institutes of

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2.2. Cell culture and transfection, stress treatments, cell lysis and immunoblotting

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HeLa cells or JNK1/2−/− murine embryonic fibroblasts (MEFs) were maintained in high glucose Dulbecco's modified Eagle's medium containing L-glutamine (Gibco) supplemented with 10% [v/v] fetal calf serum (DKSH Australia) and 100 U/ml penicillin/streptomycin (Gibco). Where indicated, transient transfection studies were carried out with GFP-tagged JNK1α1 (pEGFP-C3-JNK1α1) using Lipofectamine™ LTX with Plus™ reagent and Opti-MEM according to the manufacturer's instructions (Invitrogen). Control studies included experiments on cells expressing GFP alone, or GFP-fusion to SV40 large tumor antigen (TAg) residues 114–135, including the Impα/β1-recognized NLS (GFP-TAg) [26], to track the impact of hyperosmotic stress (0.5 M sorbitol) or sodium meta-arsenite (NaAsO2) treatment (0.5 mM) on diffusion through the nuclear pore or Impα/β-dependent nuclear import, respectively. In selected studies, HeLa cells were pretreated with 20 μM JNK inhibitor VIII (Calbiochem) for 1 h prior to treatment or transfected with ON-TARGETPlus human JNK1 siRNA pool, JNK2 siRNA pool or nontargeting siRNA (Dharmacon) using Lipofectamine™ 2000 (Invitrogen) and antibiotic-free Opti-MEM media (Invitrogen) according to the manufacturer's specifications for 48 h. Cells were exposed to 0.5 M sorbitol (Ajax FineChem) or 0.5 mM NaAsO2 (Sigma) for 0–120 min, prior to the preparation of lysates in RIPA buffer (50 mM Tris–HCl, pH 7.3, 150 mM NaCl, 0.1 mM EDTA, 1% [v/v] sodium deoxycholate, 1% [v/v] Triton X-100, 0.2% [w/v] NaF and 100 μM Na3VO4) supplemented with complete protease inhibitor mix (Roche Diagnostic). Proteins were resolved by SDS-PAGE, transferred to PVDF membranes (Amersham Life Science), and then subjected to Western analysis using the following primary antibodies: anti-JNK1/2 (BD Pharmingen #554285, 1:1000), anti-phospho-JNK1/2 (BD Biosciences #612540, 1:1000), anti-c-Jun (Cell Signaling #9165, 1:1000), anti-phosphoSer63 c-Jun (Cell Signaling #9261, 1:1000) and anti-αtubulin (Santa Cruz sc-5286, 1:2000). After incubation with horseradish peroxidase-linked secondary antibody (Thermo Scientific), immunoreactive proteins were visualized using an enhanced chemiluminescence detection system (Thermo Scientific).

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2.3. Immunofluorescence and confocal laser scanning microscopy

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Treated cells on coverslips were washed with cold phosphatebuffered saline (PBS) before fixation using 4% paraformaldehyde (15 min, 37 °C) and permeabilization in 0.2% Triton X-100/PBS (10 min, 37 °C). Non-specific binding was blocked by incubation in 1% [w/v] bovine serum albumin/PBS (30 min, room temperature), then coverslips incubated with primary antibodies [anti-JNK1/2 (BD Pharmingen #554285, 1:250 dilution), anti-phospho(Ser63) c-Jun (Cell Signaling #9261, 1:200 dilution)], in sterile-filtered 1% bovine serum albumin/PBS then washed with PBS before incubation with Cy2-/Cy3-conjugated secondary antibodies (Millipore). Nuclei were visualized using 4′,6-diamidino-2-phenylindole (DAPI; Sigma, 1:15000 in PBS). Coverslips were mounted (Biomeda Gel Mount, ProSciTech) onto glass slides and CLSM performed (Leica TCS SP2 imaging system) with a 100 × 1.35 NA objective. Quantitation of confocal images was carried out using the ImageJ 1.38 public domain software as per Section 2.1 above, by measuring an area in the nucleus and cytoplasm of cells in 10 different fields from 3 independent experiments (n = 3), with the Fn/c calculated as per Section 2.1 above.

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Health, USA). The nuclear to cytoplasmic ratio (Fn/c) was calculated from the values for the nuclear (Fn) and cytoplasmic (Fc) fluorescence subsequent to subtraction of autofluorescence, as previously [24]. Exponential curve fitting was used to estimate the Fn/cmax, the maximal level of nuclear accumulation, and t1/2, the half-time required to reach maximal accumulation as previously [24].

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within a non-canonical nuclear translocation signal (NTS: 244Ser–Pro– Ser246 in the ERK2 kinase insert domain) that facilitates interaction with Imp7 [12,13]. NTS-mediated transport of ERK1/2 raises questions regarding possible mechanisms for nuclear entry of other MAPKs such as the p38 MAPKs and the JNKs. Whereas p38 MAPKs do not contain recognized sequences corresponding to the NLS or NTS and appear to follow microtubule-dependent nuclear entry [14], an NTS motif has been suggested for JNKs [12], but site-directed mutagenesis thereof resulted in JNK1 localization in both the nucleus and the cytoplasm [15], implying that JNK1 nuclear entry relies on other sequences and mechanisms. Indeed, although JNKs are largely considered as cytosolic kinases, there have been reports of constitutive association with the nuclear pore complex [16] and JNK relocalization to the nucleus [17], mitochondria [18] or ribosomes [19] in a stress-dependent manner. Despite these observations, little is known about the mechanisms regulating JNK localization including its nuclear translocation. Here we examine the mechanism of JNK1α1 nuclear import in detail, demonstrating for the first time the requirement for Impα2/ Impβ1. Stress-stimulated global translocation of endogenous JNKs or ectopically expressed JNK1 could not be observed by standard confocal microscopy of fixed cells, but live imaging and fluorescence recovery after photobleaching (FRAP) protocols for GFP-JNK1 revealed striking decreases in its intranuclear and intracytoplasmic mobilities following cell exposure to hyperosmotic stress. Importantly, our results challenge the paradigm of stress-induced enhancement of JNK1 nuclear entry as the mechanism underlying altered phosphorylation of key nuclear substrate proteins such as c-Jun.

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Please cite this article as: M. Misheva, et al., Intracellular mobility and nuclear trafficking of the stress-activated kinase JNK1 are impeded by hyperosmotic stress, Biochim. Biophys. Acta (2013), http://dx.doi.org/10.1016/j.bbamcr.2013.10.017

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HeLa cells were seeded into 35 mm FluoroDishes (World Precision Instruments), and transiently transfected with GFP-JNK1 plasmid DNA (4 μg) using Lipofectamine 2000 (Invitrogen) and antibiotic-free OptiMEM medium (Invitrogen) according to the manufacturer's protocol. Cells were visualized 24 h post-transfection using an Olympus FluoView 1000 confocal laser scanning microscope (60× oil immersion lens). All FRAP experiments were performed with cells maintained in growth medium at 37 °C with 5% CO2 supplied in the microscope chamber. Three prebleach images were collected using 1.5% total laser power with excitation at 488 nm prior to photobleaching. A region of interest in the nucleus or cytosol was then bleached for 2 s with the laser power at 100%. To assess intranuclear mobility, we bleached a small region of the nucleus excluding the nucleoli, and followed recovery into this area from the surrounding nuclear region [23,25]. To assess cytoplasmic mobility, we bleached a small region of the cytoplasm and followed recovery into Texas Red-dextran

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this area from the surrounding cytoplasm. Finally, to assess the rate of import from cytoplasm to nucleus, we bleached a large region of the nucleus, and also followed the loss of fluorescence in the cytoplasm [23,25]. Background fluorescence was measured by quantifying a region outside the cell, and subtracted from all fluorescence values. After bleaching, the cells were immediately scanned and the recovery of fluorescence monitored by acquiring subsequent images at 5 s intervals for 6 min using detector and laser settings identical to those prior to photobleaching [23,25]. Fluorescence in the region of interest (nuclear or cytoplasm, bleached or non-bleached region) was quantitated using ImageJ software. Fractional recovery of fluorescence was expressed as a percent relative to the prebleached area, plotted against postbleach recovery time, and the resulting data fitted exponentially as described [27]. This analysis allowed the estimation of the time to reach half-maximal recovery (t1/2), as well as calculation of the initial rate of recovery of fluorescence; for pooled data, fractional recovery was corrected for post-bleach fluorescence levels.

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Fig. 1. JNK1 nuclear transport is importin-α2/importin-β1-dependent. Mechanically perforated HTC cells were incubated in the presence an ATP regenerating system with 70 kDa Texas Red dextran (TR70) to assess nuclear integrity and IAF-JNK1 protein to assess JNK1 nuclear transport. (A, B) Incubations were conducted in the presence or absence of cytosolic extract (rabbit reticulocyte lysate) as indicated. (A) Cells (shown here at 15–20 min) were imaged by confocal laser scanning microscopy (CLSM) and (B) quantitated nuclear (Fn) and cytoplasmic (Fc) fluorescence used to calculate the nuclear to cytoplasmic ratio (Fn/c); results represent the mean ± SEM from a single typical experiment, where each data point represents N3 separate measurements for Fn and Fc above background. Curves were fitted to the function: Fn/c = Fn/cmax(1−e−kt), where Fn/cmax is the maximal nuclear accumulation, k the rate constant and t time in min; see Supplementary Table 1 for pooled data. The panel on the right denotes results for steady state nuclear accumulation in the presence of the detergent CHAPS through binding to nuclear components; results are for the mean ± SEM (n = 3), with asterisks denoting a significant difference (p = 0.0035) between TR70 and JNK1 in the presence of CHAPS. (C, D) To determine the contribution of specific importins (Imps) to JNK1 nuclear uptake, assays as above in the presence of cytosolic extract were supplemented with the indicated specific antibodies, with IAF-p45 as a control. Statistically (Student's t-test) significant differences between antibody-treated and control (−) incubations are denoted by the p values. (C) Cells for no antibody addition (−) together with samples in the presence of specific antibodies as per (A) above. (D) Pooled results for Fn/cmax from experiments as per C for the various antibody additions for IAF-JNK1 (left) and IAF-p45 (right). Pooled data for % maximal nuclear accumulation (mean ± SEM: n N 2) were compared to the respective control in the absence of antibody.

Please cite this article as: M. Misheva, et al., Intracellular mobility and nuclear trafficking of the stress-activated kinase JNK1 are impeded by hyperosmotic stress, Biochim. Biophys. Acta (2013), http://dx.doi.org/10.1016/j.bbamcr.2013.10.017

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Statistical analysis was carried out using GraphPad Prism 5 software (Windows version 5.00, GraphPad Software), with the unpaired Student t-test used to compare data from control cells versus treated cells at each corresponding time point. Data are shown as the mean ± standard error of the mean (SEM), with p-values b 0.05 considered statistically significant.

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3. Results

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3.1. Nuclear entry of JNK1α1 is dependent on Impα2/β1

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We first addressed the mechanisms contributing to JNK1α1 nuclear entry using an established nuclear transport assay in combination with CLSM [24]. Specifically, using semi-intact HTC cells, we examined the nuclear import kinetics of IAF-labeled JNK1α1, alongside IAF-labeled p45 NF-E2 protein [24] as a positive control, with the integrity of the nuclear envelope monitored by exclusion of a 70 kDa Texas Red-labeled dextran (TR70). In the presence of exogenously added cytosol, JNK1α1

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showed robust and rapid nuclear accumulation (Fig. 1A), quantitative analysis indicating over 2.5 times higher levels in the nucleus than in the cytoplasm, as denoted by the nuclear to cytoplasmic ratio (Fn/c) of c. 2.6, with half-maximal accumulation attained in less than 4 min (3.7 ± 1, n = 4). That nuclear accumulation was dependent on cytosolic factors was indicated by accumulation that was significantly (p = 0.0043) reduced in the absence of cytosol (Fn/cmax of 1.4) (Fig. 1B, left panel). We also performed analysis in the presence of the detergent CHAPS, which disrupts the nuclear envelope; under these conditions, nuclear accumulation is only possible through binding to nuclear components (Fig. 1B, right panel). In contrast to the control molecule TR70, which showed equilibration between nuclear and cytoplasmic compartments (Fn/c of 1), JNK1α1 exhibited significant levels (p = 0.0035) of nuclear accumulation (an Fn/c of 1.5), underlining that JNK1α1 nuclear accumulation is in part through binding to nuclear factors. The dependence of nuclear accumulation of JNK1α1 on cytosolic factors was further assessed in this system by including specific antiimportin antibodies. Representative experiments are shown in Fig. 1C (see Supplementary Fig. 1 for the time course of study), with pooled

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Q11 Fig. 2. Endogenous JNKs phosphorylate nuclear c-Jun without altered nuclear-cytoplasmic distribution following stress exposure. HeLa cells were exposed to 0.5 mM NaAsO2 (arsenite) or 0.5 M sorbitol for 0–120 min as indicated. (A) Lysates were subjected to immunoblot analysis for activated JNK (p-JNK1/2), total JNK1/2, activated c-Jun (pS63-c-Jun), total c-Jun, or α-tubulin to indicate equivalent protein loading. (B–E) Confocal laser scanning microscopy (CLSM) analysis of cells immunostained for JNK1/2 and/or activated c-Jun (pS63-c-Jun) and costained with DAPI to indicate cell nuclei. Bars in (B), (C) and (E) represent 10 μm. (B) Pretreatment of cells with JNK1 + JNK2 siRNA, scrambled siRNA, or no siRNA (−) for 48 h prior to staining using a JNK1/2 antibody indicates detection specificity for JNK1/2. (C, D) Cells were exposed to 0.5 mM arsenite (NaAsO2) or 0.5 M sorbitol for 0–120 min before staining using the JNK1/2 antibody. (C) Cells were imaged by CLSM and (D) quantitated nuclear and cytoplasmic fluorescence for JNK was used to calculate Fn/c. Comparison of the results (mean ± SEM) in 3 independent experiments indicated no significant differences in JNK localization under the conditions evaluated. (E) Cells were preincubated with vehicle (DMSO) or JNK inhibitor VIII (20 μM) for 30 min prior to exposure for 60 min to 0.5 mM arsenite (NaAsO2) or 0.5 M sorbitol as indicated, before staining using the anti-JNK1/2 and -pS63-c-Jun antibodies as well as DAPI.

Please cite this article as: M. Misheva, et al., Intracellular mobility and nuclear trafficking of the stress-activated kinase JNK1 are impeded by hyperosmotic stress, Biochim. Biophys. Acta (2013), http://dx.doi.org/10.1016/j.bbamcr.2013.10.017

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Fig. 2 (continued).

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3.2. Stress activation does not alter JNK subcellular localization

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Stress activation of JNKs, as defined by the increased dualphosphorylation of their activation loop residues, is now widely appreciated [1,4], but detailed evaluation of how stress impacts on JNK localization under these conditions has not been performed. We decided to examine two stress pathways, treating cells with 0.5 mM sodium

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quantitative data in Fig. 1D. JNK1α1 nuclear accumulation was significantly reduced (p b 0.0001) in the presence of antibodies to importinα2 or -β1 (maximal level of nuclear accumulation ≤ 1) but not to various other importins (Fig. 1C & D). In contrast, nuclear accumulation of p45 NF-E2 was inhibited by antibodies to importin-7 (Fn/c of 1.1 compared to 2.2 in their absence; p = 0.0396), but unaffected by antibodies to importin-β1 (Fn/c of 2) (Fig. 1C & D) and nuclear accumulation of GFP-TAg was inhibited by antibodies to importin-α2, -α3 or -α4 or -β1 (see Supplementary Fig. 2). Taken together, these results indicate that JNK1α1 nuclear import is dependent on importin-α2/β1.

meta-arsenite (NaAsO2) that is associated with increased oxidative stress [28–31], or 0.5 M sorbitol, that induces hyperosmotic stress [32,33]. Both agents caused substantial and prolonged phosphorylation of JNK1/2 in HeLa cells; that this was of functional significance was indicated by the altered electrophoretic mobility/increased levels of phosphorylated c-Jun (pSer63 c-Jun) that is a direct JNK substrate (Fig. 2A). To examine how these stresses impact on localization of endogenous JNK1/2, we first validated the JNK1/2 antibody for use in immunostaining/CLSM protocols, demonstrating the loss of signal upon siRNA-mediated knockdown of JNK1/2 (Fig. 2B). When using this antibody to detect JNK1/2 during HeLa cell exposure to arsenite or sorbitol-induced stress, we observed no substantial changes in JNK1/2 localization over the 120 min time course (Fig. 2C & D). The strong links between JNK1/2 activation and c-Jun phosphorylation were confirmed in parallel experiments in which no changes in JNK1/2 localization could be observed, despite substantial increases in levels of pSer63 c-Jun (Fig. 2E, left hand panels). Importantly, the inclusion of the JNK inhibitor VIII in these incubations showed the absolute

Please cite this article as: M. Misheva, et al., Intracellular mobility and nuclear trafficking of the stress-activated kinase JNK1 are impeded by hyperosmotic stress, Biochim. Biophys. Acta (2013), http://dx.doi.org/10.1016/j.bbamcr.2013.10.017

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3.3. Live cell imaging/fluorescence recovery after photobleaching (FRAP) reveals that hyperosmotic stress slows JNK1 intra-nuclear and -cytoplasmic dynamics

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To probe the stress regulation of JNK in live cells, we firstly validated the use of a GFP-JNK1 fusion protein. Notably, with the size difference created by fusion to GFP protein, the regulation of GFP-JNK1 could be evaluated in parallel to that of endogenous JNK1/2 in transfected HeLa cells (Supplementary Fig. 3A). Indeed, GFP-JNK1 expressed in HeLa cells showed increased phosphorylation during arsenite and sorbitol exposure, which paralleled changes in the phosphorylation of endogenous JNKs (Supplementary Fig. 3A). Despite these changes, no marked alteration in the localization of GFP-JNK1 following exposure to arsenite or sorbitol was observed. Specifically, GFP-JNK1 was detected throughout the cell nuclei and cytoplasm under control conditions (when the pJNK1/2 antibody did not show any presence of phosphorylated JNK species) as well as during stress (Supplementary Fig. 3B). Importantly, GFP-JNK1 could drive c-Jun phosphorylation in JNK1/2−/− MEFs

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(Supplementary Fig. 3C). Taken together, these results confirm that endogenous JNKs and GFP-JNK1 are phosphorylated and can mediate c-Jun phosphorylation in the absence of global changes in their localization. Live cell imaging coupled with FRAP analysis allowed the evaluation of the kinetics of movement of GFP-JNK1 in the nucleus and the cytoplasm. We first evaluated the movement of GFP-JNK1 within the nucleus by bleaching a small region of the nucleus and measuring the recovery of fluorescence into this bleached area of the nucleus (Fn) and the decay of fluorescence in a non-bleached region of the nucleus (Fn non-bleached) over the ensuing 350 s postbleach (Fig. 3). As indicated for representative cells in Fig. 3Ai, GFP-JNK1 was exchanged within the nucleus under both basal and the hyperosmotic stress conditions (0.5 M sorbitol, 30 min or 60 min). Thus, as shown in Fig. 3Aii, Fn increased and Fn non-bleached decreased under all conditions tested, indicating movement of GFP-JNK1 driving fluorescence recovery within the original bleached area of the nucleus. Evaluation of the fitted exponential curve for recovery of Fn indicated a small but statistically significant c. 10% increase in the maximal extent of recovery after 30 min of hyperosmotic stress (Fig. 3B). In this situation, the failure to recover 100% fluorescence is indicative of a GFP-JNK1 fraction in the nucleus

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Fig. 3. Hyperosmotic stress slows GFP-JNK1 exchange within the nucleus. HeLa cells were transfected to express GFP-JNK1α1 then exposed to 0.5 M sorbitol for 0–60 min as indicated. A fluorescence recovery after photobleaching (FRAP) protocol with a subnuclear bleach area and measuring recovery into the bleached area (Fn) alongside the loss of fluorescence from an equivalent separate area in the same nucleus (Fn non-bleached) was utilized. (Ai) Visualization of the recovery of nuclear GFP-JNK1 in representative HeLa cells after photobleaching. Each bleached area is indicated with the red circle labeled Fn. (Aii) Recovery of nuclear fluorescence for GFP-JNK1 under basal and stress conditions over time for the representative cells displayed in (Ai). The nuclear fluorescence in the bleached area (Fn) and the fluorescence for a non-bleached region in the nucleus after background subtraction (Fn non-bleached) were plotted against time (upper panels). The percent maximal recovery of fluorescence in the bleached area was determined using fractional recovery of Fn (Fn at respective time points divided by the prebleach Fn value representing 100%) and for this data curve-fitting was applied to determine quantitative parameters defining the extent and rate of recovery (lower panels). Quantitative data analysis was undertaken to calculate (B) % recovery of fluorescence for GFP-JNK1 in the bleached area of the nucleus and (C) the time to reach half-maximal recovery of GFP-JNK1 in the bleached area (t1/2). (B, C) The results represent the mean ± SEM (8 cells per time point scored, typical results from 3 separate experiments).

Please cite this article as: M. Misheva, et al., Intracellular mobility and nuclear trafficking of the stress-activated kinase JNK1 are impeded by hyperosmotic stress, Biochim. Biophys. Acta (2013), http://dx.doi.org/10.1016/j.bbamcr.2013.10.017

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The power of FRAP is to reveal dynamic information that is not possible using fixed cell preparations. We extended our FRAP analysis to evaluate transport from the cytoplasm to the nucleus as previously [27,34–36] by bleaching a large nuclear region, and measuring the recovery of fluorescence in the bleached area of the nucleus (Fn) in

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cytoplasm. Evaluation of the fitted exponential curve for recovery of Fc indicated a c. 20% significant decrease (p b 0.05) in recovery under conditions of hyperosmotic stress (Fig. 4B). In this situation, the failure to recover 100% fluorescence is indicative of a GFP-JNK1 fraction in the cytoplasm (up to ~20% in the cytoplasm under control conditions that increases to ~45% under hyperosmotic stress conditions) that cannot be replenished by the mobile GFP-JNK1 fraction. The t1/2 was found to increase significantly (p b 0.05), up to almost 10-fold for hyperosmotic treatment (Fig. 4C). Thus, in addition to slowing intranuclear GFP-JNK1 movement, hyperosmotic stress significantly decreases the mobile population of GFP-JNK1 in the cytoplasm, as well as slowing its movement within it.

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(up to ~35% in the nucleus under control conditions that decreases to ~20% under hyperosmotic stress conditions) that cannot be replenished by the mobile GFP-JNK1 fraction, in keeping with the observations from Fig. 1B (right) indicating the ability of JNK1 ability to accumulate in the nucleus through binding to nuclear components. The time to reach half maximal recovery (t1/2) was also determined and found to increase significantly (p b 0.05) for hyperosmotic treatment (Fig. 3C); in the case of 30 min treatment with sorbitol, the t1/2 was increased over 2.5-fold, compared to in the absence of treatment. Thus, despite a slightly larger fraction of mobile GFP-JNK1, hyperosmotic stress significantly slowed intranuclear movement of GFP-JNK1. We next evaluated the movement of GFP-JNK1 within the cytoplasm, bleaching a small region in the cytoplasm, and then monitoring the recovery of fluorescence in the bleached area of the cytosol (Fc) as well as the decay of fluorescence in a non-bleached region of the cytoplasm (Fc non-bleached) over the ensuing 350 s (Fig. 4). As indicated for representative cells in Fig. 4Ai, GFP-JNK1 was exchanged within the cytoplasm under both basal and the hyperosmotic stress conditions. As shown in Fig. 4Aii, Fc increased, and Fc non-bleached decreased under all tested conditions, indicating movement of GFP-JNK1 driving fluorescence recovery within the original bleached area of the

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Fig. 4. Hyperosmotic stress slows GFP-JNK1 exchange within the cytoplasm. HeLa cells were transfected to express GFP-JNK1α1 and then exposed to 0.5 M sorbitol for 0–60 min as indicated. A FRAP protocol with a small cytoplasmic bleach area and measuring recovery into the bleached area (Fc) alongside the loss of fluorescence from an equivalent separate area in the same cell cytoplasm (Fc non-bleach) was utilized. (Ai) Visualization of the recovery of nuclear GFP-JNK1 in representative HeLa cells after photobleaching. Each bleached area is indicated with the red circle labeled Fc. (Aii) Recovery of cytoplasmic fluorescence for GFP-JNK1 under basal and stress conditions over time for the representative cells displayed in (Ai). The cytoplasmic fluorescence in the bleached area (Fc) and the fluorescence for a non-bleached region in the cytoplasm after background subtraction (Fc non-bleach) were plotted against time (upper panels). Using the same approaches as described in the legend for Fig. 3, the percent maximal recovery of fluorescence in the bleached area was determined using fractional recovery of Fc (Fc at respective time points divided by the prebleach Fc value representing 100%) (lower panels). Quantitative data analysis was undertaken to calculate (B) % recovery of fluorescence for GFP-JNK1 in the bleached area of the cytoplasm and (C) t1/2 for GFP-JNK1 in the bleached area of the cytoplasm. (B, C) The results represent the mean ± SEM (8 cells per time point scored, typical results from 3 separate experiments).

Please cite this article as: M. Misheva, et al., Intracellular mobility and nuclear trafficking of the stress-activated kinase JNK1 are impeded by hyperosmotic stress, Biochim. Biophys. Acta (2013), http://dx.doi.org/10.1016/j.bbamcr.2013.10.017

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Finally, we extended our FRAP analysis to consider how stress in- 426 duced by exposure to arsenite might impact on JNK1 trafficking. Thus, 427

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mediated nuclear entry in general, we performed similar FRAP measurements for GFP alone and the model importin-α/β cargo, GFP-TAg, respectively. As indicated for representative cells in Supplementary Fig. 4A, GFP exchanged between the cytoplasm and the nucleus under both basal and the hyperosmotic stress conditions, indicating that GFP diffuses freely between the nucleus and cytosol even under stress conditions. Quantitative analysis showed unaltered % recovery, initial rate of recovery or time to half-maximal recovery for GFP under hyperosmotic conditions (Fig. 6A–C, respectively). Similarly, as indicated for representative cells in Supplementary Fig. 4B, GFP-TAg was exchanged between the cytoplasm and the nucleus under both basal and the hyperosmotic stress conditions. Quantitative analysis showed unaltered % recovery, initial rate of recovery and time to half-maximal recovery for GFP-TAg under hyperosmotic conditions (Fig. 6D–F, respectively). Thus, hyperosmotic stress did not impact on either diffusion through the nuclear pore (as exemplified by GFP) or importinmediated nuclear transport (as exemplified by GFP-TAg). The quantitative data for GFP-JNK1, GFP and GFP-TAg are summarized in Table 1, further supporting the idea that the effects of hyperosmotic stress on nuclear import are specific to JNK1.

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parallel with the decay of fluorescence in a non-bleached region of the cytosol (Fc) over the ensuing 350 s (Fig. 5). As indicated for representative cells in Fig. 5Ai, GFP-JNK1 was exchanged between the cytoplasm and the nucleus under both basal and the hyperosmotic stress conditions. Fn increased and Fc decreased under all conditions (Fig. 5Aii), indicative of JNK movement into the nucleus under control and stress conditions to drive the recovery of fluorescence in the original nuclear bleached area. Evaluation of the fitted exponential curve for recovery of Fn indicated values of around 50%, with no significant changes in recovery during either the absence or presence of hyperosmotic stress (Fig. 5B). The time to reach half maximal recovery (t1/2), however, was observed to increase significantly (p b 0.05; over 3-fold) for the more prolonged hyperosmotic treatment (60 min 0.5 M sorbitol) (Fig. 5Ci). Additionally, estimations of the initial rates of GFP-JNK1 recovery in the nucleus revealed significantly (p b 0.05) slower initial rates during hyperosmotic stress (Fig. 5Cii; in the case of 60 min treatment with sorbitol, the rate was c. 6-fold slower). Thus, in contrast to the expectation that exposure to stress may enhance nuclear entry of a stress-activated kinase such as JNK, these analyses reveal clearly for the first time that nuclear entry of JNK1 is slowed during hyperosmotic stress. This slowed nuclear import is consistent with a greater fraction of JNK1 being immobile in the cytoplasm under stress conditions (Fig. 4B) and the slowed cytoplasmic mobility of JNK also under these stress conditions (Fig. 4C). To eliminate the possibility that hyperosmotic stress might impact on either diffusion through the nuclear pore complex or importin-

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Fig. 5. Hyperosmotic stress slows GFP-JNK1 transport into the nucleus. HeLa cells were transfected to express GFP-JNK1α1 then exposed to 0.5 M sorbitol for 0–60 min as indicated. A FRAP protocol with a large nuclear bleach area and measuring recovery into the bleached area (Fn) alongside the loss of fluorescence from the cytoplasm of the same cell (Fc) was utilized. (Ai) Visualization of the recovery of nuclear GFP-JNK1 in representative HeLa cells after photobleaching. Each bleached area is indicated with the red circle labeled Fn. (Aii) Recovery of nuclear fluorescence for GFP-JNK1 under basal and stress conditions over time for the representative cells displayed in (Ai). The nuclear fluorescence in the bleached area (Fn) and the fluorescence for a non-bleached region in the cytoplasm after background subtraction (Fc) were plotted against time (upper panels). Using the same approaches as described in the legend for Fig. 3, the percent maximal recovery of fluorescence in the bleached area was determined using fractional recovery of Fn (Fn at the respective time points divided by the prebleach Fn value representing 100%) (lower panels). Quantitative data analysis was undertaken to calculate (B) % recovery of fluorescence for GFP-JNK1 in the bleached area of the cytoplasm, (Ci) t1/2 for GFP-JNK1 in the bleached area of the cytoplasm, and (Cii) the initial rate for recovery of GFP-JNK1 in the bleached area of the nucleus. (B, C) The results represent the mean ± SEM (8 cells per time point scored, typical results from 3 separate experiments).

Please cite this article as: M. Misheva, et al., Intracellular mobility and nuclear trafficking of the stress-activated kinase JNK1 are impeded by hyperosmotic stress, Biochim. Biophys. Acta (2013), http://dx.doi.org/10.1016/j.bbamcr.2013.10.017

M. Misheva et al. / Biochimica et Biophysica Acta xxx (2013) xxx–xxx

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Fig. 6. Hyperosmotic stress does not significantly alter GFP or GFP-TAg nuclear entry. HeLa cells were transfected to express GFP or GFP-TAg and then exposed to 0.5 M sorbitol for 0–60 min as indicated. A FRAP protocol was performed as per the legend to Fig. 5. Results of typical cells are shown in Supplementary Fig. 4, with quantitative analysis performed as described in the legend to Fig. 5 to calculate (A, D) % recovery of fluorescence for GFP and GFP-TAg, respectively, in the bleached area of the cytoplasm, (B, E) the initial rate for recovery of and GFP-TAg, respectively, in the bleached area of the nucleus and (C, F) t1/2 for GFP and GFP-TAg, respectively, in the bleached area of the cytoplasm. All results represent the mean ± SEM (6 cells per time point scored, typical results from 3 separate experiments).

t1:1 t1:2 t1:3 t1:4

Table 1 Kinetics of nuclear entry of GFP-JNK1, GFP and GFP-TAg under basal and hyperosmotic stress conditions as determined by FRAP. Pooled data (mean ± SEM, n N 6) is from experiments such as those shown in Fig. 5–7; Supplementary Fig. 3.

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we bleached a large nuclear region, and measured the recovery of fluorescence in the bleached area of the nucleus (Fn) in parallel with the decay of fluorescence in a non-bleached region of the cytosol (Fc) over the ensuing 350 s (Fig. 7). As indicated in Fig. 7A, GFP-JNK1 Fn increased and Fc decreased under all conditions (Fig. 7Aii), indicative of JNK1 movement into the nucleus under control and arsenite stress conditions to drive the recovery of fluorescence in the original nuclear bleached area. Evaluation of the fitted exponential curve for recovery of Fn showed a small but significant decrease in the % recovery upon

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60 min arsenite exposure (Fig. 7B). The time to reach half maximal recovery (t1/2), was also observed to decrease significantly (p b 0.01; approximately 50%) for the more prolonged (60 min) arsenite treatment (Fig. 7Ci), but this was not reflected in the initial rates of GFP-JNK1 recovery in the nucleus (Fig. 7Cii). This decrease in the t1/2 for GFP-JNK1 prompted analysis of GFP and GFP-TAg under these conditions, revealing a parallel decrease in the calculated t1/2 for GFP without impact on GFP-TAg (Supplementary Fig. 5).

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Stress-stimulated JNK phosphorylation and activation have been typically considered as part of a key response allowing for a rapid and enhanced phosphorylation of nuclear resident transcription factors such as c-Jun [37–39]. However, additional detailed studies have revealed an important population of nuclear resident JNK interaction with the transcription factor NF-Y [40] under non-stressed conditions, and more generally that MAPKs may be either directly or indirectly associated with chromatin under a range of different physiological conditions [41], consistent with our semi-intact cell experiments shown in Fig. 1B. Our studies here examine for the first time the impact of hyperosmotic stress on JNK localization. While standard confocal microscopy studies of the bulk JNK population did not reveal significant increases in the nuclear JNK levels following stress, our detailed analysis of

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Please cite this article as: M. Misheva, et al., Intracellular mobility and nuclear trafficking of the stress-activated kinase JNK1 are impeded by hyperosmotic stress, Biochim. Biophys. Acta (2013), http://dx.doi.org/10.1016/j.bbamcr.2013.10.017

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JNK1 in living cells enabled us to assess the extent to which it may shuttle constantly under control and stress conditions, thereby accessing its nuclear substrates without the need for substantial relocalization. Indeed, we have shown using our live-imaging/FRAP that JNK1 localization is dynamic in both the cytosol and nucleus, and is in constant flux between cytoplasm and nucleus. Notably, the exposure to hyperosmotic stress induced by 0.5 M sorbitol slows JNK1 mobility in both the cell nucleus and cytoplasm, and this is accompanied by a larger fraction of immobile cytoplasmic JNK1. Contrary to expectations arising from the definition of JNKs as stress-activated kinases, these actions in combination appear to contribute to a slowed, rather than enhanced, nuclear entry of JNK1 during hyperosmotic stress. JNK1's reduced intranuclear mobility/increased binding in the nucleus upon hyperosmotic stress is clearly linked to active nuclear phosphorylation of c-Jun, as documented in Fig. 2E. A number of previous studies have indicated that the impact of stress on nuclear transport events has previously defined changes in importin localization and the Ran gradient [42,43]. It is therefore surprising that models of the stress-activated pathways culminating in JNK activation continue to allude to enhanced JNK nuclear localization following stress [44,45]. Certainly, the notion of JNK nuclear entry appears essential for JNK-mediated phosphorylation of transcription factors such as c-Jun that are considered to be nuclear resident [37–39], unlike other transcription factors that reside constitutively in the

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Fig. 7. Impact of arsenite on GFP-JNK1 transport into the nucleus. HeLa cells were transfected to express GFP-JNK1α1 then exposed to 0.5 mM arsenite for 0–60 min as indicated. A FRAP protocol with a large nuclear bleach area and measuring recovery into the bleached area (Fn) alongside the loss of fluorescence from the cytoplasm of the same cell (Fc) was utilized. (Ai) Visualization of the recovery of nuclear GFP-JNK1 in representative HeLa cells after photobleaching. Each bleached area is indicated with the red circle labeled Fn. (Aii) Recovery of nuclear fluorescence for GFP-JNK1 under basal and stress conditions over time for the representative cells displayed in (Ai). The nuclear fluorescence in the bleached area (Fn) and the fluorescence for a non-bleached region in the cytoplasm after background subtraction (Fc) were plotted against time (upper panels). Using the same approaches as described in the legend for Fig. 3, the percent maximal recovery of fluorescence in the bleached area was determined using fractional recovery of Fn (Fn at the respective time points divided by the prebleach Fn value representing 100%) (lower panels). Quantitative data analysis was undertaken to calculate (B) % recovery of fluorescence for GFP-JNK1 in the bleached area of the cytoplasm, (Ci) t1/2 for GFP-JNK1 in the bleached area of the cytoplasm, and (Cii) the initial rate for recovery of GFP-JNK1 in the bleached area of the nucleus. (B, C) The results represent the mean ± SEM (6 cells per time point scored, typical results from 3 separate experiments).

cytoplasm and only translocate to the nucleus following their activation [46–50]. Our FRAP data indicates that there is a constant exchange between the cytosolic and nuclear fractions of JNK, even under basal conditions. Our permeabilized cell nuclear transport assays have allowed our identification of importin-α2/β1-mediated nuclear entry of JNK. Notably, there have been previous suggestions of JNK1 nuclear entry potentially mediated by a novel NTS motif/importin-7-mediated mechanism [12]. Our data, however, reinforces the previous suggestions that JNK1 entry into the nucleus is not dependent on the NTS motif [15], and we have shown that antibodies to importin-7 that inhibit the nuclear entry of p45 [24] do not significantly impact JNK1 nuclear entry. Indeed, the rates of nuclear entry evaluated in our FRAP protocols for the classical importin-α/β-dependent cargo GFP-TAg [26] and GFPJNK1 are in good agreement (as summarized in Table 1). It also remains possible that alternative nuclear import mechanisms can be utilized by JNK1 under conditions of stress. That additional importin-independent pathways may make critical contributions during some forms of stress cannot be excluded [51,52], but under the conditions of our assays, the nuclear entry of both GFP only and GFP-TAg, as judged by initial rates of nuclear entry and the time to half-maximal recoveries (t1/2) of their nuclear fluorescence, was not altered significantly during hyperosmotic stress for 30–60 min. This suggests that, in contrast to GFP/GFP-TAg, there is a substantial reduction of cytosolic JNK1 population available for nuclear import in the presence of hyperosmotic stress,

Please cite this article as: M. Misheva, et al., Intracellular mobility and nuclear trafficking of the stress-activated kinase JNK1 are impeded by hyperosmotic stress, Biochim. Biophys. Acta (2013), http://dx.doi.org/10.1016/j.bbamcr.2013.10.017

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Acknowledgements

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The authors acknowledge the support of the National Health and Medical Research Council Project Grants (566804 and 628335) and scholarship support by Melbourne and Monash Universities (Melbourne International Research Scholarship, Melbourne International Fee Remission Scholarship, Melbourne Research Scholarship, Monash Graduate Scholarship, Monash Faculty of Medicine International Postgraduate Research Scholarship). DCHN is an Australian Research Council Future Fellow (FT120100193) and DAJ is an NHMRC Senior Principal Research Fellow (#APP1002486). Prof T Tao (Xiamen University) is thanked for the anti-importin-13 antibody. We are grateful for the excellent support of Cassandra David for cell culture and the Monash Micro Imaging Facility for the live-imaging facilities.

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Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.bbamcr.2013.10.017.

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In summary, this study defines a classical importin-α2/β1-dependent pathway for the constitutive nuclear entry of the stress-activated kinase JNK1 for the first time. This nucleo-cytoplasmic shuttling of JNK1 underlies its access to nuclear substrates such as c-Jun. Paradoxically, while prolonged arsenite exposure can lower the t1/2 for JNK1 nuclear accumulation, hyperosmotic stress slows the movement of both cytoplasmic and nuclear JNK1, as well as the entry of JNK1 into the nucleus. Further evaluation of the stress-induced changes in JNK1 interactions with substrate and scaffold proteins throughout the cell will define how these partners contribute to the spatial and temporal regulation of JNK1 that influences the outcomes of this critical signal transduction pathway.

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which is consistent with the reduced mobility of the cytoplasmic JNK1 revealed in our FRAP protocols targeting the cytosolic JNK1 population. We anticipate these cytoplasmic changes are likely to reflect enhanced JNK1 binding to a range of cytosolic substrates and scaffold proteins [53]. Clearly more specific evaluation of these cytoplasmic changes is required, but a less mobile JNK population is consistent with the marked increase in ribosome-associated JNK noted recently following stress [19]. It will also be important to examine the impact of additional stresses on JNK1 nuclear trafficking. Our studies here with the cytotoxic agent arsenite also indicate that its activation of JNKs is not associated with a bulk nuclear accumulation of JNKs (Fig. 2). However, FRAP studies of GFP-JNK1, as well as GFP alone, indicated that prolonged arsenite exposure (60 min, 0.5 mM) could decrease the time to half-maximal recoveries (t1/2) in the absence of significant changes in the initial rates of their nuclear import (Fig. 7 and Supplementary Fig. 5). This emphasizes the complexities in changes associated with different cell stresses, and reinforces the need to consider more broadly how nuclear transport processes, including diffusion through the nuclear pore complex, can be altered. Notably, mechanical stress has been shown to influence nuclear transport in smooth muscle cells, with increase rates of nuclear import associated with an increased expression of proteins of the nuclear pore complex [54]. Conversely, UV irradiation, heat shock and oxidative stress have been shown to disrupt nuclear transport of conventional cargoes [42], and their impact on the nucleo-cytoplasmic trafficking of JNKs remains an important area for further investigation.

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