Regulation of nuclear translocation of extracellular signal-regulated kinase 5 by active nuclear import and export mechanisms

Extracellular signal-regulated kinase 5 (ERK5), a member of the mitogen-activated protein kinase family, plays an important role in growth factor signaling to the nucleus. However, molecular mechanisms regulating subcellular localization of ERK5 have remained unclear. Here, we show that nucleocytoplasmic shuttling of ERK5 is regulated by a bipartite nuclear localization signal-dependent nuclear import mechanism and a CRM1-dependent nuclear export mechanism. Our results show that the N-terminal half of ERK5 binds to the C-terminal half and that this binding is necessary for nuclear export of ERK5. They further show that the activating phosphorylation of ERK5 by MEK5 results in the dissociation of the binding between the N- and C-terminal halves and thus inhibits nuclear export of ERK5, causing its nuclear import. These results reveal the mechanism by which the activating phosphorylation of ERK5 induces its nuclear import and suggest a novel example of a phosphorylation-dependent control mechanism for nucleocytoplasmic shuttling of proteins.     

Nuclear export of MAP kinase (ERK) involves a MAP kinase kinase (MEK)-dependent active transport mechanism

In response to extracellular stimuli, mitogen-activated protein kinase (MAPK, also known as ERK), which localizes to the cytoplasm in quiescent cells, translocates to the nucleus and then relocalizes to the cytoplasm again. The relocalization of nuclear MAPK to the cytoplasm was not inhibited by cycloheximide, confirming that the relocalization is achieved by nuclear export, but not synthesis, of MAPK. The nuclear export of MAPK was inhibited by leptomycin B (LMB), a specific inhibitor of the nuclear export signal (NES)-dependent transport. We have then shown that MAP kinase kinase (MAPKK, also known as MEK), which mostly localizes to the cytoplasm because of its having NES, is able to shuttle between the cytoplasm and the nucleus constantly. MAPK, when injected into the nucleus, was rapidly exported from the nucleus by coinjected wild-type MAPKK, but not by the NES-disrupted MAPKK. In addition, injection of the fragment corresponding to the MAPK-binding site of MAPKK into the nucleus, which would disrupt the binding of MAPK to MAPKK in the nucleus, significantly inhibited the nuclear export of endogenous MAPK. Taken together, these results suggest that the relocalization of nuclear MAPK to the cytoplasm involves a MAPKK-dependent, active transport mechanism.     

Shuttling components of nuclear import machinery involved in nuclear translocation of steroid receptors exit nucleus via exportin-1/CRM-1* independent pathway

The nucleocytoplasmic transport processes are mediated by soluble transport factors constantly navigating between nuclear and cytoplasmic compartments. Our understanding about nuclear export of general 'nuclear import factors' that deliver the cargo to the nucleus is still fragmentary. Utilizing green fluorescent protein tagged glucocorticoid receptor (GR) and relA as our working model and with judicious use of LMB, we show in living cells that all the soluble components of the nuclear import machinery exit nucleus via exportin1/CRM1 independent pathway(s).     

Dynamics of Leptomycin B-Sensitive Nucleocytoplasmic Flux of Parathyroid Hormone-Related Protein

Parathyroid hormone-related protein is responsible for hypercalcemia induced by various tumors. The similarity of its N-terminus to that of parathyroid hormone enables parathyroid hormone-related protein to share parathyroid hormone's signaling properties, but the rest of the molecule possesses distinct functions including a role in the nucleus/nucleolus in reducing apoptosis and enhancing cell proliferation. We have previously shown that parathyroid hormone-related protein nuclear import is mediated by importin β1. Here we use fluorescence recovery after photobleaching for the first time to show that, in living cells, parathyroid hormone-related protein is exported from the nucleus in a leptomycin B-sensitive manner, implicating CRM1 as the parathyroid hormone-related protein nuclear export receptor. Leptomycin B treatment significantly reduced the rate of nuclear export 4 −10-fold, thereby increasing parathyroid hormone-related protein concentration in the nucleus/nucleolus about 2-fold. Intriguingly, this also led to a 2-fold reduced nuclear import rate. Inhibiting the nuclear export of a protein able to shuttle between nucleus and cytoplasm through distinct receptors thus can also affect nuclear import, indicating that the subcellular localization of a protein containing distinct nuclear import and export signals is the product of an integrated system. Although there have been several recent studies examining the dynamics of intranuclear transport using fluorescence recovery after photobleaching, this represents, to our knowledge, the first use of the technique to examine the kinetics of nucleocytoplasmic flux in living cells.     

Nuclear extracellular signal-regulated kinase 1 and 2 translocation is mediated by casein kinase 2 and accelerated by autophosphorylation

The extracellular signal-regulated kinases (ERK) 1 and 2 (ERK1/2) are members of the mitogen-activated protein kinase [MAPK] family. Upon stimulation, these kinases translocate from the cytoplasm to the nucleus, where they induce physiological processes such as proliferation and differentiation. The mechanism of translocation of this kinase involves phosphorylation of two Ser residues within a nuclear translocation signal (NTS), which allows binding to importin7 and a subsequent penetration via nuclear pores. Here we show that the phosphorylation of both Ser residues is mediated mainly by casein kinase 2 (CK2) and that active ERK may assist in the phosphorylation of the N-terminal Ser. We also demonstrate that the phosphorylation is dependent on the release of ERK from cytoplasmic anchoring proteins. Crystal structure of the phosphomimetic ERK revealed that the NTS phosphorylation creates an acidic patch in ERK. Our model is that in resting cells ERK is bound to cytoplasmic anchors, which prevent its NTS phosphorylation. Upon stimulation, phosphorylation of the ERK TEY domain releases ERK and allows phosphorylation of its NTS by CK2 and active ERK to generate a negatively charged patch in ERK, binding to importin 7 and nuclear translocation. These results provide an important role of CK2 in regulating nuclear ERK activities.     

Active nuclear import and export pathways regulate E2F-5 subcellular localization

Epidermal keratinocyte differentiation is accompanied by differential regulation of E2F genes, including up-regulation of E2F-5 and its concomitant association with the retinoblastoma family protein p130. This complex appears to play a role in irreversible withdrawal from the cell cycle in differentiating keratinocytes. We now report that keratinocyte differentiation is also accompanied by changes in E2F-5 subcellular localization, from the cytoplasm to the nucleus. To define the molecular determinants of E2F-5 nuclear import, we tested its ability to enter the nucleus in import assays in vitro using digitonin-permeabilized cells. We found that E2F-5 enters the nucleus through mediated transport processes that involve formation of nuclear pore complexes. It has been proposed that E2F-4 and E2F-5, which lack defined nuclear localization signal (NLS) consensus sequences, enter the nucleus in association with NLS-containing DP-2 or pRB family proteins. However, we show that nuclear import of E2F-5 only requires the first N-terminal 56 amino acid residues and is not dependent on interaction with DP or pRB family proteins. Because E2F-5 is predominantly cytoplasmic in undifferentiated keratinocytes and in other intact cells, we also examined whether this protein is subjected to active nuclear export. Indeed, E2F-5 is exported from the nucleus through leptomycin B-sensitive, CRM1-mediated transport, through a region corresponding to amino acid residues 130-154. This region excludes the DNA- and the p130-binding domains. Thus, the subcellular distribution of E2F-5 is tightly regulated in intact cells, through multiple functional domains that direct nucleocytoplasmic shuttling of this protein.     

Two co-existing mechanisms for nuclear import of MAP kinase: passive diffusion of a monomer and active transport of a dimer.

In response to extracellular stimuli, mitogen-activated protein kinase (MAPK, also known as ERK) translocates from the cytoplasm to the nucleus. MAP kinase kinase (MAPKK, also know as MEK), which possesses a nuclear export signal (NES), acts as a cytoplasmic anchor of MAPK. Here we show evidence that tyrosine (Tyr190 in Xenopus MPK1/ERK2) phosphorylation of MAPK by MAPKK is necessary and sufficient for the dissociation of the MAPKK-MAPK complex, and that the dissociation of the complex is required for the nuclear translocation of MAPK. We then show that nuclear entry of MAPK through a nuclear pore occurs via two distinct mechanisms. Nuclear import of wild-type MAPK (mol. wt 42 kDa) was induced by activation of the MAPK pathway even in the presence of wheat germ agglutinin or dominant-negative Ran, whereas nuclear import of beta-galactosidase (beta-gal)-fused MAPK (mol. wt 160 kDa), which occurred in response to stimuli, was completely blocked by these inhibitors. Moreover, while a dimerization-deficient mutant of MAPK was able to translocate to the nucleus upon stimulation, this mutant MAPK, when fused to beta-gal, became unable to enter the nucleus. These results suggest that monomeric and dimeric forms of MAPK enter the nucleus by passive diffusion and active transport mechanisms, respectively.     

Nuclear export of ERK3 by a CRM1-dependent mechanism regulates its inhibitory action on cell cycle progression

Extracellular signal-regulated kinase 3 (ERK3) is an atypical member of the mitogen-activated protein kinase family of serine/threonine kinases. Little is known on the regulation of ERK3 function. Here, we report that ERK3 is constitutively localized in the cytoplasmic and nuclear compartments. In contrast to other mitogen-activated protein kinases, the cellular distribution of ERK3 remains unchanged in response to common mitogenic or stress stimuli and is independent of the enzymatic activity or phosphorylation of the kinase. The cytoplasmic localization of ERK3 is directed by a CRM1-dependent nuclear export mechanism. Treatment of cells with leptomycin B causes the nuclear accumulation of ERK3 in a high percentage of cells. Moreover, ectopic expression of CRM1 promotes the cytoplasmic relocalization of ERK3, whereas overexpression of snurportin 1, which binds CRM1 with high affinity, inhibits the nuclear export of ERK3. We also show that CRM1 binds to ERK3 in vitro. Importantly, we show that enforced localization of ERK3 in the nucleus or cytoplasm markedly attenuates the ability of the kinase to induce cell cycle arrest in fibroblasts. Our results suggest that nucleocytoplasmic shuttling of ERK3 is required for its negative regulatory effect on cell cycle progression.     

PBX1 nuclear export is regulated independently of PBX-MEINOX interaction by PKA phosphorylation of the PBC-B domain

The regulation of PBC protein function through subcellular distribution is a crucial evolutionarily conserved mechanism for appendage patterning. We investigated the processes controlling PBX1 nuclear export. Here we show that in the absence of MEINOX proteins nuclear export is not a default pathway for PBX1 subcellular localization. In different cell backgrounds, PBX1 can be imported or exported from the nucleus independently of its capacity to interact with MEINOX proteins. The cell context-specific balance between nuclear export and import of PBX1 is controlled by the PBC-B domain, which contains several conserved serine residues corresponding to phosphorylation sites for Ser/Thr kinases. PBX1 subcellular localization correlates with the phosphorylation state of these residues whose dephosphorylation induces nuclear export. Protein kinase A (PKA) specifically phosphorylates PBX1 at these serines, and stimulation of endogenous PKA activity in vivo blocks PBX1 nuclear export in distal limb mesenchymal cells. Our results reveal a novel mechanism for the control of PBX1 nuclear export in addition to the absence of MEINOX protein, which involves the inhibition of PKA-mediated phosphorylation at specific sites within the PBC-B domain.     

Dissecting the Signaling Events That Impact Classical Nuclear Import and Target Nuclear Transport Factors

Background

Signaling through MEK→ERK1/2 and PI3 kinases is implicated in many aspects of cell physiology, including the survival of oxidant exposure. Oxidants play a role in numerous physiological and pathophysiological processes, many of which rely on transport in and out of the nucleus. However, how oxidative stress impacts nuclear trafficking is not well defined.

Methodology/Principal Findings

To better understand the effect of stress on nucleocytoplasmic trafficking, we exposed cells to the oxidant diethyl maleate. This treatment activated MEK→ERK1/2 as well as PI3 kinase→Akt cascades and triggered the inhibition of classical nuclear import. To define the molecular mechanisms that regulate nuclear transport, we examined whether MEK and PI3 kinase signaling affected the localization of key transport factors. Using recently developed tools for image acquisition and analysis, the subcellular distributions of importin-α, CAS, and nucleoporins Nup153 and Nup88 were quantified in different cellular compartments. These studies identified specific profiles for the localization of transport factors in the nucleus and cytoplasm, and at the nuclear envelope. Our results demonstrate that MEK and PI3 kinase signaling as well as oxidative stress control nuclear trafficking and the localization of transport components. Furthermore, stress not only induced changes in transport factor distribution, but also upregulated post-translational modification of transport factors. Our results are consistent with the idea that the phosphorylation of importin-α, CAS, Nup153, and Nup88, and the O-GlcNAc modification of Nup153 increase when cells are exposed to oxidant.

Conclusions/Significance

Our studies defined the complex regulation of classical nuclear import and identified key transport factors that are targeted by stress, MEK, and PI3 kinase signaling.     

Nuclear-Cytoplasmic Trafficking of NTF2, the Nuclear Import Receptor for the RanGTPase, Is Subjected to Regulation

NTF2 is a cytosolic protein responsible for nuclear import of Ran, a small Ras-like GTPase involved in a number of critical cellular processes, including cell cycle regulation, chromatin organization during mitosis, reformation of the nuclear envelope following mitosis, and controlling the directionality of nucleocytoplasmic transport. Herein, we provide evidence for the first time that translocation of the mammalian NTF2 from the nucleus to the cytoplasm to collect Ran in the GDP form is subjected to regulation. Treatment of mammalian cells with polysorbitan monolaurate was found to inhibit nuclear export of tRNA and proteins, which are processes dependent on RanGTP in the nucleus, but not nuclear import of proteins. Inhibition of the export processes by polysorbitan monolaurate is specific and reversible, and is caused by accumulation of Ran in the cytoplasm because of a block in translocation of NTF2 to the cytoplasm. Nuclear import of Ran and the nuclear export processes are restored in polysorbitan monolaurate treated cells overproducing NTF2. Moreover, increased phosphorylation of a phospho-tyrosine protein and several phospho-threonine proteins was observed in polysorbitan monolaurate treated cells. Collectively, these findings suggest that nucleocytoplasmic translocation of NTF2 is regulated in mammalian cells, and may involve a tyrosine and/or threonine kinase-dependent signal transduction mechanism(s).     

Dimerization in MAP-kinase signaling

The stimulus-dependent nuclear localization of the extracellular-signal- regulated kinases ERK1 and ERK2 is required for many of their actions, including induction of neurites in PC12 cells and transformation of fibroblasts. Phosphorylation of ERK2 causes it to form dimers, and the most flexible portions of the ERK2 molecule provide the surfaces for dimerization. It is thought that dimerization promotes nuclear localization of ERK2 by its effects on import, export or retention in cytoplasmic and nuclear compartments. Dimerization might also influence substrate interactions.     

Identification of an NTF2-related factor that binds Ran-GTP and regulates nuclear protein export

Active transport of macromolecules between the nucleus and cytoplasm requires signals for import and export and their recognition by shuttling receptors. Each class of macromolecule is thought to have a distinct receptor that mediates the transport reaction. Assembly and disassembly reactions of receptor-substrate complexes are coordinated by Ran, a GTP-binding protein whose nucleotide state is regulated catalytically by effector proteins. Ran function is modulated in a noncatalytic fashion by NTF2, a protein that mediates nuclear import of Ran-GDP. Here we characterize a novel component of the Ran system that is 26% identical to NTF2, which based on its function we refer to as NTF2-related export protein 1 (NXT1). In contrast to NTF2, NXT1 preferentially binds Ran-GTP, and it colocalizes with the nuclear pore complex (NPC) in mammalian cells. These properties, together with the fact that NXT1 shuttles between the nucleus and the cytoplasm, suggest an active role in nuclear transport. Indeed, NXT1 stimulates nuclear protein export of the NES-containing protein PKI in vitro. The export function of NXT1 is blocked by the addition of leptomycin B, a compound that selectively inhibits the NES receptor Crm1. Thus, NXT1 regulates the Crm1-dependent export pathway through its direct interaction with Ran-GTP.     

Evidence for existence of a nuclear pore complex-mediated, cytosol-independent pathway of nuclear translocation of ERK MAP kinase in permeabilized cells

The classical mitogen-activated protein kinase (MAPK, also known as ERK) pathway is widely involved in eukaryotic signal transductions. In response to extracellular stimuli, MAPK becomes activated and translocates from the cytoplasm to the nucleus. At least two pathways for the nuclear import of MAPK are shown to exist; passive diffusion of a monomer and Ran-dependent active transport of a dimer, the detailed molecular mechanism of which is unknown. In this study, we have reconstituted nuclear import of MAPK in vitro by using digitonin-permeabilized cells with GFP-fused MAPK protein (GFP-MAPK), which is too large to pass through the nuclear pore by passive diffusion. GFP-MAPK was able to accumulate in the nucleus irrespective of its phosphorylation state. This import of GFP-MAPK occurred even in the absence of any soluble cytosolic factors or ATP but was inhibited by wheat germ agglutinin or an excess amount of importin-beta or at low temperatures. Moreover, MAPK directly bound to an FG repeat region of nucleoporin CAN/Nup214 in vitro. Taken together, these results suggest the third pathway for nuclear import of MAPK, in which MAPK passes through the nuclear pore by directly interacting with the nuclear pore complex.     

Signal-mediated nuclear export pathways of proteins and RNAs

Although it has been known for several years that most nuclear-encoded RNAs and some patients can be exported from the nucleus to the cytoplasm, the molecular mechanisms of these transport processes have been poorly understood. Recently, signals that can induce the rapid and active nuclear export of macromolecules have been identified in the HIV-1 Rev protein, the inhibitor of cAMP-dependent protein kinase (PKI) and the hnRNP A1 protein. Thus, nuclear export appears to be mechanistically similar to nuclear import that it requires specific signal-receptor systems.     

Nup2p, a yeast nucleoporin, functions in bidirectional transport of importin alpha

Import of proteins containing a classical nuclear localization signal (NLS) into the nucleus is mediated by importin alpha and importin beta. Srp1p, the Saccharomyces cerevisiae homologue of importin alpha, returns from the nucleus in a complex with its export factor Cse1p and with Gsp1p (yeast Ran) in its GTP-bound state. We studied the role of the nucleoporin Nup2p in the transport cycle of Srp1p. Cells lacking NUP2 show a specific defect in both NLS import and Srp1p export, indicating that Nup2p is required for efficient bidirectional transport of Srp1p across the nuclear pore complex (NPC). Nup2p is located at the nuclear side of the central gated channel of the NPC and provides a binding site for Srp1p via its amino-terminal domain. We show that Nup2p effectively releases the NLS protein from importin alpha-importin and beta and strongly binds to the importin heterodimer via Srp1p. Kap95p (importin beta) is released from this complex by a direct interaction with Gsp1p-GTP. These data suggest that besides Gsp1p, which disassembles the NLS-importin alpha-importin beta complex upon binding to Kap95p in the nucleus, Nup2p can also dissociate the import complex by binding to Srp1p. We also show data indicating that Nup1p, a relative of Nup2p, plays a similar role in termination of NLS import. Cse1p and Gsp1p-GTP release Srp1p from Nup2p, which suggests that the Srp1p export complex can be formed directly at the NPC. The changed distribution of Cse1p at the NPC in nup2 mutants also supports a role for Nup2p in Srp1p export from the nucleus.