ERK2 enters the nucleus by a carrier-independent mechanism

Abstract

In stimulated cells, the mitogen-activated protein kinase ERK2 (extracellular signal-regulated kinase 2) concentrates in the nucleus. Evidence exists for CRM1-dependent, mitogen-activated protein kinase kinase-mediated nuclear export of ERK2, but its mechanism of nuclear entry is not understood. To determine requirements for nuclear transport, we tagged ERK2 with green fluorescent protein (GFP) and examined its nuclear uptake by using an in vitro import assay. GFP-ERK2 entered the nucleus in a saturable, time-and temperature-dependent manner. Entry of GFP-ERK2, like that of ERK2, required neither energy nor transport factors and was visible within minutes. The nuclear uptake of GFP-ERK2 was inhibited by wheat germ agglutinin, which blocks nuclear entry by binding to carbohydrate moieties on nuclear pore complex proteins. The nuclear uptake of GFP-ERK2 also was reduced by excess amounts of recombinant transport factors. These findings suggest that ERK2 competes with transport factors for binding to nucleoporins, which mediate the entry and exit of transport factors. In support of this hypothesis, we showed that ERK2 binds directly to a purified nucleoporin. Our data suggest that GFP-ERK2 enters the nucleus by a saturable, facilitated mechanism, distinct from a carrier-and energy-dependent import mechanism and involves a direct interaction with nuclear pore complex proteins.mitogen-activated protein kinase ͉ import ͉ FXFG motif ͉ nucleoporins M itogen-activated protein (MAP) kinases are ubiquitous protein kinases that integrate cell surface signals by phosphorylating proteins throughout the cell. The MAP kinase ERK2 (extracellular signal-regulated kinase 2) targets proteins in multiple cell compartments after an activating stimulus. Stimuli induce the nuclear accumulation of ERK2 from its resting location in the cytoplasm of many types of cells, including fibroblasts, epithelial cells, and neuroendocrine cells (1, 2).The location of ERK2 is a significant factor in determining its ability to phosphorylate key substrates and thereby influence cell behavior. Controlling the localization of ERK2 is a mechanism by which cell function may be influenced. For example, PEA15, which promotes the cytoplasmic retention of ERK2, is overexpressed in disease states, such as type II diabetes and breast cancer (3, 4). ERK2 is active and constitutively nuclear in certain breast cancer cells, but excluded from the nucleus in certain nonsmall cell lung cancers (ref. 5, S. Muneer, personal communication). In addition to extensive data suggesting a correlation between phenotypes and ERK2 localization, some cell behaviors have been demonstrated to occur only if ERK2 accumulates in the nucleus. For example, the nuclear localization of ERK2 is essential for morphological transformation of 3T3 fibroblasts and neurite extension in PC12 cells (6, 7). An understanding of how ERK2 localization is regulated will provide important insights into its normal function and disease mechanisms.ERK2 subcellular localization is mediated by interactions w...

The Death Effector Domain Protein PEA-15 Prevents Nuclear Entry of ERK2 by Inhibiting Required Interactions

“…Protein outside the nucleus that is not tightly bound will wash away prior to visualization. Entry of GFP-ERK2 into the nucleus is inhibited by the lectin wheat germ agglutinin, which binds to N-acetylglucosamine residues on nucleoporins and occludes the NPC (19). If we included PEA-15 in these assays, we found a significant inhibition of GFP-ERK2 entry at 15 min.…”

“…Digitonin permeabilization results in the loss of soluble proteins including proteins required for conventional import and export processes, notably the small G protein Ran, but leaves the nucleus and NPC intact. We have shown previously that the control import substrate, NLS-BSA, is not imported in the absence of added transport factors and energy in this system (19). In contrast, GFP-ERK2 enters the nucleus in the absence of added soluble proteins and energy in a manner facilitated by interactions with the FXF-rich nucleoporins, which are major components of the nuclear pore (18,19).…”

“…We have shown previously that the control import substrate, NLS-BSA, is not imported in the absence of added transport factors and energy in this system (19). In contrast, GFP-ERK2 enters the nucleus in the absence of added soluble proteins and energy in a manner facilitated by interactions with the FXF-rich nucleoporins, which are major components of the nuclear pore (18,19). Protein outside the nucleus that is not tightly bound will wash away prior to visualization.…”

“…Overexpression studies have shown that ERK2 export can be facilitated by MEK1, which has a nuclear export sequence (NES) and requires the NES-binding protein CRM1 for movement out of the nucleus (16,17). Nuclear entry of ERK2 has been described recently as a facilitated mechanism in which ERK2 moves through the NPC by binding directly to nucleoporins (18,19). This process, which has been described for at least two other signaling proteins (20,21), does not rely on the classical, karyopherin-mediated import mechanism.…”

The Ras‐ERK pathway: Understanding site‐specific signaling provides hope of new anti‐tumor therapies

“…Some proteins, like PEA15, prevent ERKs nuclear translocation [69] and in doing so, promote ERK cytoplasmic activities, such as the modulation of integrin receptors, [70] while interfering with ERKs nuclear functions, like the promotion of proliferation. [71] For efficient nuclear translocation, ERKs require direct interaction with the nuclear pore complex, [72] a nuclear translocation signal within their ''insert'' domain [73] and the participation of nuclear shuttles like Importin-7 [74] or Mxi2. [75] Interestingly, ERK functions within the nucleus may also be compartmentalized further: ERKs interact with lamin A at the nuclear envelope to promote rapid, mitogen-dependent AP-1 activation, by releasing cFos from its inhibitory interaction with lamin A.…”