Identification of a Cytoplasmic-Retention Sequence in ERK2

Abstract

A key step in the signaling mechanism of the mitogenactivated protein kinase/extracellular signal-responsive kinase (ERK) cascade is its translocation into the nucleus where it regulates transcription and other nuclear processes. In an attempt to characterize the subcellular localization of ERK2, we fused it to the 3-end of the gene expressing green fluorescent protein (GFP), resulting in a GFP-ERK2 protein. The expression of this construct in CHO cells resulted in a nuclear localization of the GFP-ERK2 protein. However, coexpression of the GFP-ERK2 with its upstream activator, MEK1, resulted in a cytosolic retention of the GFP-ERK2, which was the result of its association with MEK1, and was reversed upon stimulation. We then examined the role of the C-terminal region of ERK2 in its subcellular localization. Substitution of residues 312-319 of GFP-ERK2 to alanine residues prevented the cytosolic retention of ERK2 as well as its association with MEK1, without affecting its activity. Most important for the cytosolic retention are three acidic amino acids at positions 316, 319, and 320 of ERK2. Substitution of residues 321-327 to alanines impaired the nuclear translocation of ERK2 upon mitogenic stimulation. Thus, we conclude that residues 312-320 of ERK2 are responsible for its cytosolic retention, and residues 321-327 play a role in the mechanism of ERK2 nuclear translocation. Mitogen-activated protein kinase (MAPK)1 signaling cascades are main routes of communication between the plasma membrane and regulatory intracellular targets and thus initiate a large array of cellular responses (1-4). The first MAPK cascade elucidated is the one that signals through the extracellular signal-responsive kinases 1 and 2 (ERK1/2), which are activated via a sequential phosphorylation and activation of the protein kinases Raf1 and MAPK/ERK kinase (MEK). Upon activation, ERK phosphorylates and activates several regulatory targets, which eventually culminate in regulation of proliferation, differentiation, and other cellular processes.Key steps in the signaling mechanism of the ERK cascade are the changes in localization of its components upon stimulation. In resting cells, all components of the cascade seem to be localized primarily in the cell cytosol. However, this localization is rapidly changed upon extracellular stimulation, which causes Raf1 recruitment to the plasma membrane (5) and translocation of MEK (6), ERK (7), and RSK (7) into the nucleus. After translocation, MEK seems to be rapidly exported from the nucleus by its nuclear export signal (NES; Ref. 8), although the timing and role of its translocation are still controversial (9, 10). ERK and RSK on the other hand are retained in the nucleus for longer times after stimulation, and this longer time is correlated with the effects of ERK on mitogenesis and neurite outgrowth in PC12 cells (11,12).The mechanism of nuclear translocation of the different kinases is not fully understood. Recently, it was shown that in resting cells ERK is retained in the cytosol by its assoc...

ERK2 enters the nucleus by a carrier-independent mechanism

“…However, we were unable to find effects of phosphorylation on its uptake in permeabilized cells. This result is consistent with the observation that overexpressed ERK2 accumulates in the nucleus without being phosphorylated (42). Blockade of export, by favoring the dissociation of ERK2-MEK1 complexes, may be the primary event controlled by ERK2 phosphorylation.…”

Mapping the binding interface of ERK and transcriptional repressor Capicua using photocrosslinking

“…Most ERK-interacting proteins bind to ERK at one of its two well-characterized docking domains. The first is the ERK D recruitment site (DRS) (14)(15)(16)(17), which is used by proteins containing a D site (18)(19)(20). The D site consensus sequence is (R/K) 2-3 -X 1-8 -Φ-X-Φ, where X is any amino acid and Φ is a hydrophobic amino acid.…”

Beta-amyloid disrupted synaptic vesicle endocytosis in cultured hippocampal neurons

“…For these experiments, we analyzed the distribution of the extracellular signaling-related kinase 2 (Erk 2) and the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit GluR 2/3, normally enriched in the cytosol and membrane fractions, respectively (Bennett and Dingledine 1995;Rubinfeld et al 1999). Western blot analysis showed that the majority of Erk 2 immunoreactivity was contained in the cytosol fraction obtained from two weeks in culture hippocampal neurons.…”