Abstract
ERK2 nuclear-cytoplasmic distribution is regulated in response to hormones and cellular state without the requirement for karyopherin-mediated nuclear import. One proposed mechanism for the movement of ERK2 into the nucleus is through a direct interaction between ERK2 and nucleoporins present in the nuclear pore complex. Previous reports have attributed regulation of ERK2 localization to proteins that activate or deactivate ERK2, such as the mitogen-activated protein (MAP) kinase kinase MEK1 and MAP kinase phosphatases. Recently, a small non-catalytic protein, PEA-15, has also been demonstrated to promote a cytoplasmic ERK2 localization. We found that the MAP kinase insert in ERK2 is required for its interaction with PEA-15. Consistent with its recognition of the MAP kinase insert, PEA-15 blocked activation of ERK2 by MEK1, which also requires the MAP kinase insert to interact productively with ERK2. To determine how PEA-15 influences the localization of ERK2, we used a permeabilized cell system to examine the effect of PEA-15 on the localization of ERK2 and mutants that have lost the ability to bind PEA-15. Wild type ERK2 was unable to enter the nucleus in the presence of an excess of PEA-15; however, ERK2 lacking the MAP kinase insert largely retained the ability to enter the nucleus. Binding assays demonstrated that PEA-15 interfered with the ability of ERK2 to bind to nucleoporins. These results suggest that PEA-15 sequesters ERK2 in the cytoplasm at least in part by interfering with its ability to interact with nucleoporins, presenting a potential paradigm for regulation of ERK2 localization.
Highlights
The mitogen-activated protein (MAP)1 kinase, ERK2, plays a critical role in promoting cellular changes in response to both mitogenic and non-mitogenic stimuli
To understand better the mechanism by which PEA-15 regulates ERK2 subcellular localization, we examined the behavior of ERK2 and PEA-15 using a permeabilized cell reconstitution assay, and we found that PEA-15 prevents ERK2 nuclear entry
We assessed the binding of PEA-15 to the following ERK2 mutants that are defective in interactions with other proteins, some of which were identified from an ERK2 mutant library [11]: 1) ERK2 D316A, D319A (DDAA), which lacks two aspartic acid residues required for binding to proteins with basic/hydrophobic docking (D) motifs including MEK1, the substrates p90 Rsk, MNK1, Elk1, and c-Fos, and the MAP kinase phosphatase MKP-3 [11, 14, 37,38,39]; 2) ERK2 lacking the MAP kinase insert, which is not activated by MEK1 or MEK2; and 3) four other ERK2 point mutants, Y261N/C, L235P, N236I, G243R, that are variably defective in binding to MEK1 (Fig. 1B)
Summary
The mitogen-activated protein (MAP) kinase, ERK2, plays a critical role in promoting cellular changes in response to both mitogenic and non-mitogenic stimuli. 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]. PEA-15 has an NES and accumulates in the nucleus in cells treated with leptomycin B, an inhibitor of CRM1-dependent nuclear export These data suggested that PEA-15 may influence the localization of ERK2 in a similar manner to that proposed for MEK1, by binding to ERK2 and mediating its nuclear export. These studies describe one potential mechanism for the regulation of ERK2 subcellular localization by non-catalytic proteins, which may contribute to targeting ERK2 to specific cellular compartments and substrates. PEA-15 prevents ERK2 from binding to nucleoporins in vitro, indicating it may assist in localizing ERK2 to cytoplasmic sites of action by inhibiting its movement into the nucleus
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