ERK Nuclear Translocation Is Dimerization-independent but Controlled by the Rate of Phosphorylation

Abstract

Upon activation, ERKs translocate from the cytoplasm to the nucleus. This process is required for the induction of many cellular responses, yet the molecular mechanisms that regulate ERK nuclear translocation are not fully understood. We have used a mouse embryo fibroblast ERK1-knock-out cell line expressing green fluorescent protein (GFP)-tagged ERK1 to probe the spatiotemporal regulation of ERK1. Real time fluorescence microscopy and fluorescence correlation spectroscopy revealed that ERK1 nuclear accumulation increased upon serum stimulation, but the mobility of the protein in the nucleus and cytoplasm remained unchanged. Dimerization of ERK has been proposed as a requirement for nuclear translocation. However, ERK1-⌬4, the mutant shown consistently to be dimerization-deficient in vitro, accumulated in the nucleus to the same level as wild type (WT), indicating that dimerization of ERK1 is not required for nuclear entry and retention. Consistent with this finding, energy migration Förster resonance energy transfer and fluorescence correlation spectroscopy measurements in living cells did not detect dimerization of GFP-ERK1-WT upon activation. In contrast, the kinetics of nuclear accumulation and phosphorylation of GFP-ERK1-⌬4 were slower than that of GFP-ERK1-WT. These results indicate that the differential shuttling behavior of the mutant is a consequence of delayed phosphorylation of ERK by MEK rather than dimerization. Our data demonstrate for the first time that a delay in cytoplasmic activation of ERK is directly translated into a delay in nuclear translocation.Stimulation of numerous cell surface receptors leads to activation of the Raf/MEK 7 /ERK signaling pathway. In this kinase cascade, Raf phosphorylates only MEK, and MEK phosphorylates only ERK, whereas ERK is able to phosphorylate many substrates in nearly all cell compartments (1). Noncatalytic activation of a few partners by c-Raf is well documented, but the biological outcomes of the ERK pathway are predominantly driven by the kinase activity, as evidenced, for example, by chemical inhibition (reviewed in Ref.2). ERK is primarily located in the cytoplasm of resting cells, although overexpression results in cytoplasmic and nuclear localization (3). It has long been recognized that in the course of physiological signal transduction, ERK accumulates in the nucleus after acute stimulation of the cell (3, 4). Nuclear translocation of ERK is required for cell cycle entry. Thus, retention of ERK in the cytoplasm alters neither ERK kinase activity nor phosphorylation of cytoplasmic substrates, whereas ERK-dependent transcription and cell proliferation are blocked (5). It has been demonstrated that ERK phosphorylates the Phe-Gly nucleoporins Nup50, Nup153, and Nup154, reducing importin-␤-mediated nucleocytoplasmic transport (6). This observation would expand the role of ERK nuclear entry to include the regulation of nucleocytoplasmic transport of certain classes of proteins while crossing the nucleopore.MEK functions as the cytoplasmic anchor for ERK su...

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