Active ERK1 is dimerized in vivo: bisphosphodimers generate peak kinase activity and monophosphodimers maintain basal ERK1 activity

Philipova, Rada; Whitaker, Michael

doi:10.1242/jcs.02683

Cited by 37 publications

(50 citation statements)

References 26 publications

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“…Recently, wild type ERK has been shown to migrate as an ϳ80-kDa complex, whereas the ERK dimer mutant H176E L 4 A did not form an 80-kDa complex or co-precipitate with MEK (25,49). Surprisingly, the mutant also prevented endogenous ERK from forming the complex.…”

Section: Discussionmentioning

confidence: 97%

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

Lidke

Huang

Post

et al. 2010

Journal of Biological Chemistry

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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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Section: Discussionmentioning

confidence: 97%

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

Lidke

Huang

Post

et al. 2010

Journal of Biological Chemistry

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“…This seems to be a general feature of MAPKs (Pelech, 2006). For example, mammalian ERK1 and ERK2 form symmetrical dimers through a leucine zipper at their C-termini (Khokhlatchev et al, 1998;Philipova and Whitaker, 2005). Dimerization of ERK1 and ERK2 is stimulated by phosphorylation (Khokhlatchev et al, 1998;Philipova and Whitaker, 2005;Wilsbacher et al, 2006), which appears to drive nuclear localization, at least in the case of ERK2 (Khokhlatchev et al, 1998).…”

Section: Discussionmentioning

confidence: 99%

“…Because some MAP kinases are known to homodimerize (Khokhlatchev et al, 1998;Philipova and Whitaker, 2005;Wilsbacher et al, 2006), we explored the possibility that association of the fusion proteins with endogenous Slt2p was complicating the analysis. We tested for Slt2p self-association by co-precipitation of differentially epitope-tagged forms of Slt2p.…”

Section: Slt2p Self-associatesmentioning

confidence: 99%

Dissecting the transcriptional activation function of the cell wall integrity MAP kinase

Kim

Cosano

Levin

et al. 2007

Yeast

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The cell wall integrity signalling MAP kinase of Saccharomyces cerevisiae, Slt2p/ Mpk1p, is activated in response to cell wall stress. Slt2 and its mammalian orthologue ERK5 are unusual among MAP kinases, in that they possess the ability to activate transcription of a GAL1-lacZ reporter when fused to the DNA-binding domain of the Gal4 transcription factor. In this study, we demonstrate that transcriptional activation of a Gal4-Slt2p fusion is responsive to cell wall stress and requires phosphorylation of Slt2p. We identify two neighbouring but separable transcription activation domains within the C-terminal half of Slt2p. Additionally, we present data suggesting that intramolecular interactions controlled by phosphorylation of Slt2p regulate the function of these domains, which are masked by the N-terminal catalytic domain under inactive conditions. Finally, we demonstrate that Slt2p self-associates, probably through a glutamine-rich region within the C-terminal half of the protein.

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“…A preformed complex in the nucleus could also have the purpose of facilitating a rapid ERK signaling in response to action potentials by positioning it with its substrates like p90RSK Zhao et al 2005). As dimers of phosphorylated ERK1 have the highest levels of ERK activity (Philipova et al 2005), the purpose of the complex may also be related specifically to maintain peak activity. Interestingly, stable complexes containing recombinant ERK1 dimers could be generated by incubation with extracts from sea urchin embryos but not from HeLa cells (Philipova et al 2005).…”

Section: Discussionmentioning

confidence: 99%

“…As dimers of phosphorylated ERK1 have the highest levels of ERK activity (Philipova et al 2005), the purpose of the complex may also be related specifically to maintain peak activity. Interestingly, stable complexes containing recombinant ERK1 dimers could be generated by incubation with extracts from sea urchin embryos but not from HeLa cells (Philipova et al 2005). We propose that 14-3-3 and/or transglutaminase were present in the sea urchin extract.…”

Section: Discussionmentioning

confidence: 99%

Differential activation of extracellular signal-regulated kinase 1 and a related complex in neuronal nuclei

Lundquist

Dudek

2006

Brain Cell Bio

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The Extracellular signal-Regulated Kinases 1 and 2 (ERKs 1/2) are known to participate in regulating transcription in response to moderate depolarization (synaptic stimulation), but how the same active enzyme can differentially regulate distinct transcriptional programs induced with abnormal depolarization (high potassium) is unknown. We hypothesized that ERK1 or 2 accomplishes this differential nuclear response through close association with other proteins in stable complexes. In support of this hypothesis, we have found that immunoreactivity for an apparent high molecular weight phospho-ERK1-containing complex increased in response to synaptic stimulation, but decreased in response to high potassium; p-ERK immunoreactivity at 44/42 kDa increased in both cases. Evidence supporting the conclusion that the band of interest contained ERK1 in a complex, as opposed to it being an unrelated protein crossreacting with antibodies against p-ERK, is that ERK1 (p44 MAPK) and 14-3-3 were electro-eluted from the 160 kDa band cut from a gel. We also found the nuclear complexes to be exceptionally durable, suggesting a role for the crosslinking enzyme, transglutaminase, in its stabilization. In addition, we found other components of the ERK pathway, including MEK, ERK2, p90RSK, and Elk-1, migrating at higher-than-expected weights in brain nuclei. These results describe a novel stable complex of ERK1 in neuronal nuclei that responds differentially to synaptic and depolarizing stimulation, and thus may be capable of mediating gene transcription in a way distinct from the monomeric protein.

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Active ERK1 is dimerized in vivo: bisphosphodimers generate peak kinase activity and monophosphodimers maintain basal ERK1 activity

Cited by 37 publications

References 26 publications

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

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

Dissecting the transcriptional activation function of the cell wall integrity MAP kinase

Differential activation of extracellular signal-regulated kinase 1 and a related complex in neuronal nuclei

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