Oxidative neuronal injury. The dark side of ERK1/2.

The extracellular signal regulated protein kinases (ERK1/2) are essential for normal development and functional plasticity of the central nervous system. However, a growing number of recent studies in models of cerebral ischemia, brain trauma and neurodegenerative diseases implicate a detrimental role for ERK1/2 signaling during oxidative neuronal injury. Neurons undergoing oxidative stress-related injuries typically display a biphasic or sustained pattern of ERK1/2 activation. A variety of potential targets of reactive oxygen species and reactive nitrogen species could contribute to ERK1/2 activation. These include cell surface receptors, G proteins, upstream kinases, protein phosphatases and proteasome components, each of which could be direct or indirect targets of reactive oxygen or nitrogen species, thereby modulating the duration and magnitude of ERK1/2 activation. Neuronal oxidative stress also appears to influence the subcellular trafficking and/or localization of activated ERK1/2. Differences in compartmentalization of phosphorylated ERK1/2 have been observed in diseased or injured human neurons and in their respective animal and cell culture model systems. We propose that differential accessibility of ERK1/2 to downstream targets, which is dictated by the persistent activation of ERK1/2 within distinct subcellular compartments, underlies the neurotoxic responses that are driven by this kinase.     

Novel Reporter for Faithful Monitoring of ERK2 Dynamics in Living Cells and Model Organisms

Uncoupling of ERK1/2 phosphorylation from subcellular localization is essential towards the understanding of molecular mechanisms that control ERK1/2-mediated cell-fate decision. ERK1/2 non-catalytic functions and discoveries of new specific anchors responsible of the subcellular compartmentalization of ERK1/2 signaling pathway have been proposed as regulation mechanisms for which dynamic monitoring of ERK1/2 localization is necessary. However, studying the spatiotemporal features of ERK2, for instance, in different cellular processes in living cells and tissues requires a tool that can faithfully report on its subcellular distribution. We developed a novel molecular tool, ERK2-LOC, based on the T2A-mediated coexpression of strictly equimolar levels of eGFP-ERK2 and MEK1, to faithfully visualize ERK2 localization patterns. MEK1 and eGFP-ERK2 were expressed reliably and functionally both in vitro and in single living cells. We then assessed the subcellular distribution and mobility of ERK2-LOC using fluorescence microscopy in non-stimulated conditions and after activation/inhibition of the MAPK/ERK1/2 signaling pathway. Finally, we used our coexpression system in Xenopus laevis embryos during the early stages of development. This is the first report on MEK1/ERK2 T2A-mediated coexpression in living embryos, and we show that there is a strong correlation between the spatiotemporal subcellular distribution of ERK2-LOC and the phosphorylation patterns of ERK1/2. Our approach can be used to study the spatiotemporal localization of ERK2 and its dynamics in a variety of processes in living cells and embryonic tissues.     

Transient versus sustained phosphorylation and nuclear accumulation of ERKs underlie anti-versus pro-apoptotic effects of estrogens

Sex steroids exert anti-apoptotic effects on osteoblasts/osteocytes but exert pro-apoptotic effects on osteoclasts, in both cases requiring activation of the extracellular signal-regulated kinases (ERKs). To explain the mechanistic basis of this divergence, we searched for differences in the kinetics of phosphorylation and/or in the subcellular localization of ERKs in response to 17beta-estradiol in the two cell types. In contrast to its transient effect on ERK phosphorylation in osteocytic cells (return to base line by 30 min), 17beta-estradiol-induced ERK phosphorylation in osteoclasts was sustained for at least 24 h following exposure to the hormone. Conversion of sustained ERK phosphorylation to transient, by means of cholera toxin-induced activation of the adenylate cyclase/cAMP/protein kinase A pathway, abrogated the pro-apoptotic effect of 17beta-estradiol on osteoclasts. Conversely, prolongation of ERK activation in osteocytes, by means of leptomycin B-induced inhibition of ERK export from the nucleus or overexpression of a green fluorescent protein-ERK2 mutant that resides permanently in the nucleus, converted the anti-apoptotic effect of 17beta-estradiol to a pro-apoptotic one. These findings indicate that the kinetics of ERK phosphorylation and the length of time that phospho-ERKs are retained in the nucleus are responsible for pro-versus anti-apoptotic effects of estrogen on different cell types of bone and perhaps their many other target tissues.     

Involvement of the activation loop of ERK in the detachment from cytosolic anchoring

The Extracellular signal-regulated kinases (ERKs) are translocated into the nucleus in response to mitogenic stimulation. The mechanism of translocation and the residues in ERKs that govern this process are not clear as yet. Here we studied the involvement of residues in the activation loop of ERK2 in determining its subcellular localization. Substitution of residues in the activation loop to alanines indicated that residues 173-181 do not play a significant role in the phosphorylation and activation of ERK2. However, residues 176-181 are responsible for the detachment of ERK2 from MEK1 upon mitogenic stimulation. This dissociation can be mimicked by substitution of residues 176-178 to alanines and is prevented by deletion of these residues or by substitution of residues 179-181 to alanines. On the other hand, residues 176-181, as well as residues essential for ERK2 dimerization, do not play a role in the shuttle of ERK2 through nuclear pores. Thus, phosphorylation-induced conformational rearrangement of residues in the activation loop of ERK2 plays a major role in the control of subcellular localization of this protein.     

Nerve growth factor stimulates the concentration of TrkA within lipid rafts and extracellular signal-regulated kinase activation through c-Cbl-associated protein

Nerve growth factor (NGF) acts through its receptor, TrkA, to elicit the neuronal differentiation of PC12 cells through the action of extracellular signal-regulated kinase 1 (ERK1) and ERK2. Upon NGF binding, TrkA translocates and concentrates in cholesterol-rich membrane microdomains or lipid rafts, facilitating formation of receptor-associated signaling complexes, activation of downstream signaling pathways, and internalization into endosomes. We have investigated the mechanisms responsible for the localization of TrkA within lipid rafts and its ability to activate ERK1 and ERK2. We report that NGF treatment results in the translocation of activated forms of TrkA to lipid rafts, and this localization is important for efficient activation of the ERKs. TrkA is recruited and retained within lipid rafts through its association with flotillin, an intrinsic constituent of these membrane microdomains, via the adapter protein, c-Cbl associated protein (CAP). Mutant forms of CAP that lack protein interaction domains block TrkA localization to lipid rafts and attenuate ERK activation. Importantly, suppression of endogenous CAP expression inhibited NGF-stimulated neurite outgrowth from primary dorsal root ganglion neurons. These data provide a mechanism for the lipid raft localization of TrkA and establish the importance of the CAP adaptor protein for NGF activation of the ERKs and neuronal differentiation.     

Differential subcellular membrane recruitment of Src may specify its downstream signalling

Most Src family members are diacylated and constitutively associate with membrane "lipid rafts" that coordinate signalling. Whether the monoacylated Src, frequently hyperactive in carcinomas, also localizes at "rafts" remains controversial. Using polarized MDCK cells expressing the thermosensitive v-Src/tsLA31 variant, we here addressed how Src tyrosine-kinase activation may impact on its (i) membrane recruitment, in particular to "lipid rafts"; (ii) subcellular localization; and (iii) signalling. The kinetics of Src-kinase thermoactivation correlated with its recruitment from the cytosol to sedimentable membranes where Src largely resisted solubilisation by non-ionic detergents at 4 degrees C and floated into sucrose density gradients like caveolin-1 and flotillin-2, i.e. "lipid rafts". By immunofluorescence, activated Src showed a dual localization, at apical endosomes/macropinosomes and at the apical plasma membrane. The plasma membrane Src pool did not colocalize with caveolin-1 and flotillin-2, but extensively overlapped GM1 labelling by cholera toxin. Severe ( approximately 70%) cholesterol extraction with methyl-beta-cyclodextrin (MbetaCD) did not abolish "rafts" floatation, but strongly decreased Src association with floating "rafts" and abolished its localization at the apical plasma membrane. Src activation independently activated first the MAP-kinase - ERK1/2 pathway, then the PI3-kinase - Akt pathway. MAP-kinase - ERK1/2 activation was insensitive to MbetaCD, which suppressed Akt phosphorylation and apical endocytosis induced by Src, both depending on the PI3-kinase pathway. We therefore suggest that activated Src is recruited at two membrane compartments, allowing differential signalling, first via ERK1/2 at "non-raft" domains on endosomes, then via PI3-kinase-Akt on a distinct set of "rafts" at the apical plasma membrane. Whether this model is applicable to c-Src remains to be examined.     

Different domains of the mitogen-activated protein kinases ERK3 and ERK2 direct subcellular localization and upstream specificity in vivo.

Extracellular signal-regulated kinase 3 (ERK3) is a member of the mitogen-activated protein (MAP) kinase family. ERK3 is most similar in its kinase catalytic domain to ERK2, yet it displays many unique properties. Among these, unlike ERK2, which translocates to the nucleus following activation, ERK3 is constitutively localized to the nucleus, despite the lack of a defined nuclear localization sequence. We created two chimeras between ERK2 and the catalytic domain of ERK3 (ERK3DeltaC), and some mutants of these chimeras, to examine the basis for the different behaviors of these two MAP kinase family members. We find the following: 1) the N-terminal folding domain of ERK3 functions in phosphoryl transfer reactions with the C-terminal folding domain of ERK2; 2) the C-terminal halves of ERK2 and ERK3DeltaC are primarily responsible for their subcellular localization in resting cells; and 3) the N-terminal folding domain of ERK2 is required for its activation in cells, its interaction with MEK1, and its accumulation in the nucleus.     

A novel mechanism for mitogen-activated protein kinase localization

Mitogen-activated protein kinases/extracellular signal regulated kinases (MAPKs/ERKs) are typically thought to be soluble cytoplasmic enzymes that translocate to the nucleus subsequent to their phosphorylation by their activating kinases or mitogen-activated protein/extracellular signal regulated kinase kinase. We report here the first example of nuclear translocation of a MAPK that occurs via temporally regulated exit from a membranous organelle. Confocal microscopy examining the subcellular localization of ERK3 in several cell lines indicated that this enzyme was targeted to the Golgi/endoplasmic reticulum Golgi intermediate compartment. Deletion analysis of green fluorescent protein (GFP)-ERK3 uncovered a nuclear form that was carboxy-terminally truncated and established a Golgi targeting motif at the carboxy terminus. Immunoblot analysis of cells treated with the proteasome inhibitor MG132 further revealed two cleavage products, suggesting that in vivo, carboxy-terminal cleavage of the full-length protein controls its subcellular localization. In support of this hypothesis, we found that deletion of a small region rich in acidic residues within the carboxy terminus eliminated both the cleavage and nuclear translocation of GFP-ERK3. Finally, cell cycle synchronization studies revealed that the subcellular localization of ERK3 is temporally regulated. These data suggest a novel mechanism for the localization of an MAPK family member, ERK3, in which cell cycle-regulated, site-specific proteolysis generates the nuclear form of the protein.     

Distinct subcellular localization and substrate specificity of extracellular signal-regulated kinase in B cells upon stimulation with IgM and CD40

We and others previously observed that IgM and CD40 stimulation in murine B cells resulted in activation of extracellular signal-regulated kinase (ERK), a subfamily of mitogen-activated protein kinase. The present study demonstrated that ERK was rapidly phosphorylated and translocated to the nucleus in murine B cells upon stimulation with CD40, whereas it was preferentially localized within the cytosol after stimulation with IgM, suggesting that signaling through CD40 and IgM differentially regulates ERK subcellular localization. Costimulation with CD40 and IgM (CD40/IgM) resulted in subcellular localization of ERK within the cytosol, supporting the notion that stimulation with IgM delivers the signal responsible for inhibition of ERK nuclear transport. Consistent with these observations, IgM and CD40/IgM stimulation resulted in activation of ribosomal S6 kinase, which is a cytoplasmic substrate for ERK, whereas CD40 stimulation had little effect on its activity. Disruption of the microtubule by colchicine in WEHI231 cells resulted in reduction of ERK activity in IgM signaling, but not in CD40 signaling, compatible with the notion that the microtubule network may hold cytoplasmic ERK activity mediated by IgM stimulation. These results support the notion that ERK could mediate different effector functions in B cells upon stimulation with IgM and CD40.     

ERK1b, a 46-kDa ERK isoform that is differentially regulated by MEK

We identified a 46-kDa ERK, whose kinetics of activation was similar to that of ERK1 and ERK2 in most cell lines and conditions, but showed higher fold activation in response to osmotic shock and epidermal growth factor treatments of Ras-transformed cells. We purified and cloned this novel ERK (ERK1b), which is an alternatively spliced form of ERK1 with a 26-amino acid insertion between residues 340 and 341 of ERK1. When expressed in COS7 cells, ERK1b exhibited kinetics of activation and kinase activity similar to those of ERK1. Unlike the uniform pattern of expression of ERK1 and ERK2, ERK1b was detected only in some of the tissues examined and seems to be abundant in the rat and human heart. Interestingly, in Ras-transformed Rat1 cells, there was a 7-fold higher expression of ERK1b, which was also more responsive than ERK1 and ERK2 to various extracellular treatments. Unlike ERK1 and ERK2, ERK1b failed to interact with MEK1 as judged from its nuclear localization in resting cells overexpressing ERK1b together with MEK1 or by lack of coimmunoprecipitation of the two proteins. Thus, ERK1b is a novel 46-kDa ERK isoform, which seems to be the major ERK isoform that responds to exogenous stimulation in Ras-transformed cells probably due to its differential regulation by MEK.     

Phosphorylation regulates KSR1 stability, ERK activation, and cell proliferation

Kinase suppressor of Ras (KSR) is a molecular scaffold that interacts with the components of the Raf/MEK/ERK kinase cascade and positively regulates ERK signaling. Phosphorylation of KSR1, particularly at Ser(392), is a critical regulator of KSR1 subcellular localization and ERK activation. We examined the role of phosphorylation of both Ser(392) and Thr(274) in regulating ERK activation and cell proliferation. We hypothesized that KSR1 phosphorylation is involved in generating signaling specificity through the Raf/MEK/ERK kinase cascade in response to stimulation by different growth factors. In fibroblasts, platelet-derived growth factor stimulation induces sustained ERK activation and promotes S-phase entry. Treatment with epidermal growth factor induces transient ERK activation but fails to drive cells into S phase. Mutation of Ser(392) and Thr(274) (KSR1.TVSA) promotes sustained ERK activation and cell cycle progression with either platelet-derived growth factor or epidermal growth factor treatment. KSR1(-/-) mouse embryo fibroblasts expressing KSR1.TVSA proliferate two times faster and grow to a higher density than cells expressing the same level of wild-type KSR1. In addition, KSR1.TVSA is more stable than wild-type KSR1. These data demonstrate that phosphorylation and stability of the molecular scaffold KSR1 are critical regulators of growth factor-specific responses that promote cell proliferation.     

Reversible oxidation of ERK-directed protein phosphatases drives oxidative toxicity in neurons

Oxidative stress links diverse neuropathological conditions that include stroke, Parkinson's disease, and Alzheimer's disease and has been modeled in vitro with various paradigms that lead to neuronal cell death following the increased accumulation of reactive oxygen species. For example, immortalized neurons and immature primary cortical neurons undergo cell death in response to depletion of the antioxidant glutathione, which can be elicited by administration of glutamate at high concentrations. We have demonstrated previously that this glutamate-induced oxidative toxicity requires activation of the mitogen-activated protein kinase member ERK1/2, but the mechanisms by which this activation takes place in oxidatively stressed neurons are still not fully known. In this study, we demonstrate that during oxidative stress, ERK-directed phosphatases of both the serine/threonine- and tyrosine-directed classes are selectively and reversibly inhibited via a mechanism that is dependent upon the oxidation of cysteine thiols. Furthermore, the impact of ERK-directed phosphatases on ERK1/2 activation and oxidative toxicity in neurons was tested in a neuronal cell line and in primary cortical cultures. Overexpression of the highly ERK-specific phosphatase MKP3 and its catalytic mutant, MKP3 C293S, were neuroprotective in transiently transfected HT22 cells and primary neurons. The neuroprotective effect of the MKP3 C293S mutant, which enhances ERK1/2 phosphorylation but blocks its nuclear translocation, demonstrates the necessity for active ERK1/2 nuclear localization for oxidative toxicity in neurons. Together, these data implicate the inhibition of endogenous ERK-directed phosphatases as a mechanism that leads to aberrant ERK1/2 activation and nuclear accumulation during oxidative toxicity in neurons.     

Eukaryotic translation initiation factor 3, subunit a, regulates the extracellular signal-regulated kinase pathway

The extracellular signal-regulated kinase (ERK) pathway participates in the control of numerous cellular processes, including cell proliferation. Since its activation kinetics are critical for to its biological effects, they are tightly regulated. We report that the protein translation factor, eukaryotic translation initiation factor 3, subunit a (eIF3a), binds to SHC and Raf-1, two components of the ERK pathway. The interaction of eIF3a with Raf-1 is increased by β-arrestin2 expression and transiently decreased by epidermal growth factor (EGF) stimulation in a concentration-dependent manner. The EGF-induced decrease in Raf-1-eIF3a association kinetically correlates with the time course of ERK activation. eIF3a interferes with Raf-1 activation and eIF3a downregulation by small interfering RNA enhances ERK activation, early gene expression, DNA synthesis, expression of neuronal differentiation markers in PC12 cells, and Ras-induced focus formation in NIH 3T3 cells. Thus, eIF3a is a negative modulator of ERK pathway activation and its biological effects.     

Gab2 requires membrane targeting and the Met binding motif to promote lamellipodia, cell scatter, and epithelial morphogenesis downstream from the Met receptor

Gab1 and Gab2 are conserved scaffolding proteins that amplify and integrate signals stimulated by many growth factor receptors including the Met receptor. Gab1 acts to diversify the signal downstream from Met through the recruitment of multiple signaling proteins, and is essential for epithelial morphogenesis. However, whereas Gab1 and Gab2 are both expressed in epithelial cells, Gab2 fails to support a morphogenic response. We demonstrate that Gab1 and Gab2 are divergent in their function whereby Gab1, but not Gab2, promotes lamellipodia formation, and is localized to the membrane of lamellipodia upon Met activation. We have identified activation of ERK1/2 as a requirement for lamellipodia formation. Moreover, activated ERK1/2 are localized to lamellipodia in Gab1 expressing cells but not in cells that overexpress Gab2. By structure-function studies, we identify that enhanced membrane localization conferred through the addition of a myristoylation signal, together with the addition of the direct Met binding motif (MBM) from Gab1, are required to promote lamellipodia and confer a morphogenic signaling response to Gab2. Moreover, the morphogenesis competent myristoylated Gab2MBM promotes localization of activated ERK1/2 to the leading edge of lamellipodia in a similar manner to Gab1. Hence, subcellular localization of the Gab scaffold, as well as the ability of Gab to interact directly with the Met receptor, are both essential components of the morphogenic signaling response which involves lamellipodia formation and the localization of ERK1/2 activation in membrane ruffles.