Cloning and characterization of mouse extracellular-signal-regulated protein kinase 3 as a unique gene product of 100 kDa. (1/65)

MAP (mitogen-activated protein) kinases are a family of serine/threonine kinases that have a pivotal role in signal transduction. Here we report the cloning and characterization of a mouse homologue of extracellular-signal-regulated protein kinase (ERK)3. The mouse Erk3 cDNA encodes a predicted protein of 720 residues, which displays 94% identity with human ERK3. Transcription and translation of this cDNA in vitro generates a 100 kDa protein similar to the human gene product ERK3. Immunoblot analysis with an antibody raised against a unique sequence of ERK3 also recognizes a 100 kDa protein in mouse tissues. A single transcript of Erk3 was detected in every adult mouse tissue examined, with the highest expression being found in the brain. Interestingly, expression of Erk3 mRNA is acutely regulated during mouse development, with a peak of expression observed at embryonic day 11. The mouse Erk3 gene was mapped to a single locus on central mouse chromosome 9, adjacent to the dilute mutation locus and in a region syntenic to human chromosome 15q21. Finally, we provide several lines of evidence to support the existence of a unique Erk3 gene product of 100 kDa in mammalian cells.  (+info)

Different domains of the mitogen-activated protein kinases ERK3 and ERK2 direct subcellular localization and upstream specificity in vivo. (2/65)

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.  (+info)

Induction of iNOS expression in skeletal muscle by IL-1beta and NFkappaB activation: an in vitro and in vivo study. (3/65)

OBJECTIVE: The intracellular pathway and the regulation of inducible nitric oxide synthase (iNOS) expression in skeletal muscle is incompletely understood. In vitro studies, using different cell types, suggest that inflammatory cytokines are potential triggers to induce iNOS expression. METHODS: To analyze intracellular pathways leading to iNOS induction, rat skeletal myoblasts were incubated with inflammatory cytokines and assessed for iNOS expression by Western blot and Griess reaction. To confirm the in vitro findings, local cytokine levels were determined in skeletal muscle biopsies of patients with chronic heart failure (CHF) and correlated with iNOS expression. RESULTS: Nitrite accumulation in the myoblast culture supernatant or iNOS protein in the cell pellet was significantly increased after incubation with IL-1beta in combination with gamma-IFN. Priming experiments revealed that gamma-IFN elevated the expression of IL-1beta receptor mRNA, whereby IL-1beta was able to induce iNOS expression. The cytokine-mediated iNOS induction was significantly reduced by blocking ERK1/ERK2 activation and completely abolished by the inhibition of NFkappaB. In skeletal muscle biopsies of CHF patients the local content of IL-1beta was significantly increased as compared to healthy controls. Furthermore, a linear correlation between IL-1beta content and iNOS expression in the skeletal muscle was detected. CONCLUSIONS: These data demonstrate that IL-1beta, together with the priming effect of gamma-IFN, induces iNOS expression in skeletal muscle via activation of ERK1/ERK2 and NFkappaB.  (+info)

Rapid turnover of extracellular signal-regulated kinase 3 by the ubiquitin-proteasome pathway defines a novel paradigm of mitogen-activated protein kinase regulation during cellular differentiation. (4/65)

Mitogen-activated protein (MAP) kinases are stable enzymes that are mainly regulated by phosphorylation and subcellular targeting. Here we report that extracellular signal-regulated kinase 3 (ERK3), unlike other MAP kinases, is an unstable protein that is constitutively degraded in proliferating cells with a half-life of 30 min. The proteolysis of ERK3 is executed by the proteasome and requires ubiquitination of the protein. Contrary to other protein kinases, the catalytic activity of ERK3 is not responsible for its short half-life. Instead, analysis of ERK1/ERK3 chimeras revealed the presence of two destabilization regions (NDR1 and -2) in the N-terminal lobe of the ERK3 kinase domain that are both necessary and sufficient to target ERK3 and heterologous proteins for proteasomal degradation. To assess the physiological relevance of the rapid turnover of ERK3, we monitored the expression of the kinase in different cellular models of differentiation. We observed that ERK3 markedly accumulates during differentiation of PC12 and C2C12 cells into the neuronal and muscle lineage, respectively. The accumulation of ERK3 during myogenic differentiation is associated with the time-dependent stabilization of the protein. Terminal skeletal muscle differentiation is accompanied by cell cycle withdrawal. Interestingly, we found that expression of stabilized forms of ERK3 causes G(1) arrest in NIH 3T3 cells. We propose that ERK3 biological activity is regulated by its cellular abundance through the control of protein stability.  (+info)

Nuclear export of ERK3 by a CRM1-dependent mechanism regulates its inhibitory action on cell cycle progression. (5/65)

Extracellular signal-regulated kinase 3 (ERK3) is an atypical member of the mitogen-activated protein kinase family of serine/threonine kinases. Little is known on the regulation of ERK3 function. Here, we report that ERK3 is constitutively localized in the cytoplasmic and nuclear compartments. In contrast to other mitogen-activated protein kinases, the cellular distribution of ERK3 remains unchanged in response to common mitogenic or stress stimuli and is independent of the enzymatic activity or phosphorylation of the kinase. The cytoplasmic localization of ERK3 is directed by a CRM1-dependent nuclear export mechanism. Treatment of cells with leptomycin B causes the nuclear accumulation of ERK3 in a high percentage of cells. Moreover, ectopic expression of CRM1 promotes the cytoplasmic relocalization of ERK3, whereas overexpression of snurportin 1, which binds CRM1 with high affinity, inhibits the nuclear export of ERK3. We also show that CRM1 binds to ERK3 in vitro. Importantly, we show that enforced localization of ERK3 in the nucleus or cytoplasm markedly attenuates the ability of the kinase to induce cell cycle arrest in fibroblasts. Our results suggest that nucleocytoplasmic shuttling of ERK3 is required for its negative regulatory effect on cell cycle progression.  (+info)

N-Terminal ubiquitination of extracellular signal-regulated kinase 3 and p21 directs their degradation by the proteasome. (6/65)

Extracellular signal-regulated kinase 3 (ERK3) is an unstable mitogen-activated protein kinase homologue that is constitutively degraded by the ubiquitin-proteasome pathway in proliferating cells. Here we show that a lysineless mutant of ERK3 is still ubiquitinated in vivo and requires a functional ubiquitin conjugation pathway for its degradation. Addition of N-terminal sequence tags of increasing size stabilizes ERK3 by preventing its ubiquitination. Importantly, we identified a fusion peptide between the N-terminal methionine of ERK3 and the C-terminal glycine of ubiquitin in vivo by tandem mass spectrometry analysis. These findings demonstrate that ERK3 is conjugated to ubiquitin via its free NH(2) terminus. We found that large N-terminal tags also stabilize the expression of the cell cycle inhibitor p21 but not that of substrates ubiquitinated on internal lysine residues. Consistent with this observation, lysineless p21 is ubiquitinated and degraded in a ubiquitin-dependent manner in intact cells. Our results suggests that N-terminal ubiquitination is a more prevalent modification than originally recognized.  (+info)

A novel mechanism for mitogen-activated protein kinase localization. (7/65)

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.  (+info)

Characterization of a novel low-molecular-mass dual-specificity phosphatase-3 (LDP-3) that enhances activation of JNK and p38. (8/65)

We have isolated a mouse cDNA for a novel dual-specificity phosphatase designated LDP-3 (low-molecular-mass dual-specificity phosphatase 3). The 450 bp open reading frame encodes a protein of 150 amino acids with a predicted molecular mass of 16 kDa. Northern blot and reverse transcription-PCR analyses show that LDP-3 transcripts are expressed in almost all mouse tissues examined. In vitro analyses using several substrates and inhibitors indicate that LDP-3 possesses intrinsic dual-specificity phosphatase activity. When expressed in mammalian cells, LDP-3 protein is localized mainly to the apical submembrane area. Forced expression of LDP-3 does not alter activation of ERK (extracellular-signal-regulated kinase), but rather enhances activation of JNK (c-Jun N-terminal kinase) and p38 and their respective upstream kinases MKK4 (mitogen-activated protein kinase kinase 4) and MKK6 in cells treated with 0.4 M sorbitol. By screening with a variety of stimuli, we found that LDP-3 specifically enhances the osmotic stress-induced activation of JNK and p38.  (+info)