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Activation of MK5/PRAK by the atypical MAP kinase ERK3 defines a novel signal transduction pathway.

ABSTRACT: Extracellular signal-regulated kinase 3 (ERK3) is an atypical mitogen-activated protein kinase (MAPK), which is regulated by protein stability. However, its function is unknown and no physiological substrates for ERK3 have yet been identified. Here we demonstrate a specific interaction between ERK3 and MAPK-activated protein kinase-5 (MK5). Binding results in nuclear exclusion of both ERK3 and MK5 and is accompanied by ERK3-dependent phosphorylation and activation of MK5 in vitro and in vivo. Endogenous MK5 activity is significantly reduced by siRNA-mediated knockdown of ERK3 and also in fibroblasts derived from ERK3-/- mice. Furthermore, increased levels of ERK3 protein detected during nerve growth factor-induced differentiation of PC12 cells are accompanied by an increase in MK5 activity. Conversely, MK5 depletion causes a dramatic reduction in endogenous ERK3 levels. Our data identify the first physiological protein substrate for ERK3 and suggest a functional link between these kinases in which MK5 is a downstream target of ERK3, while MK5 acts as a chaperone for ERK3. Our findings provide valuable tools to further dissect the regulation and biological roles of both ERK3 and MK5.

SUBMITTER: Seternes OM

PROVIDER: S-EPMC535098 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Docking of PRAK/MK5 to the atypical MAPKs ERK3 and ERK4 defines a novel MAPK interaction motif.

Project description:ERK3 and ERK4 are atypical MAPKs in which the canonical TXY motif within the activation loop of the classical MAPKs is replaced by SEG. Both ERK3 and ERK4 bind, translocate, and activate the MAPK-activated protein kinase (MK) 5. The classical MAPKs ERK1/2 and p38 interact with downstream MKs (RSK1-3 and MK2-3, respectively) through conserved clusters of acidic amino acids, which constitute the common docking (CD) domain. In contrast to the classical MAPKs, the interaction between ERK3/4 and MK5 is strictly dependent on phosphorylation of the SEG motif of these kinases. Here we report that the conserved CD domain is dispensable for the interaction of ERK3 and ERK4 with MK5. Using peptide overlay assays, we have defined a novel MK5 interaction motif (FRIEDE) within both ERK4 and ERK3 that is essential for binding to the C-terminal region of MK5. This motif is located within the L16 extension lying C-terminal to the CD domain in ERK3 and ERK4 and a single isoleucine to lysine substitution in FRIEDE totally abrogates binding, activation, and translocation of MK5 by both ERK3 and ERK4. These findings are the first to demonstrate binding of a physiological substrate via this region of the L16 loop in a MAPK. Furthermore, the link between activation loop phosphorylation and accessibility of the FRIEDE interaction motif suggests a switch mechanism for these atypical MAPKs in which the phosphorylation status of the activation loop regulates the ability of both ERK3 and ERK4 to bind to a downstream effector.

| S-EPMC2740564 | biostudies-literature

Elimination of protein kinase MK5/PRAK activity by targeted homologous recombination.

Project description:MK5 (mitogen-activated protein kinase [MAPK]-activated protein kinase 5), also designated PRAK (p38-regulated and -activated kinase), was deleted from mice by homologous recombination. Although no MK5 full-length protein and kinase activity was detected in the MK5 knockout mice, the animals were viable and fertile and did not display abnormalities in tissue morphology or behavior. In addition, these mice did not show increased resistance to endotoxic shock or decreased lipopolysaccharide-induced cytokine production. Hence, MK5 deletion resulted in a phenotype very different from the complex inflammation-impaired phenotype of mice deficient in MK2, although MK2 and MK5 exhibit evolutional, structural, and apparent extensive functional similarities. To explain this discrepancy, we used wild-type cells and embryonic fibroblasts from both MK2 and MK5 knockout mice as controls to reexamine the mechanism of activation, the interaction with endogenous p38 MAPK, and the substrate specificity of both enzymes. In contrast to MK2, which shows interaction with and chaperoning properties for p38 MAPK and which is activated by extracellular stresses such as arsenite or sorbitol treatment, endogenous MK5 did not show these properties. Furthermore, endogenous MK5 is not able to phosphorylate Hsp27 in vitro and in vivo. We conclude that the differences between the phenotypes of MK5- and MK2-deficient mice result from clearly different functional properties of both enzymes.

| S-EPMC207624 | biostudies-literature

The diterpenoid alkaloid noroxoaconitine is a Mapkap kinase 5 (MK5/PRAK) inhibitor.

Project description:The mitogen-activated protein kinase-activated protein kinase MK5 is ubiquitously expressed in vertebrates and is implicated in cell proliferation, cytoskeletal remodeling, and anxiety behavior. This makes MK5 an attractive drug target. We tested several diterpenoid alkaloids for their ability to suppress MK5 kinase activity. We identified noroxoaconitine as an ATP competitor that inhibited the catalytic activity of MK5 in vitro (IC₅₀ = 37.5 μM; K(i) = 0.675 μM) and prevented PKA-induced nuclear export of MK5, a process that depends on kinase active MK5. MK5 is closely related to MK2 and MK3, and noroxoaconitine inhibited MK3- and MK5- but not MK2-mediated phosphorylation of the common substrate Hsp27. Molecular docking of noroxoaconitine into the ATP binding sites indicated that noroxoaconitine binds more strongly to MK5 than to MK3. Noroxoaconitine and derivatives may help in elucidating the precise biological functions of MK5 and may prove to have therapeutic values.

| S-EPMC3016492 | biostudies-literature

The non-classical MAP kinase ERK3 controls T cell activation.

Project description:The classical mitogen-activated protein kinases (MAPKs) ERK1 and ERK2 are activated upon stimulation of cells with a broad range of extracellular signals (including antigens) allowing cellular responses to occur. ERK3 is an atypical member of the MAPK family with highest homology to ERK1/2. Therefore, we evaluated the role of ERK3 in mature T cell response. Mouse resting T cells do not transcribe ERK3 but its expression is induced in both CD4⁺ and CD8⁺ T cells following T cell receptor (TCR)-induced T cell activation. This induction of ERK3 expression in T lymphocytes requires activation of the classical MAPK ERK1 and ERK2. Moreover, ERK3 protein is phosphorylated and associates with MK5 in activated primary T cells. We show that ERK3-deficient T cells have a decreased proliferation rate and are impaired in cytokine secretion following in vitro stimulation with low dose of anti-CD3 antibodies. Our findings identify the atypical MAPK ERK3 as a new and important regulator of TCR-induced T cell activation.

| S-EPMC3903551 | biostudies-literature

Scaffolding by ERK3 regulates MK5 in development.

Project description:Extracellular-regulated kinase 3 (ERK3, MAPK6) is an atypical member of the ERKs, lacking the threonine and tyrosine residues in the activation loop, carrying a unique C-terminal extension and being mainly regulated by its own protein stability and/or by autophosphorylation. Here we show that ERK3 specifically interacts with the MAPK-activated protein kinase 5 (MK5 or PRAK) in vitro and in vivo. Expression of ERK3 in mammalian cells leads to nuclear-cytoplasmic translocation and activation of MK5 and to phosphorylation of both ERK3 and MK5. Remarkably, activation of MK5 is independent of ERK3 enzymatic activity, but depends on its own catalytic activity as well as on a region in the C-terminal extension of ERK3. In mouse embryonic development, mRNA expression patterns of ERK3 and MK5 suggest spatiotemporal coexpression of both kinases. Deletion of MK5 leads to strong reduction of ERK3 protein levels and embryonic lethality at about stage E11, where ERK3 expression in wild-type mice is maximum, indicating a role of this signalling module in development.

| S-EPMC535084 | biostudies-literature

Regulation of atypical MAP kinases ERK3 and ERK4 by the phosphatase DUSP2.

Project description:The atypical MAP kinases ERK3 and ERK4 are activated by phosphorylation of a serine residue lying within the activation loop signature sequence S-E-G. However, the regulation of ERK3 and ERK4 phosphorylation and activity is poorly understood. Here we report that the inducible nuclear dual-specificity MAP kinase phosphatase (MKP) DUSP2, a known regulator of the ERK and p38 MAPKs, is unique amongst the MKP family in being able to bind to both ERK3 and ERK4. This interaction is mediated by a conserved common docking (CD) domain within the carboxyl-terminal domains of ERK3 and ERK4 and the conserved kinase interaction motif (KIM) located within the non-catalytic amino terminus of DUSP2. This interaction is direct and results in the dephosphorylation of ERK3 and ERK4 and the stabilization of DUSP2. In the case of ERK4 its ability to stabilize DUSP2 requires its kinase activity. Finally, we demonstrate that expression of DUSP2 inhibits ERK3 and ERK4-mediated activation of its downstream substrate MK5. We conclude that the activity of DUSP2 is not restricted to the classical MAPK pathways and that DUSP2 can also regulate the atypical ERK3/4-MK5 signalling pathway in mammalian cells.

| S-EPMC5333157 | biostudies-literature

Comparative Analysis of Two Gene-Targeting Approaches Challenges the Tumor-Suppressive Role of the Protein Kinase MK5/PRAK.

Project description:MK5 (MAPK-activated protein kinase 5) or PRAK (p38-regulated and -activated kinase) are alternative names for a serine/threonine protein kinase downstream to ERK3/4 and p38 MAPK. A previous gene targeting approach for MK5/PRAK (termed here MK5/PRAK-Δex8) revealed a seemingly tumor-suppressive role of MK5/PRAK in DMBA-induced one step skin carcinogenesis and Ras-induced transformation. Here we demonstrate that an alternative targeting strategy of MK5/PRAK (termed MK5/PRAK-Δex6) increased neither tumor incidence in the one step skin carcinogenesis model, nor Ras-induced transformation in primary cells. Interestingly, due to the targeting strategies and exon skipping both knockouts do not completely abolish the generation of MK5/PRAK protein, but express MK5/PRAK deletion mutants with different biochemical properties depending on the exon targeted: Targeting of exon 6 leads to expression of an unstable cytoplasmic catalytically inactive MK5/PRAK-Δex6 mutant while targeting of exon 8 results in a more stable nuclear MK5/PRAK-Δex8 mutant with residual catalytic activity. The different properties of the MK5/PRAK deletion mutants could be responsible for the observed discrepancy between the knockout strains and challenge the role of MK5/PRAK in p53-dependent tumor suppression. Further MK5/PRAK knockout and knock-in mouse strains will be necessary to assign a physiological function to MK5/PRAK in this model organism.

| S-EPMC4546416 | biostudies-literature

Subcellular location defines GPCR signal transduction

Project description:G protein-coupled receptors in intracellular organelles can be activated in response to membrane permeant ligands, which contributes to the diversity and specificity of agonist action. The opioid receptors (ORs) provide a striking example, where small molecule opioid drugs activate ORs in the Golgi apparatus within seconds of drug addition. To date, our knowledge on the signaling of intracellular GPCRs remains incomplete and it is unknown if the downstream events triggered by ORs in plasma membrane and Golgi apparatus differ. To address this gap, we analyzed OR-mediated gene expression changes upon treatment with impermeant peptide ligand DPDPE, permeant ligand SNC80 or ICI-SNC80 (Golgi-restricted signaling). We show that DOR activation in the Golgi does not trigger any gene transcription at these two time points as compare to plasma membrane receptors. The study delineates OR signal transduction with unprecedented resolution and reveals that the subcellular location defines the signaling effect promoted by opioid drugs.

2023-04-24 | GSE221762 | GEO

PRAK, a novel protein kinase regulated by the p38 MAP kinase.

Project description:We have identified and cloned a novel serine/ threonine kinase, p38-regulated/activated protein kinase (PRAK). PRAK is a 471 amino acid protein with 20-30% sequence identity to the known MAP kinase-regulated protein kinases RSK1/2/3, MNK1/2 and MAPKAP-K2/3. PRAK was found to be expressed in all human tissues and cell lines examined. In HeLa cells, PRAK was activated in response to cellular stress and proinflammatory cytokines. PRAK activity was regulated by p38alpha and p38beta both in vitro and in vivo and Thr182 was shown to be the regulatory phosphorylation site. Activated PRAK in turn phosphorylated small heat shock protein 27 (HSP27) at the physiologically relevant sites. An in-gel kinase assay demonstrated that PRAK is a major stress-activated kinase that can phosphorylate small heat shock protein, suggesting a potential role for PRAK in mediating stress-induced HSP27 phosphorylation in vivo.

| S-EPMC1170675 | biostudies-other

Subcellular location defines GPCR signal transduction

Project description:G protein-coupled receptors in intracellular organelles can be activated in response to membrane permeant ligands, which contributes to the diversity and specificity of agonist action. The opioid receptors (ORs) provide a striking example, where small molecule opioid drugs activate ORs in the Golgi apparatus within seconds of drug addition. To date, our knowledge on the signaling of intracellular GPCRs remains incomplete and it is unknown if the downstream events triggered by ORs in plasma membrane and Golgi apparatus differ. To address this gap, we analyzed OR-mediated phosphorylation changes in cells treated with SNC80 (signaling control) or ICI-SNC80 (Golgi-restricted signaling) for 5 min or 25 min. We show that OR activation in the plasma membrane or Golgi apparatus have strikingly different downstream effects on protein phosphorylation. The study delineates OR signal transduction with unprecedented resolution and reveals that the subcellular location defines the signaling effect promoted by opioid drugs.

2023-04-25 | PXD038921 | Pride

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