Regulation of atypical MAP kinases ERK3 and ERK4 by the phosphatase DUSP2.
ABSTRACT: 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.
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.
Project description:Classical mitogen-activated protein (MAP) kinases are activated by dual phosphorylation of the Thr-Xxx-Tyr motif in their activation loop, which is catalyzed by members of the MAP kinase kinase family. The atypical MAP kinases extracellular signal-regulated kinase 3 (ERK3) and ERK4 contain a single phospho-acceptor site in this segment and are not substrates of MAP kinase kinases. Previous studies have shown that ERK3 and ERK4 are phosphorylated on activation loop residue Ser-189/Ser-186, resulting in their catalytic activation. However, the identity of the protein kinase mediating this regulatory event has remained elusive. We have used an unbiased biochemical purification approach to isolate the kinase activity responsible for ERK3 Ser-189 phosphorylation. Here, we report the identification of group I p21-activated kinases (PAKs) as ERK3/ERK4 activation loop kinases. We show that group I PAKs phosphorylate ERK3 and ERK4 on Ser-189 and Ser-186, respectively, both in vitro and in vivo, and that expression of activated Rac1 augments this response. Reciprocally, silencing of PAK1/2/3 expression by RNA interference (RNAi) completely abolishes Rac1-induced Ser-189 phosphorylation of ERK3. Importantly, we demonstrate that PAK-mediated phosphorylation of ERK3/ERK4 results in their enzymatic activation and in downstream activation of MAP kinase-activated protein kinase 5 (MK5) in vivo. Our results reveal that group I PAKs act as upstream activators of ERK3 and ERK4 and unravel a novel PAK-ERK3/ERK4-MK5 signaling pathway.
Project description: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.
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.
Project description:The mitogen-activated protein kinase-activated protein kinase-5 (MK5) resides predominantly in the nucleus of resting cells, but p38(MAPK), extracellular signal-regulated kinases-3 and -4 (ERK3 and ERK4), and protein kinase A (PKA) induce nucleocytoplasmic redistribution of MK5. The mechanism by which PKA causes nuclear export remains unsolved. In the study reported here we demonstrated that Ser-115 is an in vitro PKA phosphoacceptor site, and that PKA, but not p38(MAPK), ERK3 or ERK4, is unable to redistribute MK5 S115A to the cytoplasm. However, the phospho-mimicking MK5 S115D mutant resides in the cytoplasm in untreated cells. While p38(MAPK), ERK3 and ERK4 fail to trigger nuclear export of the kinase dead T182A and K51E MK5 mutants, S115D/T182A and K51E/S115D mutants were able to enter the cytoplasm of resting cells. Finally, we demonstrated that mutations in Ser-115 affect the biological properties of MK5. Taken together, our results suggest that Ser-115 plays an essential role in PKA-regulated nuclear export of MK5, and that it also may regulate the biological functions of MK5.
Project description:Erk4 and Erk3 are atypical members of the mitogen-activated protein (MAP) kinase family. The high sequence identity of Erk4 and Erk3 proteins and the similar organization of their genes imply that the two protein kinases are paralogs. Recently, we have shown that Erk3 function is essential for neonatal survival and critical for the establishment of fetal growth potential and pulmonary function. To investigate the specific functions of Erk4, we have generated mice with a targeted disruption of the Mapk4 gene. We show that Erk4-deficient mice are viable and fertile and exhibit no gross morphological or physiological anomalies. Loss of Erk4 is not compensated by changes in Erk3 expression or activity during embryogenesis or in adult tissues. We further demonstrate that additional loss of Erk4 does not exacerbate the fetal growth restriction and pulmonary immaturity phenotypes of Erk3(-/-) mice and does not compromise the viability of Erk3(+/-) neonates. Interestingly, behavioral phenotyping revealed that Erk4-deficient mice manifest depression-like behavior in the forced-swimming test. Our analysis indicates that the MAP kinase Erk4 is dispensable for mouse embryonic development and reveals that Erk3 and Erk4 have acquired specialized functions through evolutionary diversification.
Project description:MK5, a member of the MAPK-activated protein kinase family, is highly expressed in the heart. Whereas MK2 and MK3 are activated by p38 MAPK, MK5 has also been shown to be activated by ERK3 and ERK4. We studied the regulation of MK5 in mouse heart. mRNA for 5 splice variants (MK5.1-5.5), including the original form (MK5.1), was detected. MK5 comprises 14 exons: exon 12 splicing was modified in MK5.2, MK5.3, and MK5.5. MK5.2 and MK5.5 lacked 6 bases at the 3'-end of exon 12, whereas MK5.3 lacked exon 12, resulting in a frame shift and premature termination of translation at codon 3 of exon 13. MK5.4 and MK5.5 lacked exons 2-6, encoding kinase subdomains I-VI, and were kinase-dead. All 5 MK5 variants were detected at the mRNA level in all mouse tissues examined; however, their relative abundance was tissue-specific. Furthermore, the relative abundance of variant mRNA was altered both during hypertrophy and postnatal cardiac development, suggesting that the generation or the stability of MK5 variant mRNAs is subject to regulation. When expressed in HEK293 cells, MK5.1, MK5.2 and MK5.3 were nuclear whereas MK5.4 and MK5.5 were cytoplasmic. A p38 MAPK activator, anisomycin, induced the redistribution of each variant. In contrast, MK5 co-immunoprecipitated ERK3, but not ERK4 or p38 alpha, in control and hypertrophying hearts. GST pull-down assays revealed unbound ERK4 and p38 alpha but no free MK5 or ERK3 in heart lysates. Hence, 1) in heart MK5 complexes with ERK3 and 2) MK5 splice variants may mediate distinct effects thus increasing the functional diversity of ERK3-MK5 signaling.
Project description:Mitogen-activated protein kinase 6/extracellular signal-regulated kinase 3 (MAPK6/ERK3) is an atypical member of the MAPKs. An essential role has been suggested by the perinatal lethal phenotype of ERK3 knockout mice carrying a lacZ insertion in exon 2 due to pulmonary dysfunction and by defects in function, activation, and positive selection of T cells. To study the role of ERK3 in vivo, we generated mice carrying a conditional Erk3 allele with exon 3 flanked by loxP sites. Loss of ERK3 protein was validated after deletion of Erk3 in the female germ line using zona pellucida 3 (Zp3)-cre and a clear reduction of the protein kinase MK5 is detected, providing the first evidence for the existence of the ERK3/MK5 signaling complex in vivo In contrast to the previously reported Erk3 knockout phenotype, these mice are viable and fertile and do not display pulmonary hypoplasia, acute respiratory failure, abnormal T-cell development, reduction of thymocyte numbers, or altered T-cell selection. Hence, ERK3 is dispensable for pulmonary and T-cell functions. The perinatal lethality and lung and T-cell defects of the previous ERK3 knockout mice are likely due to ERK3-unrelated effects of the inserted lacZ-neomycin resistance cassette. The knockout mouse of the closely related atypical MAPK ERK4/MAPK4 is also normal, suggesting redundant functions of both protein kinases.
Project description:Obesity-induced diabetes affects >400 million people worldwide. Uncontrolled lipolysis (free fatty acid release from adipocytes) can contribute to diabetes and obesity. To identify future therapeutic avenues targeting this pathway, we performed a high-throughput screen and identified the extracellular-regulated kinase 3 (ERK3) as a hit. We demonstrated that β-adrenergic stimulation stabilizes ERK3, leading to the formation of a complex with the cofactor MAP kinase-activated protein kinase 5 (MK5), thereby driving lipolysis. Mechanistically, we identified a downstream target of the ERK3/MK5 pathway, the transcription factor FOXO1, which promotes the expression of the major lipolytic enzyme ATGL. Finally, we provide evidence that targeted deletion of ERK3 in mouse adipocytes inhibits lipolysis, but elevates energy dissipation, promoting lean phenotype and ameliorating diabetes. Thus, ERK3/MK5 represents a previously unrecognized signaling axis in adipose tissue and an attractive target for future therapies aiming to combat obesity-induced diabetes.
Project description:Extracellular signal-regulated kinase 3 (ERK3) is an atypical member of the mitogen-activated protein kinase (MAPK) family. It harbors a kinase domain in the N-terminus and a long C-terminus extension. The C-terminus extension comprises a conserved in ERK3 and ERK4 (C34) region and a unique C-terminus tail, which was shown to be required for the interaction of ERK3 with the cytoskeletal protein septin 7. Recent studies have elucidated the role of ERK3 signaling in promoting the motility and invasiveness of cancer cells. However, little is known about the intramolecular regulation of the enzymatic activity and cellular functions of ERK3. In this study, we investigated the role of the elongated C-terminus extension in regulating ERK3 kinase activity and its ability to promote cancer cell migration and invasion. Our study revealed that the deletion of the C-terminus tail greatly diminishes the ability of ERK3 to promote the migration and invasion of lung cancer cells. We identified two molecular mechanisms underlying this effect. Firstly, the deletion of the C-terminus tail decreases the kinase activity of ERK3 towards substrates, including the oncogenic protein steroid receptor co-activator 3 (SRC-3), an important downstream target for ERK3 signaling in cancer. Secondly, in line with the previous finding that the C-terminus tail mediates the interaction of ERK3 with septin 7, we found that the depletion of septin 7 abolished the ability of ERK3 to promote migration, indicating that septin 7 acts as a downstream effector for ERK3-induced cancer cell migration. Taken together, the findings of this study advance our understanding of the molecular regulation of ERK3 signaling by unraveling the role of the C-terminus tail in regulating ERK3 kinase activity and functions in cancer cells. These findings provide useful insights for the development of therapeutic agents targeting ERK3 signaling in cancer.