Extracellular signal-regulated kinase 3 (Erk3) is an atypical member of the mitogen-activated protein (MAP) kinase family. No function has yet been ascribed to this MAP kinase. Here we show that targeted disruption of the Mapk6 gene (encoding Erk3) leads to intrauterine growth restriction, associated with marked pulmonary hypoplasia, and early neonatal death during the first day of life. Around 40% of Erk3 ؊/؊ neonates die within minutes after birth from acute respiratory failure. Erk3-deficient mice have normal lung-branching morphogenesis, but show delayed lung maturation characterized by decreased sacculation, atelectasis, and defective type II pneumocyte differentiation. Interestingly, in utero administration of glucocorticoid promoted fetal lung maturity and rescued differentiation of type II cells, but failed to alter the neonatal lethality. We observed that loss of Erk3 retards intrauterine growth, as reflected by a marked reduction in fetal lung, heart, and liver weights, and by low body weight at birth. Importantly, we found that insulin-like growth factor (IGF)-2 levels are decreased in the serum of Erk3-deficient mice. Our findings reveal a critical role for Erk3 in the establishment of fetal growth potential and pulmonary function in the mouse.AP kinases are a family of serine/threonine kinases that play a key role in transducing environmental stimuli into a wide range of intracellular responses (1). In mammals, 14 MAP kinase genes have been identified that define seven distinct MAP kinase signaling pathways (2). The classical MAP kinases are phosphorylated and activated by members of the MAP kinase kinase/Mek family and comprise the well-characterized Erk1/ Erk2, Jnk1/2/3, p38␣/␤/␥/␦, and Erk5 enzymes. Atypical MAP kinases include Erk3/Erk4, NLK, and Erk7. Much less is known about the mechanisms of regulation, substrate specificity, and physiological functions of this latter group of MAP kinases.The Erk3 gene was originally identified by homology-based cloning using the Erk1 cDNA as probe (3). Erk3 exists as a 100-kDa protein consisting of a kinase domain at the N terminus followed by a unique C-terminal extension of unknown function (3-5). Despite the significant homology of their kinase domains, several properties distinguish Erk3 from Erk1/Erk2 and other classical MAP kinases. Erk3 contains a single phospho-acceptor site in the activation loop instead of the canonical dual phosphorylation motif Thr-Xaa-Tyr. Unlike classical MAP kinases, activation loop phosphorylation of Erk3 is detected in resting cells and is minimally modulated by mitogenic or stress stimuli (6). Functionally, Erk3 does not phosphorylate generic MAP kinase substrates such as myelin basic protein [(7); unpublished data], indicating that it has different or more restricted substrate specificity. The only substrate of Erk3 that has been identified so far is the MAP kinase-activated protein kinase MK5 (8, 9). Structurally, Erk3 is most closely related to the MAP kinase Erk4. The two proteins display 73% amino acid identity in the ...

OBJECTIVE-Glucagon-like peptide-1 (GLP-1) is a growth and differentiation factor for mature ␤-cells and their precursors. However, the overall effect of GLP-1 on increasing ␤-cell mass in both in vivo and in vitro conditions is relatively small, and augmenting this effect would be beneficial for the treatment or prevention of type 1 and type 2 diabetes. Here, we searched for cellular mechanisms that may limit the proliferative effect of GLP-1 and tested whether blocking them could increase ␤-cell proliferation. RESEARCH DESIGN AND METHODS-We examined GLP-1-regulated genes in ␤TC-Tet cells by cDNA microarrays. To assess the effect of some of these gene on cell proliferation, we reduced their expression using small heterogenous RNA in ␤-cell lines and primary mouse islets and measured [ 3 H]thymidine or 5Ј-bromo-2Ј-deoxyuridine incorporation.RESULTS-We identified four negative regulators of intracellular signaling that were rapidly and strongly activated by GLP-1: the regulator of G-protein-signaling RGS2; the cAMP response element-binding protein (CREB) antagonists cAMP response element modulator (CREM)-␣ and ICERI; and the dual specificity phosphatase DUSP14, a negative regulator of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase 1/2 (ERK1/2) pathway. We show that knockdown of CREM␣ or DUSP14 or expression of a dominant-negative form of DUSP14 increased ␤-cell line proliferation and enhanced the GLP-1-induced proliferation of primary ␤-cells.CONCLUSIONS-Together, our data show that 1) the cAMP/ protein kinase A/CREB and MAPK/ERK1/2 pathways can additively control ␤-cell proliferation, 2) ␤-cells have evolved several mechanisms limiting GLP-1-induced cellular proliferation, and 3) blocking these mechanisms increases the positive effect of GLP-1 on ␤-cell mass. Diabetes 57:584-593, 2008 A reduction in pancreatic ␤-cell mass and function triggers the overt phase of type 2 diabetes (1,2), whereas the destruction of ␤-cells by autoimmune attack causes type 1 diabetes (3). Thus, finding means to restore ␤-cell mass by stimulating their replication or their regeneration from precursor cells is a major goal of current research. At present, the molecular mechanisms that control these processes are poorly understood. However, we know that the glucoincretin hormone glucagon-like peptide-1 (GLP-1) can act as a growth and differentiation factor for mature ␤-cells and their precursors. Chronic administration of GLP-1 receptor agonists or inhibition of the GLP-1 degrading enzyme dipeptidylpeptidase IV increases ␤-cell mass in mice or rats (4 -9). This can result from a direct effect on the stimulation of mature ␤-cell proliferation (10 -12) but also from an increased differentiation of pancreatic precursors into insulin-producing cells (13-15) or a combination of both. The effect on ␤-cell mass is, however, relatively small; augmenting it may pave the way to reach the therapeutic goal of restoring a sufficient number of functional ␤-cells.The ␤-cell GLP-1 receptor is a G s -protein-coupled rec...

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.Mitogen-activated protein (MAP) kinases are core components of evolutionarily conserved signaling pathways that play a key role in eukaryotic signal transduction. These enzymes process information from a wide variety of extracellular stimuli and cellular perturbations to control multiple physiological processes required to maintain normal cellular and tissue homeostasis (4,6,13,26,41). The MAP kinase family is composed of seven distinct subfamilies in mammals: Erk1/Erk2, Jnk1/2/3, p38␣/␤/␦/␥, Erk5, Erk3/Erk4, Nlk, and Erk7 (4). Among these, Erk4 remains the least characterized member of the family.Erk4 (originally known as p63 mapk ) is a 70-kDa protein kinase that is most closely related to the MAP kinase Erk3, with 73% amino acid identity within the kinase catalytic domain (1, 10). In addition to their kinase domain located at the N terminus, the two proteins contain a noncatalytic C-terminal region of unknown function. The first 150 residues of the Cterminal extension display nearly 50% identity between the two kinases, whereas the extreme C terminus differs both in length and in sequence. Erk4 and Erk3 also contain a single phosphoacceptor site (SEG) in the activation loop and the unique sequence SPR instead of APE in subdomain VIII of the kinase domain, two features that distinguish them from other MAP kinases. At the genomic level, the genes encoding Erk4 (Mapk4) and Erk3 (Mapk6) share a similar organization of exon/intron boundaries, suggesting that they arose by duplication of a common ancestor (37). All these observations imply that Erk4 and Erk3 are true paralogous proteins.The cellular and physiological functions of Erk4 remain to be defined. Erk4 and Erk3 have a more restricted substrate specificity than conventional MAP kinases...

The physiological functions of the atypical mitogen-activated protein kinase extracellular signal-regulated kinase 3 (ERK3) remain poorly characterized. Previous analysis of mice with a targeted insertion of the lacZ reporter in the Mapk6 locus (Mapk6 lacZ ) showed that inactivation of ERK3 in Mapk6 lacZ mice leads to perinatal lethality associated with intrauterine growth restriction, defective lung maturation, and neuromuscular anomalies. To further explore the role of ERK3 in physiology and disease, we generated novel mouse models expressing a catalytically inactive (Mapk6 KD ) or conditional (Mapk6 ⌬ ) allele of ERK3. Surprisingly, we found that mice devoid of ERK3 kinase activity or expression survive the perinatal period without any observable lung or neuromuscular phenotype. ERK3 mutant mice reached adulthood, were fertile, and showed no apparent health problem. However, analysis of growth curves revealed that ERK3 kinase activity is necessary for optimal postnatal growth. To gain insight into the genetic basis underlying the discrepancy in phenotypes of different Mapk6 mutant mouse models, we analyzed the regulation of genes flanking the Mapk6 locus by quantitative PCR. We found that the expression of several Mapk6 neighboring genes is deregulated in Mapk6 lacZ mice but not in Mapk6 KD or Mapk6 ⌬ mutant mice. Our genetic analysis suggests that off-target effects of the targeting construct on local gene expression are responsible for the perinatal lethality phenotype of Mapk6 lacZ mutant mice.