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 ...

ERK1 and ERK2 are the effector kinases of the ERK1/2 MAP-kinase signaling pathway, which plays a central role in transducing signals controlling cell proliferation, differentiation, and survival. Deregulated activity of the ERK1/2 pathway is linked to a group of developmental syndromes and contributes to the pathogenesis of various human diseases. One fundamental question that remains unaddressed is whether ERK1 and ERK2 have evolved unique physiological functions or whether they are used redundantly to reach a threshold of global ERK activity. Here, we show that the extent of development of the mouse placenta and embryo bearing different combinations of Erk1 and Erk2 alleles is strictly correlated with total ERK1/2 activity. We further demonstrate that transgenic expression of ERK1 fully rescues the embryonic and placental developmental defects associated with the loss of ERK2. We conclude that ERK1 and ERK2 exert redundant functions in mouse development.

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.

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38The physiological functions of the atypical MAP kinase ERK3 remain poorly characterized. 39Previous analysis of mice with a targeted insertion of the lacZ reporter in the Mapk6 locus 40 (Mapk6 lacZ ) showed that inactivation of ERK3 in Mapk6 lacZ mice leads to perinatal lethality 41 associated with intrauterine growth restriction, defective lung maturation, and neuromuscular 42 anomalies. To further explore the role of ERK3 in physiology and disease, we generated novel 43 mouse models expressing a catalytically-inactive (Mapk6 KD ) or conditional (Mapk6  ) allele of 44 ERK3. Surprisingly, we found that mice devoid of ERK3 kinase activity or expression survive 45 the perinatal period without any observable lung or neuromuscular phenotype. ERK3 mutant 46 mice reached adulthood, were fertile and showed no apparent health problem. However, analysis 47 56 57 Extracellular signal-regulated kinase 3 (ERK3) and ERK4 are atypical members of the mitogen-58 activated protein (MAP) kinase family (1). Despite their identification more than 25 years ago, 59 very little is known about their physiological functions. The ERK3 gene (MAPK6 in human) is 60 expressed ubiquitously in adult mammalian tissues, while ERK4 gene (MAPK4) shows a 61 restricted expression profile being detected mainly in brain tissue (2-4) 62 (www.gtexportal.org/home). During mouse embryogenesis, ERK3 mRNA and protein levels 63 peak around embryonic day 11, after the onset of organogenesis, and then decline thereafter (5). 64Expression of ERK3 is also up-regulated during in vitro differentiation of cell line models along 65 the neuronal or muscle lineage (2, 6). These early observations have suggested a potential role 66 for ERK3 signaling in cell differentiation and tissue development. 67 68 Analogous to classical MAP kinases, ERK3 is activated by phosphorylation of the activation 69 loop, which stimulates its intrinsic catalytic activity and affinity for the substrate MK5 (7). 70Activation loop phosphorylation is mediated by Group I p21-activated kinases (8, 9), and is 71 reversed by the action of the MAP kinase phosphatase DUSP2 (10). Of note, it has been 72 previously proposed that ERK3 enzymatic activity is dispensable for MK5 activation and that 73 ERK3 may exert a scaffolding function (11). However, other experiments using coupled in vitro 74 kinase assays did not suppport this hypothesis (7). A more recent study reported that 75 overexpression of ERK3 induces rounding of MDA-MB-231 breast cancer cells by an unknown 76 mechanism independent of its kinase activity (12). The physiological importance of catalytic and 77 non-catalytic functions of ERK3 remains an open question. 78 * Mixed C57Bl/6J-129/Sv genetic background

Most patients with hepatocellular carcinoma (HCC) are diagnosed at a late stage and have few therapeutic options and a poor prognosis. This is due to the lack of clearly defined underlying mechanisms or a dominant oncogene that can be targeted pharmacologically, unlike in other cancer types. Here, we report the identification of a previously uncharacterized oncogenic signaling pathway in HCC that is mediated by the tyrosine kinase Yes. Using genetic and pharmacological interventions in cellular and mouse models of HCC, we showed that Yes activity was necessary for HCC cell proliferation. Transgenic expression of activated Yes in mouse hepatocytes was sufficient to induce liver tumorigenesis. Yes phosphorylated the transcriptional coactivators YAP and TAZ (YAP/TAZ), promoting their nuclear accumulation and transcriptional activity in HCC cells and liver tumors. We also showed that YAP/TAZ were effectors of the Yes-dependent oncogenic transformation of hepatocytes. Src family kinase activation correlated with the tyrosine phosphorylation and nuclear localization of YAP in human HCC and was associated with increased tumor burden in mice. Specifically, high Yes activity predicted shorter overall survival in patients with HCC. Thus, our findings identify Yes as a potential therapeutic target in HCC.