Benjamin Turgeon

NeuroD1/beta2 contributes to cell-specific transcription of the proopiomelanocortin gene.

NeuroD1/beta2 is a basic helix-loop-helix (bHLH) factor expressed in the endocrine cells of the pancreas and in a subset of neurons as they undergo terminal differentiation. We now show that NeuroD1 is expressed in corticotroph cells of the pituitary gland and that it is involved in cell-specific transcription of the proopiomelanocortin (POMC) gene. It was previously shown that corticotroph-specific POMC transcription depends in part on the action of cell-restricted bHLH factors that were characterized as the CUTE (corticotroph upstream transcription element) (M. Therrien and J. Drouin, Mol. Cell. Biol. 13:2342-2353, 1993) complexes. We now demonstrate that these complexes contain NeuroD1 in association with various ubiquitous bHLH dimerization partners. The NeuroD1-containing heterodimers specifically recognize and activate transcription from the POMC promoter E box that confers transcriptional specificity. Interestingly, the NeuroD1 heterodimers activate transcription in synergy with Ptx1, a Bicoid-related homeodomain protein, which also contributes to corticotroph specificity of POMC transcription. In the adult pituitary gland, NeuroD1 transcripts are detected in POMC-expressing corticotroph cells. Taken together with the restricted pattern of Ptx1 expression, these results suggest that these two factors establish the basis of a combinatorial code for the program of corticotroph-specific gene expression.

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

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.

Interpreting Neonatal Lethal Phenotypes in Mouse Mutants: Insights Into Gene Function and Human Diseases

The mouse represents the model of choice to study the biological function of mammalian genes through mutation of its genome. However, the biggest challenge of mouse geneticists remains the phenotypic analysis of mouse mutants. A survey of mouse mutant databases reveals a surprisingly high number of gene mutations leading to neonatal death. These genetically modified mouse mutants have been instrumental in elucidating gene function and have become important models of congenital human diseases. The main complication when phenotyping mutant mice dying during the neonatal period is the large spectrum of physiological systems whose defects can challenge neonatal survival. Here, we present a comprehensive review of gene mutations leading to neonatal lethality and discuss the impact of these mutations on the major physiological processes critical to mouse newborn survival: parturition, breathing, suckling, and homeostasis. Selected examples of mouse mutants are highlighted to illustrate how the precise identification of the timing and cause of death associated with these physiological processes allows for a more profound understanding of the underlying cellular and molecular defects. This review provides a guide for the analysis of neonatal lethal phenotypes in mutant mice that will be helpful for dissecting out the function of specific genes during mouse development.

Loss of Erk3 function in mice leads to intrauterine growth restriction, pulmonary immaturity, and neonatal lethality.

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.

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

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.

The Extracellular Signal-Regulated Kinase 3 (Mitogen-Activated Protein Kinase 6 [MAPK6])–MAPK-Activated Protein Kinase 5 Signaling Complex Regulates Septin Function and Dendrite Morphology

ABSTRACT Mitogen-activated protein kinase-activated protein (MAPKAP) kinase 5 (MK5) deficiency is associated with reduced extracellular signal-regulated kinase 3 (ERK3) (mitogen-activated protein kinase 6) levels, hence we utilized the MK5 knockout mouse model to analyze the physiological functions of the ERK3/MK5 signaling module. MK5-deficient mice displayed impaired dendritic spine formation in mouse hippocampal neurons in vivo. We performed large-scale interaction screens to understand the neuronal functions of the ERK3/MK5 pathway and identified septin7 (Sept7) as a novel interacting partner of ERK3. ERK3/MK5/Sept7 form a ternary complex, which can phosphorylate the Sept7 regulators Binders of Rho GTPases (Borgs). In addition, the brain-specific nucleotide exchange factor kalirin-7 (Kal7) was identified as an MK5 interaction partner and substrate protein. In transfected primary neurons, Sept7-dependent dendrite development and spine formation are stimulated by the ERK3/MK5 module. Thus, the regulation of neuronal morphogenesis is proposed as the first physiological function of the ERK3/MK5 signaling module.

Targeted Inactivation of Mapk4 in Mice Reveals Specific Nonredundant Functions of Erk3/Erk4 Subfamily Mitogen-Activated Protein Kinases

ABSTRACT 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.

The Non-Classical MAP Kinase ERK3 Controls T Cell Activation

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.

The Catalytic Activity of the Mitogen-Activated Protein Kinase Extracellular Signal-Regulated Kinase 3 Is Required To Sustain CD4+ CD8+ Thymocyte Survival

ABSTRACT Extracellular signal-regulated kinase 3 (ERK3) is an atypical member of the mitogen-activated protein kinase (MAPK) family whose function is largely unknown. Given the central role of MAPKs in T cell development, we hypothesized that ERK3 may regulate thymocyte development. Here we have shown that ERK3 deficiency leads to a 50% reduction in CD4+ CD8+ (DP) thymocyte number. Analysis of hematopoietic chimeras revealed that the reduction in DP thymocytes is intrinsic to hematopoietic cells. We found that early thymic progenitors seed the Erk3−/− thymus and can properly differentiate and proliferate to generate DP thymocytes. However, ERK3 deficiency results in a decrease in the DP thymocyte half-life, associated with a higher level of apoptosis. As a consequence, ERK3-deficient DP thymocytes are impaired in their ability to make successful secondary T cell receptor alpha (TCRα) gene rearrangement. Introduction of an already rearranged TCR transgene restores thymic cell number. We further show that knock-in of a catalytically inactive allele of Erk3 fails to rescue the loss of DP thymocytes. Our results uncover a unique role for ERK3, dependent on its kinase activity, during T cell development and show that this atypical MAPK is essential to sustain DP survival during RAG-mediated rearrangements.