Hervé Enslen

Selective Activation of p38 Mitogen-activated Protein (MAP) Kinase Isoforms by the MAP Kinase Kinases MKK3 and MKK6

The cellular response to treatment with proinflammatory cytokines or exposure to environmental stress is mediated, in part, by the p38 group of mitogen-activated protein (MAP) kinases. We report the molecular cloning of a novel isoform of p38 MAP kinase, p38β2. This p38 MAP kinase, like p38α, is inhibited by the pyridinyl imidazole drug SB203580. The p38 MAP kinase kinase MKK6 is identified as a common activator of p38α, p38β2, and p38γ MAP kinase isoforms, while MKK3 activates only p38α and p38γ MAP kinase isoforms. The MKK3 and MKK6 signal transduction pathways are therefore coupled to distinct, but overlapping, groups of p38 MAP kinases.

Interferon-gamma expression by Th1 effector T cells mediated by the p38 MAP kinase signaling pathway.

Signal transduction via MAP kinase pathways plays a key role in a variety of cellular responses, including growth factor‐induced proliferation, differentiation and cell death. In mammalian cells, p38 MAP kinase can be activated by multiple stimuli, such as pro‐inflammatory cytokines and environmental stress. Although p38 MAP kinase is implicated in the control of inflammatory responses, the molecular mechanisms remain unclear. Upon activation, CD4+ T cells differentiate into Th2 cells, which potentiate the humoral immune response or pro‐inflammatory Th1 cells. Here, we show that pyridinyl imidazole compounds (specific inhibitors of p38 MAP kinase) block the production of interferon‐γ (IFNγ) by Th1 cells without affecting IL‐4 production by Th2 cells. These drugs also inhibit transcription driven by the IFNγ promoter. In transgenic mice, inhibition of the p38 MAP kinase pathway by the expression of dominant‐negative p38 MAP kinase results in selective impairment of Th1 responses. In contrast, activation of the p38 MAP kinase pathway by the expression of constitutivelyactivated MAP kinase kinase 6 in transgenic mice caused increased production of IFNγ during the differentiation and activation of Th1 cells. Together, these data demonstrate that the p38 MAP kinase is relevant for Th1 cells, not Th2 cells, and that inhibition of p38 MAP kinase represents a possible site of therapeutic intervention in diseases where a predominant Th1 immune response leads to a pathological outcome. Moreover, our study provides an additional mechanism by which the p38 MAP kinase pathway controls inflammatory responses.

Molecular determinants that mediate selective activation of p38 MAP kinase isoforms

The p38 mitogen‐activated protein kinase (MAPK) group is represented by four isoforms in mammals (p38α, p38β2, p38γ and p38δ). These p38 MAPK isoforms appear to mediate distinct functions in vivo due, in part, to differences in substrate phosphorylation by individual p38 MAPKs and also to selective activation by MAPK kinases (MAPKKs). Here we report the identification of two factors that contribute to the specificity of p38 MAPK activation. One mechanism of specificity is the selective formation of functional complexes between MAPKK and different p38 MAPKs. The formation of these complexes requires the presence of a MAPK docking site in the N‐terminus of the MAPKK. The second mechanism that confers signaling specificity is the selective recognition of the activation loop (T‐loop) of p38 MAPK isoforms. Together, these processes provide a mechanism that enables the selective activation of p38 MAPK in response to activated MAPKK.

A phosphatase cascade by which rewarding stimuli control nucleosomal response

Dopamine orchestrates motor behaviour and reward-driven learning. Perturbations of dopamine signalling have been implicated in several neurological and psychiatric disorders, and in drug addiction. The actions of dopamine are mediated in part by the regulation of gene expression in the striatum, through mechanisms that are not fully understood. Here we show that drugs of abuse, as well as food reinforcement learning, promote the nuclear accumulation of 32-kDa dopamine-regulated and cyclic-AMP-regulated phosphoprotein (DARPP-32). This accumulation is mediated through a signalling cascade involving dopamine D1 receptors, cAMP-dependent activation of protein phosphatase-2A, dephosphorylation of DARPP-32 at Ser 97 and inhibition of its nuclear export. The nuclear accumulation of DARPP-32, a potent inhibitor of protein phosphatase-1, increases the phosphorylation of histone H3, an important component of nucleosomal response. Mutation of Ser 97 profoundly alters behavioural effects of drugs of abuse and decreases motivation for food, underlining the functional importance of this signalling cascade.

MODULATION OF HIV-1 INFECTIVITY BY MAPK, A VIRION-ASSOCIATED KINASE

Infection of a cell by human immunodeficiency virus type 1 (HIV‐1) results in the formation of a reverse transcription complex in which viral nucleic acids are synthesized. Efficient disengagement of the reverse transcription complex from the cell membrane and subsequent nuclear translocation require phosphorylation of reverse transcription complex components by a virion‐associated kinase. In this study, we identify the virion‐associated kinase as mitogen‐activated protein kinase (ERK/MAPK). Upon density gradient fractionation, MAPK, but not its activating kinase MEK, co‐sedimented with viral particles. Expression of a constitutively active, but not kinase‐inactive, MEK1 in virus producer cells was able to activate virion‐associated MAPK in trans. Stimulation of virion‐associated MAPK activity in trans by the mitogen phorbol myristate acetate (PMA) increased viral infectivity. Conversely, suppression of virion‐associated MAPK by specific inhibitors of the MAPK cascade markedly impaired viral infectivity. These studies demonstrate regulation of an early step in HIV‐1 infection by the host cell MAPK signal transduction pathway.

Phosphorylation of NFATc4 by p38 mitogen-activated protein kinases.

ABSTRACT Nuclear factor of activated T cells (NFAT) is implicated in multiple biological processes, including cytokine gene expression, cardiac hypertrophy, and adipocyte differentiation. A conserved NFAT homology domain is identified in all NFAT members. Dephosphorylation of the NFAT homology region is critical for NFAT nuclear translocation and transcriptional activation. Here we demonstrate that NFATc4 is phosphorylated by p38 mitogen-activated protein (MAP) kinase but not by JNK. The p38 MAP kinase phosphorylates multiple residues, including Ser168 and Ser170, in the NFAT homology domain of NFATc4. Replacement of Ser168,170 with Ala promotes nuclear localization of NFATc4 and increases NFAT-mediated transcription activity. Stable expression of Ala168,170 NFATc4, but not of wild-type NFATc4, in NIH 3T3 cells promotes adipocyte formation under differentiation conditions. Molecular analysis indicates that peroxisome proliferator-activated receptor γ2 (PPARγ2) is a target of NFAT. Two distinct NFAT binding elements are located in the PPARγ2 gene promoter. Stable expression of Ala168,170 NFATc4, but not of wild-type NFATc4, increases the expression of PPARγ, which contributes in part to increased adipocyte formation. Thus, NFAT regulates PPARγ gene expression and has a direct role in adipocyte differentiation.

Trio Mediates Netrin-1-Induced Rac1 Activation in Axon Outgrowth and Guidance

ABSTRACT The chemotropic guidance cue netrin-1 promotes neurite outgrowth through its receptor Deleted in Colorectal Cancer (DCC) via activation of Rac1. The guanine nucleotide exchange factor (GEF) linking netrin-1/DCC to Rac1 activation has not yet been identified. Here, we show that the RhoGEF Trio mediates Rac1 activation in netrin-1 signaling. We found that Trio interacts with the netrin-1 receptor DCC in mouse embryonic brains and that netrin-1-induced Rac1 activation in brain is impaired in the absence of Trio. Trio−/− cortical neurons fail to extend neurites in response to netrin-1, while they are able to respond to glutamate. Accordingly, netrin-1-induced commissural axon outgrowth is reduced in Trio−/− spinal cord explants, and the guidance of commissural axons toward the floor plate is affected by the absence of Trio. The anterior commissure is absent in Trio-null embryos, and netrin-1/DCC-dependent axonal projections that form the internal capsule and the corpus callosum are defective in the mutants. Taken together, these findings establish Trio as a GEF that mediates netrin-1 signaling in axon outgrowth and guidance through its ability to activate Rac1.

Differential involvement of p38 mitogen‐activated protein kinase kinases MKK3 and MKK6 in T‐cell apoptosis

The p38 mitogen‐activated protein kinase (p38MAPK) is activated in response to various stimuli, including cellular stress, inflammatory cytokines and cell surface receptors. The activation of p38MAPK is predominantly mediated by the two upstream MAPK kinases MKK3 and MKK6. To study the role of the p38MAPK pathway in vivo, we generated Mkk6−/− mice. We examined whether T‐cell apoptosis is affected in these mice and in our previously reported Mkk3−/− mice. Strikingly, in vivo deletion of double positive thymocytes in Mkk6−/− mice was impaired, whereas Mkk3−/− mice showed no apparent abnormality. Conversely, CD4+T cells from Mkk3−/− but not from Mkk6−/− mice were resistant to activation‐induced cell death and cytokine‐withdrawal‐induced apoptosis. In peripheral CD4+T cells, MKK3 is induced upon stimulation, whereas MKK6 is downregulated. These results suggest a novel mechanism regulating T‐cell apoptosis differentially through the p38MAPK pathway by MKK3 and MKK6.

Activation of p38 Mitogen-Activated Protein Kinase In Vivo Selectively Induces Apoptosis of CD8+ but Not CD4+ T Cells

ABSTRACT CD4+ and CD8+ T cells play specific roles during an immune response. Different molecular mechanisms could regulate the proliferation, death, and effector functions of these two subsets of T cells. The p38 mitogen-activated protein (MAP) kinase pathway is induced by cytokines and environmental stress and has been associated with cell death and cytokine expression. Here we report that activation of the p38 MAP kinase pathway in vivo causes a selective loss of CD8+ T cells due to the induction of apoptosis. In contrast, activation of p38 MAP kinase does not induce CD4+T-cell death. The apoptosis of CD8+ T cells is associated with decreased expression of the antiapoptotic protein Bcl-2. Regulation of the p38 MAP kinase pathway in T cells is therefore essential for the maintenance of CD4/CD8 homeostasis in the peripheral immune system. Unlike cell death, gamma interferon production is regulated by the p38 MAP kinase pathway in both CD4+ and CD8+ T cells. Thus, specific aspects of CD4+and CD8+ T-cell function are differentially controlled by the p38 MAP kinase signaling pathway.

Distinct Roles of c-Jun N-Terminal Kinase Isoforms in Neurite Initiation and Elongation during Axonal Regeneration

c-Jun N-terminal kinases (JNKs) (comprising JNK1–3 isoforms) are members of the MAPK (mitogen-activated protein kinase) family, activated in response to various stimuli including growth factors and inflammatory cytokines. Their activation is facilitated by scaffold proteins, notably JNK-interacting protein-1 (JIP1). Originally considered to be mediators of neuronal degeneration in response to stress and injury, recent studies support a role of JNKs in early stages of neurite outgrowth, including adult axonal regeneration. However, the function of individual JNK isoforms, and their potential effector molecules, remained unknown. Here, we analyzed the role of JNK signaling during axonal regeneration from adult mouse dorsal root ganglion (DRG) neurons, combining pharmacological JNK inhibition and mice deficient for each JNK isoform and for JIP1. We demonstrate that neuritogenesis is delayed by lack of JNK2 and JNK3, but not JNK1. JNK signaling is further required for sustained neurite elongation, as pharmacological JNK inhibition resulted in massive neurite retraction. This function relies on JNK1 and JNK2. Neurite regeneration of jip1−/− DRG neurons is affected at both initiation and extension stages. Interestingly, activated JNKs (phospho-JNKs), as well as JIP1, are also present in the cytoplasm of sprouting or regenerating axons, suggesting a local action on cytoskeleton proteins. Indeed, we have shown that JNK1 and JNK2 regulate the phosphorylation state of microtubule-associated protein MAP1B, whose role in axonal regeneration was previously characterized. Moreover, lack of MAP1B prevents neurite retraction induced by JNK inhibition. Thus, signaling by individual JNKs is differentially implicated in the reorganization of the cytoskeleton, and neurite regeneration.