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Cellular Responses to Stress
By C.P. Downes, C.R. Wolf, D.P. Lane PRINCETON UNIVERSITY PRESS
Copyright © 1999 The Biochemical Society, London
All rights reserved.
ISBN: 978-0-691-00951-3
CHAPTER 1
Signal transduction by the c-Jun N-terminal kinase
Roger J. Davis
Howard Hughes Medical Institute and Program in Molecular Medicine, Department of Biochemistry and Molecular Biology, University of Massachusetts Medical School, 373 Plantation Street, Worcester, MA 01605, U .S.A.
Abstract
The c-Jun N-terminal kinase (JNK) group of mitogen-activated protein kinases (MAP kinases) is activated by exposure of cells to environmental stress and by the treatment of cells with cytokines. The mechanism of activation of JNK is mediated by dual phosphorylation within kinase subdomain VIII on the motif Thr-Pro-Tyr. This phosphorylation is mediated by the MAP kinase kinases MKK4 and MKK7. These MAP kinase kinases serve as signalling molecules that integrate a wide array of stimuli into the activation of the JNK signalling pathway. Studies of the physiological function of JNK have been facilitated by the molecular genetic analysis of JNK signalling in Drosophila and by the creation of mice with targeted disruption of components of the JNK pathway. These studies demonstrate that the JNK pathway regulates AP-I (activator protein-1) transcriptional activity in vivo and indicate that JNK is required for embryonic morphogenesis, the regulation of cellular proliferation and apoptosis, and the response of cells to immunological stimuli.
Introduction
Mitogen-activated protein kinases (MAP kinases) are established to be important mediators of intracellular signalling within cells. These protein kinases function within signalling pathways that are initiated by multiple mechanisms, including the activation of cell surface receptors. A major target of MAP kinase signalling is the regulation of gene expression. These properties implicate MAP kinases in developmental processes and in the response of cells to their environment, for example growth factors, cytokines or exposure to stress. Indeed, studies using both genetic and biochemical approaches have demonstrated the essential role of MAP kinases in mammals, insects, nematodes and plants.
In mammals, three groups of MAP kinases have been identified: the extracellular-signal-regulated kinases (ERKs); the p38 MAP kinases; and the c-Jun N-terminal kinases (JNKs; also known as stress-activated protein kinases, or SAPKs). These MAP kinases are activated by dual phosphorylation within protein kinase subdomain VIII. This phosphorylation is mediated by a protein kinase cascade that consists of a MAP kinase, a MAP kinase kinase and a MAP kinase kinase kinase. Individual MAP kinases are activated by different signalling modules that are regulated bv different stimuli. For example, the ERK group is activated by the MAP kinase kinases MKKl and MKK2; the p38 MAP kinase group is activated by MKK3, MKK4 and MKK6; and the JNK group is activated by MKK4 and MKK7 (Fig. 1). These separate signalling modules allow the integrated response of MAP kinase pathways to different stimuli.
The JNK group of MAP kinases
Three genes that encode JNKs have been identified by molecular cloning. The human genes are JNK1, JNK2 and JNK3. The corresponding genes in the rat have also been identified, Transcripts of each of these genes are alternatively spliced to create mRNAs that encode 46 kDa and 55 kDa JNK isoforms. The presence of a C-terminal extension on the 55 kDa isoforms serves to distinguish these isoforms from the 46 kDa JNK isoforms. An additional site of alternative splicing has been identified within the kinase domains of JNK1 and JNK2, but not JNK3. This pattern of alternative splicing is illustrated in Fig. 2. No functional differences have been detected in experiments designed to compare the 46 kDa and 55 kDa J NK isoforms, In contrast, the alternative splicing of JNKl and JNK2 within the kinase domain causes changes in the substrate specificity of these protein kinases.
Substrate recognition by JNKs is mediated by a binding interaction between a site on the substrate and the JNK. This binding site is independent of the sites of phosphorylation by JNK. Deletion of the JNK binding site prevents phosphorylation of the substrate by JNK. The alternative splicing of JNK1 and JNK2 within the kinase domain changes the specificity of the binding interaction between JNK and its substrates. This observation suggests that indivivdual JNK isoforms target different groups of JNK substrates in vivo.
The JNK MAP kinases are activated by exposure of cells to environmental stress or by treatment of cells with pro-inflammatory cytokines. Targets of the JNK signal transduction pathway include the transcription factors ATF2 (activating transcription factor 2) and c-Jun, These transcription factors are members of the bZIP (basic region leucine zipper) group that bind as homo- and hetero-dimeric complexes to AP-1 and AP-1-like sites in the promoters of many genes. JNK binds to an N-terminal region of ATF2 and c-Jun, and phosphorylates two sites within the activation domain of each transcription factor. This phosphorylation leads to increased transcriptional activity, AP-1 transcriptional activity is also increased by the JNK pathway through increased expression of c-Fos and c-Jun. An increase in c-Fos expression is mediated by activation of the serum response element in the c-Fos promoter. Increased expression of c-Jun is mediated by at least two mechanisms. First, JNK causes increased AP-1 activity, which increases c-Jun expression through the AP-1-like sites in the c-Jun promoter. Secondly, the phosphorylation of c-Jun by JNK causes decreased ubiquitin-mediated degradation of c-Jun and an increase in the half-life of the c-Jun protein.
Together, these biochemical studies indicate that the JNK signal transduction pathway contributes to the regulation of AP-1 transcriptional activity in response to cytokines and environmental stress. Strong support for this hypothesis is provided by genetic evidence indicating that the JNK signalling pathway is required for the normal regulation of AP-1 transcriptional activity.
MKK4 is an activator of the JNK and p38 MAP kinases
JNK is activated by dual phosphorylation on Thr-183 and Tyr-185. This phosphorylation is mediated, in part, by the MAP kinase kinase MKK4 (also known as SEK1). The relationship between the primary sequences of MKK4 and other members of the MAP kinase kinase group is illustrated in Fig. 3. MKK4 phosphorylates and activates JNK in vitro. Furthermore, transfection studies demonstrate that MKK4 activates JNK in vivo. In addition, transfection of cells with dominant-negative MKK4 inhibits JNK activation in vivo. These studies provide biochemical evidence for a role for MKK4 as an activator of JNK.
In vitro protein kinase assays demonstrate that MKK4 is also able to activate the p38 MAP kinases. Transfection assays indicate that MKK4 can act as an activator of the p38 MAP kinase pathway. MKK4 is therefore a candidate activator of the p38 MAP kinase pathway in vivo. However, two lines of evidence suggest that the role of MKK4 as a regulator of the p38 MAP kinase pathway requires further evaluation. First, dominant-negative MKK4 inhibits JNK activity more potently than it inhibits p38 MAP kinase. This differential inhibition of JNK and p38 MAP kinases mav be mediated by the stronger binding interaction that is observed between MKK4 and JNK than between MKK4 and p38 MAP kinase. Secondly, an upstream activator of MKK4, MEKKl (MEK kinase 1), causes selective activation of the JNK signal transduction pathwav in transfection experiments. Together, these data indicate that, while MKK4 does activate p38 MAP kinase in vitro, the role of MKK4 in the regulation of p38 MAP kinase in vivo is unclear. Other studies have implicated the MAP kinase kinases MKK3 and MKK6 as major activators of the p38 MAP kinase in vivo.
Recently, two studies using homologous recombination to create null alleles of the MKK4 gene in murine cells have been reported. Disruption of the MKK4 gene caused embryonic death in mice. Analysis of cells with a homozygous deficiency in MKK4 demonstrated that MKK4 is required for the normal regulation of JNK by environmental stress . These data provide strong genetic evidence that MKK4 does function as an activator of the JNK signal transduction pathway in vivo. No defects in the regulation of the p38 MAP kinase signalling pathway were detected in MKK4 (–/–) cells. The normal regulation of p38 MAP kinase activity in MKK4 (–/–) cells may be caused either: (1) by the absence of a role for MKK4 in p38 MAP kinase regulation in vivo; or (2) by complementation of the MKK4 defect by MKK3 and MKK6. Further studies are required to discriminate between these two possible mechanisms.
MKK7 is an activator of the JNK signal transduction pathway
The existence of a specific activator of JNK that is independent of MKK4 has been proposed previously. Indeed, biochemical studies support the conclusion that MKK4 is not the only activator of JNK in mammalian cells. Furthermore, genetic evidence for this novel JNK activator was obtained from the results of experiments in which the MKK4 gene was disrupted by homologous recombination. These studies demonstrated that, although MKK4 (–/–) cells are defective in JNK regulation, the loss of MKK4 does not block JNK activation completely. They provide definitive evidence that MKK4 represents only one mechanism of activation of the JNK protein kinase in vivo.
Recently, the molecular cloning of a new member of the mammalian MAP kinase kinase group, MKK7, was reported. Comparison of MKK7 with other members of the mammalian MAP kinase kinase group indicates that it is related to the JNK activator MKK4 (Figs. 3 and 4). However, MKK7 is most closely related to the Drosophila protein kinase hemipterous (HEP).
The MAP kinase kinase MKK7 activates JNK, but not the ERK or p38 MAP kinases, in vitro. Transfection assays confirm that MKK7 activates JNK, but not the ERK or p38 MAP kinases, in vivo. MKK7 therefore appears to be the specific J NK activator that has been proposed in previous studies. Thus mammalian cells express two activators of the JNK MAP kinases, MKK4 and MKK7 (Fig. 1).
MKK4 and MKK7 integrate signals initiated at the cell surface
MKK4 and MKK7 function as activators of JNK. Northern blot analysis demonstrates that both MKK4 and MKK7 are widely expressed in human and murine tissues. Although MKK4 and MKK7 are co-expressed, the relative abundance of each MAP kinase kinase differs between tissues. These data indicate that MKK4 and MKK7 serve to integrate the divergent signals that lead to activation of the JNKs in vivo. Further studies are required to identify the signalling mechanisms that lead to the activation of MKK4, MKK7, and MKK4 together with MKK7.
Multiple protein kinases have been reported to function as upstream elements of the J NK signalling pathway (reviewed in). The mixed-lineage kinases MLK2, MLK3 and DLK/MUK (dual leucine-zipper kinase) function as activators of the JNK pathway that phosphorylate and activate MKK4. The protein kinases ASK1 (apoptosis-stimulating kinase 1) and Tpl-2/Cot also activate the MKK4/JNK pathway. In addition, members of the MEKK family, including MEKK1, MEKK2, MEKK3 and MEKK4, activate the JNK signalling pathway by phosphorylation and activation of MKK4. The primary role for MKK4 inferred in each of these studies will need to be re-evaluated in light of the recent identification of the second JNK activator, MKK7.
Other protein kinases have also been implicated as activators of the JNK pathway, although the mechanism by which they activate JNK is unclear. Examples of these kinases include members of the large group of Ste20-related protein kinases, e.g. PAK (p21-activated kinase), GCK (germinal centre kinase), Krs (kinase responsive to stress) and NIK (Nck-interacting kinase). One additional Ste20-related protein kinase that activates the JNK pathway, HPKl (haematopoietic progenitor kinase 1), appears to activate JNK through interaction with the mixed-lineage kinase MLK3.
The mechanism by which environmental stress causes activation of the JNK signalling pathway is unclear, but it has been proposed that this response is mediated by the clustering of cell surface receptors. The signalling mechanisms employed by cell surface receptors to activate the JNK pathway are poorly understood. However, recent studies have led to the elucidation of receptor-proximal components that lead to the activation of the JNK signalling pathway. For example, the adaptor protein TRAF2 (tumour necrosis factor-receptor-associated factor) and the protein kinase RIP (receptor-interacting protein) couple tumour necrosis factor receptor signalling to the JNK pathway. The effect of TRAF2 appears to be mediated by a MAP kinase kinase kinase, which binds to TRAF2. In the case of Fas signalling, the protein Daxx binds to the death domain of Fas and mediates activation of the JNK pathway. Other upstream components of the JNK signalling pathway include the small GTPases Racl and Cdc42 and heterotrimeric GTPases.
Function of the JNK signal transduction pathway
Comparison of the primary sequence of MKK7 with those of other members of the mammalian MAP kinase kinase group demonstrates that MKK7 is most similar to MKK4 (Fig. 1). This similarity in primary sequence reflects the common enzymic property of MKK4 and MKK7 as activators of JNK. However, MKK7 is most similar to the Drosophila MAP kinase kinase HEP. Biochemical analysis of HEP demonstrates that it is a potent activator of Drosophila JNK in vitro. The function of HEP as a physiological JNK activator is supported by genetic analysis. Loss-of-function alleles of the MAP kinase kinase (hep) and JNK (bsk) cause the same embryonic-lethal phenotype. Detailed studies of Drosophila development demonstrate that JNK is required for morphogenetic cell movement during embryogenesis. Similarly, the JNK pathway is required for embryonic viability in mice. It is therefore possible that, as in Drosophila, the JNK signalling pathway is required for embryonic morphogenesis in mammals. The specific targets of the JNK signalling pathway during embryonic development remain to be identified.
The function of the JNK signal transduction pathway in vivo is poorly understood. Genc disruption experiments in mice demonstrate that the JNK pathway is required for the normal regulation of AP-I transcriptional activity. The biological significance of AP-I regulation by the JNK pathway is unclear, but this pathway has been implicated in the stress-induced apoptosis of neurons and other cell types, transformation of pre-B-cells by Bcr-Abl, transformation of fibroblasts by the Met oncogene, survival signals in cells exposed to apoptotic stimuli, co-stimulatory signalling in the immune response of T-cells, and the inflammation-associated expression of E-selectin by endothelial cells. Further studies of the physiological role of the JNK signal transduction pathway in mammals ,will be greatly facilitated by the creation of animals with specific defects in JNK signalling. Targeted disruption of the MKK4 gene causes only a partial defect in JNK signalling. Progress towards understanding the physiological role of the JNK signalling pathway will require analysis of the effects of targeted disruption of the genes encoding MKK7, JNK1, JNK2 and JNK3.
I thank K. Gemme for administrative assistance. The studies performed in this laboratory were supported by grants from the National Cancer Institute. R.J.D. is an investigator of the Howard Hughes Medical Institute.
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Excerpted from Cellular Responses to Stress by C.P. Downes, C.R. Wolf, D.P. Lane. Copyright © 1999 The Biochemical Society, London. Excerpted by permission of PRINCETON UNIVERSITY PRESS.
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