Apoptosis plays an important role during neuronal development, and defects in apoptosis may underlie various neurodegenerative disorders. To characterize molecular mechanisms that regulate neuronal apoptosis, the contributions to cell death of mitogen-activated protein (MAP) kinase family members, including ERK (extracellular signal-regulated kinase), JNK (c-JUN NH2-terminal protein kinase), and p38, were examined after withdrawal of nerve growth factor (NGF) from rat PC-12 pheochromocytoma cells. NGF withdrawal led to sustained activation of the JNK and p38 enzymes and inhibition of ERKs. The effects of dominant-interfering or constitutively activated forms of various components of the JNK-p38 and ERK signaling pathways demonstrated that activation of JNK and p38 and concurrent inhibition of ERK are critical for induction of apoptosis in these cells. Therefore, the dynamic balance between growth factor-activated ERK and stress-activated JNK-p38 pathways may be important in determining whether a cell survives or undergoes apoptosis.

Protein kinases activated by dual phosphorylation on Tyr and Thr (MAP kinases) can be grouped into two major classes: ERK and JNK. The ERK group regulates multiple targets in response to growth factors via a Ras-dependent mechanism. In contrast, JNK activates the transcription factor c-Jun in response to pro-inflammatory cytokines and exposure of cells to several forms of environmental stress. Recently, a novel mammalian protein kinase (p38) that shares sequence similarity with mitogen-activated protein (MAP) kinases was identified. Here, we demonstrate that p38, like JNK, is activated by treatment of cells with pro-inflammatory cytokines and environmental stress. The mechanism of p38 activation is mediated by dual phosphorylation on Thr-180 and Tyr-182. Immunofluorescence microscopy demonstrated that p38 MAP kinase is present in both the nucleus and cytoplasm of activated cells. Together, these data establish that p38 is a member of the mammalian MAP kinase group.

Mammalian mitogen-activated protein (MAP) kinases include extracellular signal-regulated protein kinase (ERK), c-Jun amino-terminal kinase (JNK), and p38 subgroups. These MAP kinase isoforms are activated by dual phosphorylation on threonine and tyrosine. Two human MAP kinase kinases (MKK3 and MKK4) were cloned that phosphorylate and activate p38 MAP kinase. These MKK isoforms did not activate the ERK subgroup of MAP kinases, but MKK4 did activate JNK. These data demonstrate that the activators of p38 (MKK3 and MKK4), JNK (MKK4), and ERK (MEK1 and MEK2) define independent MAP kinase signal transduction pathways.

The p38 mitogen-activated protein (MAP) kinase signal transduction pathway is activated by proinflammatory cytokines and environmental stress. The detection of p38 MAP kinase in the nucleus of activated cells suggests that p38 MAP kinase can mediate signaling to the nucleus. To test this hypothesis, we constructed expression vectors for activated MKK3 and MKK6, two MAP kinase kinases that phosphorylate and activate p38 MAP kinase. Expression of activated MKK3 and MKK6 in cultured cells caused a selective increase in p38 MAP kinase activity. Cotransfection experiments demonstrated that p38 MAP kinase activation causes increased reporter gene expression mediated by the transcription factors ATF2 and Elk-1. These data demonstrate that the nucleus is one target of the p38 MAP kinase signal transduction pathway.Several mitogen-activated protein (MAP) kinase signal transduction pathways have been detected in mammalian cells (15). Three groups of MAP kinases have been molecularly cloned: ERK (7, 8), JNK (16,22,31,34,55), and p38 (27,35,47). These MAP kinases are activated by dual phosphorylation on Thr and Tyr within the motif Thr-Xaa-Tyr in subdomain VIII (15). The sequence of this dual phosphorylation motif differs for each MAP kinase group as follows: p38, Thr-GlyTyr; JNK, Thr-Pro-Tyr; and ERK, Thr-Glu-Tyr. Each MAP kinase group has a distinct substrate specificity and is regulated by a separate signal transduction pathway (15). Mammalian cells therefore contain multiple MAP kinase signal transduction pathways that mediate the effects of extracellular stimuli on a wide array of biological processes.Detailed studies of the JNK and ERK groups of MAP kinase have led to significant insight into the physiological function of these signaling pathways (6, 13-15, 40, 45). In contrast, the role of the p38 MAP kinase signal transduction pathway is poorly understood (20,27,35,44,47). p38 MAP kinase is weakly activated by protein kinase C and receptor tyrosine kinases but is strongly activated by the treatment of cells with inflammatory cytokines (e.g., tumor necrosis factor and interleukin-1) and environmental stress (e.g., osmotic shock and UV radiation) (20,27,35,44,47). The contribution of the p38 MAP kinase pathway to the cellular response to these stimuli has not been established. However, recent studies have implicated p38 MAP kinase in the phosphorylation of the small heat shock protein Hsp27 (20, 47), in increased cytokine expression (35), and in programmed cell death (61). Furthermore, in vitro protein kinase assays demonstrate that p38 MAP kinase phosphorylates MAPKAP kinase-2 (20, 47) and the transcription factor ATF2 (17, 44).The mechanism of p38 MAP kinase activation is mediated by dual phosphorylation on Thr and Tyr within the motif ThrGly-Tyr located in subdomain VIII (44). The p38 MAP kinase activator MKK3 has been molecularly cloned (17). MKK3 is a protein kinase that phosphorylates and activates p38 MAP kinase but does not phosphorylate the related JNK or ERK MAP kinases (17). MKK3 is therefore a specific act...

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, p38␤2. This p38 MAP kinase, like p38␣, is inhibited by the pyridinyl imidazole drug SB203580. The p38 MAP kinase kinase MKK6 is identified as a common activator of p38␣, p38␤2, and p38␥ MAP kinase isoforms, while MKK3 activates only p38␣ and p38␥ MAP kinase isoforms. The MKK3 and MKK6 signal transduction pathways are therefore coupled to distinct, but overlapping, groups of p38 MAP kinases. Mitogen-activated protein (MAP)1 kinases are proline-directed protein kinases that mediate the effects of numerous extracellular stimuli on a wide array of biological processes, such as cellular proliferation, differentiation, and death (1). Three groups of mammalian MAP kinases have been studied in detail: the extracellular signal-regulated kinases (ERK) (2), the c-Jun NH 2 -terminal kinases (JNK) (3), and the p38 MAP kinases (3). The ERKs are robustly activated by growth factors and phorbol ester, but are only weakly activated by cytokines and environmental stress. In contrast, JNK and p38 MAP kinases are strongly activated by cytokines and environmental stress, but are poorly activated by growth factors and phorbol ester.Recently, progress toward understanding the physiological role of the p38 MAP kinases has been achieved through the use of drugs that bind p38 MAP kinase (4, 5). These drugs are pyridinyl imidazole derivatives that inhibit p38 MAP kinase activity (4, 5). Studies using these drugs indicate that p38 MAP kinase is required for lipopolysaccharide-induced production of IL-1 and TNF in monocytes (4), the induction of IL-6 and granulocyte-macrophage/colony-stimulating factor transcription by TNF (6), the proliferation of T cells in response to IL-2 and IL-7 (7), and the stress-induced transcription of c-jun and c-fos in fibroblasts (8). The targets of p38 MAP kinase that mediate these responses are poorly characterized. However, biochemical studies indicate that p38 MAP kinase signaling pathway activates the transcription factors CREB and ATF1 (9, 10), ATF2 (11, 12), CHOP (13), and MEF-2C (14). The p38 MAP kinase also activates other protein kinases, such as Mapkap-2 (15-17), 19), and Mnk1/2 (20, 21).The p38 MAP kinase group includes the isoforms p38␣ (4, 12, 16, 17, 22), p38␤ (23), and p38␥ (24 -27). Recent studies indicate the presence of a fourth p38 MAP kinase isoform, p38␦ (28, 29). These p38 MAP kinases are widely expressed in many tissues and are activated by dual phosphorylation on Thr and Tyr within the motif Thr-Gly-Tyr located in kinase subdomain VIII (12). This phosphorylation is mediated by a protein kinase cascade (1). Components of this signaling pathway include the MAP kinase kinases MKK3 (30) and MKK6 (11,(31)(32)(33). It is also possible that MKK4 contributes to the activation of p38 MAP kinase. In vitro studies demonstrate that MKK4 activates b...

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 ϩ T cells differentiate into Th2 cells, which potentiate the humoral immune response or pro-inflammatory Th1 cells. Here, we show that pyridinyl imidazole compounds

The Fas receptor mediates a signalling cascade resulting in programmed cell death (apoptosis) within hours of receptor cross-linking. In this study Fas activated the stress-responsive mitogen-activated protein kinases, p38 and JNK, within 2 h in Jurkat T lymphocytes but not the mitogen-responsive kinase ERK1 or pp70 S6k . Fas activation of p38 correlated temporally with the onset of apoptosis, and transfection of constitutively active MKK3(glu), an upstream regulator of p38, potentiated Fas-induced cell death, suggesting a potential involvement of the MKK3/p38 activation pathway in Fas-mediated apoptosis. Fas has been shown to require ICE (interleukin-1␤-converting enzyme) family proteases to induce apoptosis from studies utilizing the cowpox ICE inhibitor protein CrmA, the synthetic tetrapeptide ICE inhibitor YVAD-CMK, and the tripeptide pan-ICE inhibitor Z-VAD-FMK. In this study, crmA antagonized, and YVAD-CMK and Z-VAD-FMK completely inhibited, Fas activation of p38 kinase activity, demonstrating that Fas-dependent activation of p38 requires ICE/CED-3 family members and conversely that the MKK3/p38 activation cascade represents a downstream target for the ICE/CED-3 family proteases. Intriguingly, p38 activation by sorbitol and etoposide was resistant to YVAD-CMK and Z-VAD-FMK, suggesting the existence of an additional mechanism(s) of p38 regulation. The ICE/CED-3 family-p38 regulatory relationship described in the current work indicates that in addition to the previously described destructive cleavage of substrates such as poly(ADP ribose) polymerase, lamins, and topoisomerase, the apoptotic cysteine proteases also function to regulate stress kinase signalling cascades.Fas (APO-1/CD95) encodes a transmembrane type I receptor belonging to the tumor necrosis factor (TNF) receptor superfamily which includes TNF receptors 1 and 2 (TNFR1 and TNFR2), nerve growth factor (NGF) receptor, CD27, CD30, CD40, and OX40 (47, 67; reviewed in references 65 and 80). The cognate Fas ligand is a type II transmembrane protein belonging to the TNF family (83). The Fas receptor system has been extensively studied as a model of apoptosis, since crosslinking of the Fas receptor with Fas ligand or specific agonist antibodies results in rapid programmed cell death (47,89,97).Fas-induced apoptosis plays an important role in T-and B-cell homeostasis in the immune system and participates in T-cell activation-induced cell death (5,22,48), the elimination of autoreactive B cells (71), and the maintenance of sites of immune privilege (1). lpr mice defective in Fas (93), gld mice defective in Fas ligand (84), and humans with mutations in Fas (34, 73) all develop an autoimmune, lymphoproliferative disorder, indicating that Fas-induced death plays an important role in the peripheral deletion of lymphocytes.The essential signalling events linking Fas receptor crosslinking to apoptosis have been the subject of intense investigation. The intracellular domain of Fas contains an approximately 70-amino-acid "death domain" required for the induction...

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