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...