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.
deviation from topological equilibrium for relaxation predicted by the model agrees with our experimental results and provides an explanation for the puzzling observation that type II topoisomerases are much better at decatenation and unknotting than relaxation (10,13,14).The ability of type II topoisomerases to directionally simplify DNA topology is in accord with their physiological role in DNA replication and chromosome segregation (25). Every turn of the double helix that is replicated introduces a positive supercoil or catenane link into topologically constrained DNA, which must be faithfully and rapidly removed by topoisomerases. However, given the high intracellular DNA concentration (26) and the presence of DNA condensing agents, the topoisomerases might instead be expected to ensnarl chromosomes (27). Our discovery that type II topoisomerases untangle DNA molecules against the thermal drive may help solve this problem. Biochem. 65, 635 (1996). 8. L. F. Liu, C.-C. Liu, B. M. Alberts, Cell 19, 697 (1980). 9. J.-i. Kato, H. Suzuki, H. Ikeda, J. Biol. Chem. 267, 25676 (1992). 10. H. Peng and K. J. Marians, ibid. 268, 24481 (1993). 11. M. A. Krasnow and N. R. Cozzarelli, ibid. 257, 2687 (1982. 12. To obtain mixtures with twice the equilibrium amount of heterodimeric catenanes, we cyclized P4 DNA (4 g/ml) in the presence of pAB4 DNA (100 g/ml). To obtain mixtures with no heterodimeric catenanes, we cyclized P4 DNA in the absence of pAB4. After cyclization, the concentration of DNA substrates in both mixtures was adjusted to 2 g/ml for P4 DNA and 50 g/ml for pAB4 DNA. The reaction mixtures also contained 20 mM tris-Cl (pH 7.8), 10 mM MgCl 2 , 1 mM dithiothreitol, bovine serum albumin (50 g/ml), 1 mM ATP, and either 80 mM potassium acetate for E. coli topoisosomerases III and IV and bacteriophage T2 topo II or 200 mM potassium acetate for eukaryotic topo II, so that each enzyme was assayed under its optimal ionic conditions. The reactions were carried out at 30°C for 60 min, quenched by adding 20 mM EDTA, 0.5% SDS, and 100 g/ml proteinase K, and incubated for an additional 60 min at 30°C. Because topo III requires single-stranded regions of DNA for optimal activity, this enzyme was assayed on pAB4 DNA containing a 25-nucleotidelong gap generated by exonuclease III from E. coli. 13. J. Roca and J. C. Wang, Genes Cells 1, 17 (1996). 14. C. J. Ullsperger and N. R. Cozzarelli, J. Biol. Chem. 271, 31549 (1996 REFERENCES AND NOTES ___________________________
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