MEKK1/JNK signaling stabilizes and activates p53 (original) (raw)
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Molecular and cellular biology, 2001
The p53 tumor suppressor protein plays a key role in the regulation of stress-mediated growth arrest and apoptosis. Stress-induced phosphorylation of p53 tightly regulates its stability and transcriptional activities. Mass spectrometry analysis of p53 phosphorylated in 293T cells by active Jun NH 2 -terminal kinase (JNK) identified T81 as the JNK phosphorylation site. JNK phosphorylated p53 at T81 in response to DNA damage and stress-inducing agents, as determined by phospho-specific antibodies to T81. Unlike wild-type p53, in response to JNK stimuli p53 mutated on T81 (T81A) did not exhibit increased expression or concomitant activation of transcriptional activity, growth inhibition, and apoptosis. Forced expression of MKP5, a JNK phosphatase, in JNK kinase-expressing cells decreased T81 phosphorylation while reducing p53 transcriptional activity and p53-mediated apoptosis. Similarly transfection of antisense JNK 1 and -2 decreased T81 phosphorylation in response to UV irradiation. More than 180 human tumors have been reported to contain p53 with mutations within the region that encompasses T81 and the JNK binding site (amino acids 81 to 116). Our studies identify an additional mechanism for the regulation of p53 stability and functional activities in response to stress.
Oncogene, 2004
c-Jun N-terminal kinase (JNK) is activated by diverse cell stimuli, including stress, growth factors, and cytokines. Traditionally, activation of JNK by stress treatment is thought to induce cell death. However, our recent data indicate that JNK's ability to sensitize cells to apoptosis may be, in part, cell cycle dependent. Here, we show that the majority of both paclitaxel-and UV-induced apoptosis can be inhibited by the pharmacological JNK inhibitor, SP600125, in MCF-7 cells. However, inhibition of JNK does little to reverse doxorubicin-induced apoptosis in MCF-7 cells or doxorubicin-and UV-mediated death in MDA MB-231 cells. SP treatment causes G2/M arrest of three breast cancer cell lines and results in the endoreduplication (cellular DNA content 44N) of MCF-7 and MDA MB-231 cells. These effects on cell cycle and apoptosis are not significantly altered by the inhibition of p53, indicating that JNK is functioning independently of p53. Lastly, inhibition of JNK using both SP and antisense oligonucleotides targeted to JNK1 and JNK2 reduced proliferation of all three breast cancer cell lines. Taken together, these results suggest that the activation of JNK is important for the induction of apoptosis following stresses that function at different cell cycle phases, and that basal JNK activity is necessary to promote proliferation and maintain diploidy in breast cancer cells.
JNK targets p53 ubiquitination and degradation in nonstressed cells
Genes & …, 1998
In this study we elucidated the role of nonactive JNK in regulating p53 stability. The amount of p53-JNK complex was inversely correlated with p53 level. A peptide corresponding to the JNK binding site on p53 efficiently blocked ubiquitination of p53. Similarly, p53 lacking the JNK binding site exhibits a longer half-life than p53 wt. Outcompeting JNK association with p53 increased the level of p53, whereas overexpression of a phosphorylation mutant form of JNK inhibited p53 accumulation. JNK-p53 and Mdm2-p53 complexes were preferentially found in G 0 /G 1 and S/G 2 M phases of the cell cycle, respectively. Altogether, these data indicate that JNK is an Mdm2-independent regulator of p53 stability in nonstressed cells.
Journal of Biological Chemistry, 2000
The stress-activated protein kinase JNK plays an important role in the stability and activities of key regulatory proteins, including c-Jun, ATF2, and p53. To better understand mechanisms underlying the regulation of JNK activities, we studied the effect of expression of the amino-terminal JNK fragment (N-JNK; amino acids 1-206) on the stability and activities of JNK substrates under nonstressed growth conditions, as well as after exposure to hydrogen peroxide. Mouse fibroblasts that express N-JNK under tetracycline-off (tet-off) inducible promoter exhibited elevated expression of c-Jun, ATF2, and p53 upon tetracycline removal. This increased coincided with elevated transcriptional activities of p53, but not of c-Jun or ATF2, as reflected in luciferase activities of p21 Waf1/Cip1-Luc, AP1-Luc, and Jun2-Luc, respectively. Expression of N-JNK in cells that were treated with H 2 O 2 impaired transcriptional output as reflected in a delayed and lower level of c-Jun-, limited ATF2-, and reduced p53-transcriptional activities. N-JNK elicited an increase in H 2 O 2-induced cell death, which is p53-dependent, because it was not seen in p53 null cells yet could be observed upon coexpression of p53 and N-JNK. The ability to alter the activity of ATF2, c-Jun, and p53 and the degree of stress-induced cell death by a JNKderived fragment identifies new means to elucidate the nature of JNK regulation and to alter the cellular response to stress.
The stress kinase MKK7 couples oncogenic stress to p53 stability and tumor suppression
Nature Genetics, 2011
Most preneoplastic lesions are quiescent and do not progress to form overt tumors. It has been proposed that oncogenic stress activates the DNA damage response and the key tumor suppressor p53, which prohibits tumor growth. However, the molecular pathways by which cells sense a premalignant state in vivo are largely unknown. Here we report that tissue-specific inactivation of the stress signaling kinase MKK7 in KRas G12D -driven lung carcinomas and NeuT-driven mammary tumors markedly accelerates tumor onset and reduces overall survival. Mechanistically, MKK7 acts through the kinases JNK1 and JNK2, and this signaling pathway directly couples oncogenic and genotoxic stress to the stability of p53, which is required for cell cycle arrest and suppression of epithelial cancers. These results show that MKK7 functions as a major tumor suppressor in lung and mammary cancer in mouse and identify MKK7 as a vital molecular sensor to set a cellular anti-cancer barrier.
Cell Death and Differentiation, 2014
Rescue of the p53 tumor suppressor is an attractive cancer therapy approach. However, pharmacologically activated p53 can induce diverse responses ranging from cell death to growth arrest and DNA repair, which limits the efficient application of p53-reactivating drugs in clinic. Elucidation of the molecular mechanisms defining the biological outcome upon p53 activation remains a grand challenge in the p53 field. Here, we report that concurrent pharmacological activation of p53 and inhibition of thioredoxin reductase followed by generation of reactive oxygen species (ROS), result in the synthetic lethality in cancer cells. ROS promote the activation of c-Jun N-terminal kinase (JNK) and DNA damage response, which establishes a positive feedback loop with p53. This converts the p53-induced growth arrest/senescence to apoptosis. We identified several survival oncogenes inhibited by p53 in JNK-dependent manner, including Mcl1, PI3K, eIF4E, as well as p53 inhibitors Wip1 and MdmX. Further, we show that Wip1 is one of the crucial executors downstream of JNK whose ablation confers the enhanced and sustained p53 transcriptional response contributing to cell death. Our study provides novel insights for manipulating p53 response in a controlled way. Further, our results may enable new pharmacological strategy to exploit abnormally high ROS level, often linked with higher aggressiveness in cancer, to selectively kill cancer cells upon pharmacological reactivation of p53.
Cell Stress and MEKK1-mediated c-Jun Activation Modulate NFκB Activity and Cell Viability
Molecular Biology of the Cell, 2002
Chemotherapeutic agents such as cisplatin induce persistent activation of N-terminal c-Jun Kinase, which in turn mediates induction of apoptosis. By using a common MAPK Kinase, MEKK1, cisplatin also activates the survival transcription factor NFκB. We have found a cross-talk between c-Jun expression and NFκB transcriptional activation in response to cisplatin. Fibroblast derived from c-jun knock out mice are more resistant to cisplatin-induced cell death, and this survival advantage is mediated by upregulation of NFκB-dependent transcription and expression of MIAP3. This process can be reverted by ectopic expression of c-Jun in c-jun−/−fibroblasts, which decreases p65 transcriptional activity back to normal levels. Negative regulation of NFκB-dependent transcription by c-jun contributes to cisplatin-induced cell death, which suggests that inhibition of NFκB may potentiate the antineoplastic effect of conventional chemotherapeutic agents.
Analysis of JNK, Mdm2 and p14ARF contribution to the regulation of mutant p53 stability
Journal of Molecular Biology, 2000
Identi®cation of Mdm2 and JNK as proteins that target degradation of wt p53 prompted us to examine their effect on mutant p53, which exhibits a prolonged half-life. Of ®ve mutant p53 forms studied for association with the targeting molecules, two no longer bound to Mdm2 and JNK. Three mutant forms, which exhibit high expression levels, showed lower af®nity for association with Mdm2 and JNK in concordance with greater af®nity to p14 ARF , which is among the stabilizing p53 molecules. Monitoring mutant p53 stability in vitro con®rmed that, while certain forms of mutant p53 are no longer affected by either JNK or Mdm2, others are targeted for degradation by JNK/Mdm2, albeit at lower ef®ciency when compared with wt p53. Expression of wt p53 in tumor cells revealed a short half-life, suggesting that the targeting molecules are functional. Forced expression of mutant p53 in p53 null cells con®rmed pattern of association with JNK/Mdm2 and prolonged half-life, as found in the tumor cells. Over-expression of Mdm2 in either tumor (which do express endogenous functional Mdm2) or in p53 null cells decreased the stability of mutant p53 suggesting that, despite its expression, Mdm2/ JNK are insuf®cient (amount/af®nity) for targeting mutant p53 degradation. Based on both in vitro and in vivo analyses, we conclude that the prolonged half-life of mutant p53 depends on the nature of the mutation, which either alters association with targeting molecules, ratio between p53 and targeting/stabilizing molecules or targeting ef®ciency.