ASK1-signaling promotes c-Myc protein stability during apoptosis

We previously reported that JNK is involved in the regulation of c-Myc-mediated apoptosis triggered by UV irradiation and anticancer drug treatment. Here we show that ASK1 is an upstream regulator for c-Myc-mediated apoptosis triggered by UV, and we found a direct role for Ser-62 and Ser-71 in the regulation of protein stability and function of c-Myc. The ASK1-JNK pathway enhanced the protein stability of c-Myc through phosphorylation at Ser-62 and Ser-71, which was required for c-Myc-dependent apoptosis by ASK1-signaling. Interestingly, ASK1-signaling attenuated the degradation of ubiquitinated c-Myc without affecting the ubiquitination process. Together, these findings indicate that the ASK1-JNK pathway promotes the proapoptotic activity of c-Myc by modulating c-Myc protein stability through phosphorylation at Ser-62 and Ser-71.     

Phosphorylation of BAD at Ser-128 during mitosis and paclitaxel-induced apoptosis

Phosphorylation of BCL-2 family member BAD at different residues triggers different physiological effects, either inhibiting or promoting apoptosis. The recently identified phosphorylation site at Ser-128 enhances the apoptotic activity of BAD. We here show that BAD becomes phosphorylated at Ser-128 in the mitotic phase of the cell cycle in NIH3T3 cells. We also show that BAD-S128 is phosphorylated in taxol-treated mouse fibroblasts and MDA-MB-231 human breast cancer cells. However, expression of a phosphorylation-defective dominant negative BAD mutant did not block taxol-induced apoptosis. These data support the view that the phosphorylation of BAD Serine 128 exerts cell-specific effects on apoptosis. Whereas the BAD Serine 128 phosphorylation induces apoptosis in neuronal cells, it does not appear to promote apoptosis in proliferating non-neural cells during mitosis or upon exposure to the antineoplastic agent taxol.     

p38 MAP kinase mediates apoptosis through phosphorylation of BimEL at Ser-65

The stress-activated c-Jun N-terminal protein kinase (JNK) and p38 mitogen-activated protein (MAP) kinase (p38) regulate apoptosis induced by several forms of cellular insults. Potential targets for these kinases include members of the Bcl-2 family proteins, which mediate apoptosis generated through the mitochondria-initiated, intrinsic cell death pathway. Indeed, the activities of several Bcl-2 family proteins, both pro- and anti-apoptotic, are controlled by JNK phosphorylation. For example, the pro-apoptotic activity of Bim(EL), a member of the Bcl-2 family, is stimulated by JNK phosphorylation at Ser-65. In contrast, there is no reported evidence that p38-induced apoptosis is due to direct phosphorylation of Bcl-2 family proteins. Here we report evidence that sodium arsenite-induced apoptosis in PC12 cells may be due to direct phosphorylation of Bim(EL) at Ser-65 by p38. This conclusion is supported by data showing that ectopic expression of a wild type, but not a non-phosphorylatable S65A mutant of Bim(EL), potentiates sodium arsenite-induced apoptosis and by experiments showing direct phosphorylation of Bim(EL) at Ser-65 by p38 in vitro. Furthermore, sodium arsenite induced Bim(EL) phosphorylation at Ser-65, which was blocked by p38 inhibition. This study provides the first example whereby p38 induces apoptosis by phosphorylating a member of the Bcl-2 family and illustrates that phosphorylation of Bim(EL) on Ser-65 may be a common regulatory point for cell death induced by both JNK and p38 pathways.     

Differential role of the JNK and p38 MAPK pathway in c-Myc- and s-Myc-mediated apoptosis

The s-Myc is similar to c-Myc in its ability to induce apoptosis requiring caspase activation. However, s-Myc is distinct from c-Myc in that it has activity to suppress tumor growth and does not require wild-type p53 to induce apoptosis. These facts suggest differential regulation between s-Myc and c-Myc. Here we showed that s-Myc-mediated apoptosis triggered by UV was not inhibited by the inactive form mutant JNK (APF), though c-Myc-mediated apoptosis was. Moreover, we found that JNK did not affect the transactivation activity of s-Myc, but stimulated that of c-Myc. In contrast, both Myc-mediated apoptosis and caspase-3-like protease activation were suppressed by kinase-negative MKK6 and an inactive form mutant p38(AGF). Our results indicate that s-Myc does not require the JNK signaling unlike c-Myc during UV-triggered apoptosis, but the MKK6/p38MAPK pathway might regulate common apoptotic machinery for both s-Myc and c-Myc upstream of caspase.     

Differential FAK phosphorylation at Ser-910, Ser-843 and Tyr-397 induced by angiotensin II, LPA and EGF in intestinal epithelial cells

A rapid increase in the tyrosine phosphorylation of the non-receptor tyrosine kinase FAK is a prominent early event in fibroblasts stimulated by a variety of signaling molecules. However, a variety of epithelial cells, including intestinal epithelial cells, show a high basal level of tyrosine phosphorylated FAK that is only slightly further increased by addition of G protein-coupled receptor (GPCR) agonists or growth factors. In this study, we determined whether these stimuli could elicit FAK phosphorylation at serine residues, including Ser-910 and Ser-843. Our results show that multiple agonists including angiotensin II (ANGII), lysophosphatidic acid (LPA), phorbol esters and EGF induced a striking stimulation of FAK phosphorylation at Ser-910 in rat intestinal epithelial IEC-18 cells via an ERK-dependent pathway. In striking contrast, none of these stimuli promoted a significant further increase in FAK phosphorylation at Tyr-397 in these cells. These results were extended using cultures of polarized human colonic epithelial T84 cells. We found that either carbachol or EGF promoted a striking ERK-dependent phosphorylation of FAK at Ser-910, but these agonists caused only slight stimulation of FAK at Tyr-397 in T84 cells. In addition, we demonstrated that GPCR agonists also induced a dramatic increase of FAK phosphorylation at Ser-843 in either IEC-18 or T84 cells. Our results indicate that Ser-910 and Ser-843, rather than Tyr-397, are prominent sites differentially phosphorylated in response to neurotransmitters, bioactive lipids, tumor promoters and growth factors in intestinal epithelial cells.     

Control of death receptor and mitochondrial-dependent apoptosis by c-Jun N-terminal kinase in hippocampal CA1 neurones following global transient ischaemia

c-Jun N-terminal kinase (JNK), a member of the mitogen-activated protein kinase family, is activated in response to a number of extracellular stimuli, including inflammatory cytokines, UV irradiation and ischaemia. A large body of evidence supports a role for JNK signalling in stress-induced apoptosis. It has been hypothesized that JNK may contribute to the apoptotic response by regulating the intrinsic cell death pathway involving the mitochondria. Here, we examined the role of the JNK signalling pathway in hippocampal CA1 apoptotic neurones following transient ischaemia in gerbils. We showed early activation of death receptor-dependent apoptosis (caspase-8 activation 2 days after ischaemia) and a biphasic activation of caspase-3 and caspase-9 after ischaemia. Activation of the mitochondrial pathway, as measured by cytochrome c release, appeared as a late event (5-7 days after ischaemia). AS601245, a novel JNK inhibitor, antagonized activation of both pathways and significantly protected CA1 neurones from cell death. Our results suggest a key role of JNK in the control of death receptor and mitochondrial-dependent apoptosis after transient ischaemia.     

E2F1 and c-Myc potentiate apoptosis through inhibition of NF-kappaB activity that facilitates MnSOD-mediated ROS elimination

Overexpression of c-Myc or E2F1 sensitizes host cells to various types of apoptosis. Here, we found that overexpressed c-Myc or E2F1 induces accumulation of reactive oxygen species (ROS) and thereby enhances serum-deprived apoptosis in NIH3T3 and Saos-2. During serum deprivation, MnSOD mRNA was induced by NF-kappaB in mock-transfected NIH3T3, while this induction was inhibited in NIH3T3 overexpressing c-Myc or E2F1. In these clones, E2F1 inhibited NF-kappaB activity by binding to its subunit p65 in competition with a heterodimeric partner p50. In addition to overexpressed E2F1, endogenous E2F1 released from Rb was also found to inhibit NF-kappaB activity in a cell cycle-dependent manner by using E2F1(+/+) and E2F1(-/-) murine embryonic fibroblasts. These results indicate that E2F1 promotes apoptosis by inhibiting NF-kappaB activity.     

Apoptosis induced by the fungal pathogen gliotoxin requires a triple phosphorylation of Bim by JNK

We previously reported that gliotoxin (GT), the major virulence factor of the mold Aspergillus fumigatus causing invasive aspergillosis (IA) in immunocompromised patients, induces apoptosis in a Bak-dependent manner. The signaling pathway leading to Bak activation and subsequent mitochondrial outer membrane permeabilization (MOMP) is elusive. Here, we show that GT and the supernatant of A. fumigatus (but not its GT-defective mutant) activate the JNK pathway and require a co-operative JNK-mediated Bim_EL phosphorylation at three sites (S100, T112 and S114) to induce apoptosis in mouse fibroblasts, human bronchial and mouse alveolar epithelial cells. Cells (i) treated with the JNK inhibitor SP600125, (ii) deleted or knocked down for JNK1/2 or Bim or (iii) carrying the Bim_EL triple phosphomutant S100A/T112A/S114A instead of wild-type Bim_EL are similarly resistant to GT-induced apoptosis. Triple-phosphorylated Bim_EL is more stable, redistributes from a cytoskeletal to a membrane fraction, better interacts with Bcl-2 and Bcl-x_L and more effectively activates Bak than the unphosphorylated mutant. These data indicate that JNK-mediated Bim_EL phosphorylation at S100, T112 and S114 constitutes a novel regulatory mechanism to activate Bim in response to apoptotic stimuli.     

Oncogenic ras mediates apoptosis in response to protein kinase C inhibition through the generation of reactive oxygen species

Ras is a well established modulator of apoptosis. Suppression of protein kinase C (PKC) activity can selectively induce apoptosis in cells expressing a constitutively activated Ras protein. We wished to determine whether reactive oxygen species serve as an effector of Ras-mediated apoptosis. Ras-transformed NIH/3T3 cells contained higher basal levels of intracellular H(2)O(2) compared with normal NIH/3T3 cells, and PKC inhibition up-regulated ROS to 5-fold greater levels in Ras-transformed cells than in normal cells. Treatment with N-acetyl-l-cysteine reduced both the basal and inducible levels of intracellular H(2)O(2) in NIH/3T3-Ras cells and antagonized the induction of apoptosis by PKC inhibition. Culturing NIH/3T3-Ras cells in low oxygen conditions, which prevents ROS generation, also inhibited the apoptotic response to PKC inhibition. These results suggest that reactive oxygen species are necessary as downstream effectors of the Ras-mediated apoptotic response to PKC inhibition. However, the generation of ROS alone is not sufficient to induce apoptosis in Ras-transformed cells because inhibition of cell cycle progression prevented the induction of apoptosis in NIH/3T3-Ras cells without inhibiting the generation of intracellular H(2)O(2) observed after PKC inhibition. These findings suggest that continued cell cycle progression of Ras-transformed cells during PKC inhibition is also necessary for the induction of apoptosis.     

c-Myc overexpression sensitizes Bim-mediated Bax activation for apoptosis induced by histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) through regulating Bcl-2/Bcl-xL expression

Overexpression of the oncogene c-Myc sensitizes many apoptotic signals through the activation of mitochondrial apoptosis pathway. However, the underling mechanism has not been clearly defined. Here, we investigated the effect of c-Myc expression on histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA)-induced apoptosis in rat fibroblast cells possessing various c-Myc levels. In Rat 1a cells overexpressing c-Myc, SAHA-induced enhanced the cell death response relative to the parental cells; whereas Rat 1a cells lacking c-Myc were refractory to SAHA treatment. We demonstrated that SAHA selectively induced the expression of pro-apoptotic BH3-only protein Bim, leading to Bax activation in c-Myc-expressing cells. Where c-Myc was absent, Bim, despite its induction by SAHA, failed to activate Bax and was unable to induce apoptosis. These results indicate that c-Myc is dispensable for Bim induction by SAHA, but is required for subsequent Bax activation. We further show that the expression levels of anti-apoptotic Bcl-2/Bcl2-xL were much elevated in Myc-null cells compared with the c-Myc-expressing cells; furthermore, depletion of Bcl-2/Bcl-xL in these cells restored the ability of SAHA to induce apoptosis by enhancing Bax activation. These data indicate that SAHA induces apoptosis through Bim-triggered Bax activation and that c-Myc regulates this process by modulating Bcl-2/Bcl-xL. Our results provide novel insight into the mechanism whereby Myc sensitizes the apoptotic signals; furthermore, our data suggest that cancer cells with deregulated Myc might be more sensitive to SAHA treatment.     

BAD与JNK协同调节UV诱导的细胞凋亡

JNK和BAD(bcl-2相关死亡启动子)都是参与细胞凋亡的重要调控蛋白. 然而,二者在功能上的联系及其在细胞凋亡中的相互作用尚未见报导. 本研究证明, BAD可作为JNK的磷酸化底物, 与JNK相互作用, 协同调节紫外线（UV）诱导的细胞凋亡. 蛋白质印迹检测PARP (聚ADP核糖聚合酶)裂解, 以及流式细胞术检测细胞凋亡结果揭示, UV诱导的MEF细胞凋亡依赖JNK的激酶活性. siRNA敲降BAD的蛋白表达,可增加MEF细胞对UV 诱导的细胞凋亡的敏感性. UV处理的野生型MEF细胞抽提液（含JNK激酶活性）可催化GST-BAD底物发生磷酸化修饰, 而UV未处理的细胞抽提液却不能. 结果提示, UV激活的JNK活性可催化BAD磷酸化|体外合成的持续活化的JNK与GST-BAD体外共孵育结合质谱分析证明, JNK 可催化BAD蛋白的Thr-201磷酸化. 提示BAD是JNK的底物. 此外,野生型和T201A突变的BAD质粒转染BAD-/-细胞结果显示, BAD的T201磷酸化可抑制JNK激酶活性及其底物c-Jun的磷酸化, 提示BAD磷酸化对JNK具有负反馈调节作用. 上述结果证明,BAD作为底物可被UV激活的JNK激酶磷酸化|磷酸化BAD反过来又可抑制JNK的激酶活性, 负性调节细胞凋亡. 综上所述, BAD与JNK能够相互影响, 协同调控UV诱导的细胞凋亡.     

Hierarchical phosphorylation at N-terminal transformation-sensitive sites in c-Myc protein is regulated by mitogens and in mitosis.

The N-terminal domain of the c-Myc protein has been reported to be critical for both the transactivation and biological functions of the c-Myc proteins. Through detailed phosphopeptide mapping analyses, we demonstrate that there is a cluster of four regulated and complex phosphorylation events on the N-terminal domain of Myc proteins, including Thr-58, Ser-62, and Ser-71. An apparent enhancement of Ser-62 phosphorylation occurs on v-Myc proteins having a mutation at Thr-58 which has previously been correlated with increased transforming ability. In contrast, phosphorylation of Thr-58 in cells is dependent on a prior phosphorylation of Ser-62. Hierarchical phosphorylation of c-Myc is also observed in vitro with a specific glycogen synthase kinase 3 alpha, unlike the promiscuous phosphorylation observed with other glycogen synthase kinase 3 alpha and 3 beta preparations. Although both p42 mitogen-activated protein kinase and cdc2 kinase specifically phosphorylate Ser-62 in vitro and cellular phosphorylation of Thr-58/Ser-62 is stimulated by mitogens, other in vivo experiments do not support a role for these kinases in the phosphorylation of Myc proteins. Unexpectedly, both the Thr-58 and Ser-62 phosphorylation events, but not other N-terminal phosphorylation events, can occur in the cytoplasm, suggesting that translocation of the c-Myc proteins to the nucleus is not required for phosphorylation at these sites. In addition, there appears to be an unusual block to the phosphorylation of Ser-62 during mitosis. Finally, although the enhanced transforming properties of Myc proteins correlates with the loss of phosphorylation at Thr-58 and an enhancement of Ser-62 phosphorylation, these phosphorylation events do not alter the ability of c-Myc to transactivate through the CACGTG Myc/Max binding site.     

Calphostin C-induced apoptosis is mediated by a tissue transglutaminase-dependent mechanism involving the DLK/JNK signaling pathway

A role for tissue transglutaminase (TG2) and its substrate dual leucine zipper-bearing kinase (DLK), an upstream component of the c-Jun N-terminal kinase (JNK) signaling pathway, has been previously suggested in the apoptotic response induced by calphostin C. In the current study, we directly tested this hypothesis by examining via pharmacological and RNA-interference approaches whether inhibition of expression or activity of TG2, DLK and JNK in mouse NIH 3T3 fibroblasts and human MDA-MB-231 breast cancer epithelial cells affects calphostin C-induced apoptosis. Our experiments with the selective JNK inhibitor SP600125 reveal that calphostin C is capable of causing JNK activation and JNK-dependent apoptosis in both cell lines. Small interfering RNA-mediated depletion of TG2 alone strongly reduces calphostin C action on JNK activity and apoptosis. Consistent with an active role for DLK in this cascade of event, cells deficient in DLK demonstrate a substantial delay of JNK activation and poly-ADP-ribose polymerase (PARP) cleavage in response to calphostin C, whereas overexpression of a recombinant DLK resistant to silencing, but sensitive to TG2-mediated oligomerization, reverses this effect. Importantly, combined depletion of TG2 and DLK further alters calphostin C effects on JNK activity, Bax translocation, caspase-3 activation, PARP cleavage and cell viability, demonstrating an obligatory role for TG2 and DLK in calphostin C-induced apoptosis.     

Phosphorylation of mammalian target of rapamycin (mTOR) at Ser-2448 is mediated by p70S6 kinase

The mammalian target of rapamycin (mTOR) coordinates cell growth with the growth factor and nutrient/energy status of the cell. The phosphatidylinositol 3-kinase-AKT pathway is centrally involved in the transmission of mitogenic signals to mTOR. Previous studies have shown that mTOR is a direct substrate for the AKT kinase and identified Ser-2448 as the AKT target site in mTOR. In this study, we demonstrate that rapamycin, a specific inhibitor of mTOR function, blocks serum-stimulated Ser-2448 phosphorylation and that this drug effect is not explained by the inhibition of AKT. Furthermore, the phosphorylation of Ser-2448 was dependent on mTOR kinase activity, suggesting that mTOR itself or a protein kinase downstream from mTOR was responsible for the modification of Ser-2448. Here we show that p70S6 kinase phosphorylates mTOR at Ser-2448 in vitro and that ectopic expression of rapamycin-resistant p70S6 kinase restores Ser-2448 phosphorylation in rapamycin-treated cells. In addition, we show that cellular amino acid status, which modulates p70S6 kinase (S6K1) activity via the TSC/Rheb pathway, regulates Ser-2448 phosphorylation. Finally, small interfering RNA-mediated depletion of p70S6 kinase reduces Ser-2448 phosphorylation in cells. Taken together, these results suggest that p70S6 kinase is a major effector of mTOR phosphorylation at Ser-2448 in response to both mitogen- and nutrient-derived stimuli.     

c-Myc Blazing a Trail of Death: Coupling of the Mitochondrial and Death Receptor Apoptosis Pathways by c-Myc

TRAIL ligand induces selectively apoptosis in tumor cells by binding to two death receptors (DR4 and DR5) and holds promise as a potential therapeutic agent against cancer. While it has been known for long time that TRAIL receptors are commonly expressed in wide variety of normal tissues, it is not well understood why TRAIL kills tumor cells but leaves normal cells unharmed. The prototypic oncogene c-Myc promotes the cell cycle and simultaneously primes activation of the Bcl-2 family controlled mitochondria apoptosis pathway. A striking reflection of the c-Myc-dependent apoptotic sensitization is the dramatic c-Myc-induced vulnerability of cells to TRAIL and other death receptor ligands. Here we summarize the recent findings regarding the death mechanisms of TRAIL/TRAIL receptor system and the connection of c-Myc to the mitochondrial apoptosis pathway, focusing on our work that couples c-Myc via Bak to the TRAIL death receptor pathway. Finally, we present a mitochondria-priming model to explain how c-Myc-Bak interaction amplifies the TRAIL-induced caspase 8-Bid pathway to induce fullblown apoptosis. We discuss the implications of these findings for understanding the selective cytotoxicity of TRAIL and for the therapeutic exploitation of the death receptor pathway.