The E3 ubiquitin ligases β-TrCP and FBXW7 cooperatively mediates GSK3-dependent Mcl-1 degradation induced by the Akt inhibitor API-1, resulting in apoptosis (original) (raw)
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API-1 is a novel small molecule inhibitor of Akt, which acts by binding to Akt and preventing its membrane translocation, and has promising preclinical antitumor activity. In this study, we reveal a novel function of API-1 in regulation of c-FLIP levels and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, independent of Akt inhibition. API-1 effectively induced apoptosis in tested cancer cell lines including activation of caspase-8 and caspase-9. It reduced the levels of c-FLIP without increasing the expression of DR4 or DR5. Accordingly, it synergized with TRAIL to induce apoptosis. Enforced expression of ectopic c-FLIP did not attenuate API-1-induced apoptosis, but inhibited its ability to enhance TRAILinduced apoptosis. These data indicate that downregulation of c-FLIP mediates enhancement of TRAIL-induced apoptosis by API-1, but is not sufficient for API-1-induced apoptosis. API-1induced reduction of c-FLIP could be blocked by the proteasome inhibitor MG132. Moreover, API-1 increased c-FLIP ubiquitination and decreased c-FLIP stability. These data together suggest that API-1 downregulates c-FLIP by facilitating its ubiquitination and proteasome-mediated degradation. Since other Akt inhibitors including API-2 and MK2206 had minimal effects on reducing c-FLIP and enhancement of TRAIL-induced apoptosis, it is likely that API-1 reduces c-FLIP and enhances TRAIL-induced apoptosis independent of its Akt-inhibitory activity.
2021
SummaryApoptosis inhibitor 5 (Api5) is an inhibitor of apoptosis, which is found to be upregulated in several cancers and promotes invasion as well as metastasis. Over-expression of Api5 is positively co-related with poor survival of cancers and inhibition of DNA damage induced apoptosis in cancerous cells. Acetylation at lysine 251 (K251) on Api5 facilitates the stability of the protein and thus functionally provides resistance to cancer cells against chemotherapeutic or anti-cancerous agents. However, the regulation of Api5 upon DNA damage is not yet known. In this study, we demonstrate that Api5 undergoes degradation following DNA damage via the ubiquitin-proteasome system. Upon DNA damage, ATR was observed to phosphorylate Api5 at serine 138 which led to the cytoplasmic localisation of Api5. The E3-ubiquitin ligase, SCF-FBXW2 ubiquitinates Api5 leading to its proteasomal degradation.
Cell, 2005
members of the family promote the release of apoptogenic proteins from mitochondria. The fate of a cell un-and Xiaodong Wang* Howard Hughes Medical Institute and der apoptotic stimulation is determined by the balance between the pro-and antiapoptotic members of the Department of Biochemistry University of Texas Southwestern Medical Center Bcl-2 family. Therefore, studying the regulation of such balance will be critically important for our understand-at Dallas Dallas, Texas 75390 ing of apoptosis regulation. Among the antiapoptotic members of the Bcl-2 family proteins, Mcl-1 is unique in that it is an early-response Summary gene that can be rapidly induced and turned over (Kozopas et al., 1993; Yang et al., 1995; Yang et al., 1996). The elimination of Mcl-1, an anti-apoptotic Bcl-2 fam-This property enables Mcl-1 to function at an early step ily member, is an early and required step for DNA in a signaling cascade, consisting of Bcl-2 family prodamage-induced apoptosis. The degradation of Mcl-1 teins, and provides an acute protective function against can be blocked by proteasome inhibitors, suggesting apoptosis induced by a variety of stimuli, including a role for the ubiquitin proteasome pathway in apopto-DNA damage, adenoviral infection, growth factors' sis. Here, we demonstrate that Mcl-1 is ubiquinated at withdrawal, and treatment of cytotoxic agents (Cucofive lysines. Biochemical fractionation of cell extracts nati et al., 2003; Derouet et al., 2004; Huang et al., 2000; allowed us to identify a 482 kDa HECT-domain-contain-Le Gouill et al., 2004; Nijhawan et al., 2003; Piret et al., ing ubiquitin ligase named Mule (Mcl-1 ubiquitin ligase 2004; Zhang et al., 2002; Zhou et al., 1997). Consis-E3) that is both required and sufficient for the polytently, disappearance of Mcl-1 is associated with the ubiquitination of Mcl-1. Mule also contains a region onset of apoptosis and is achieved by the combination similar to the Bcl-2 homology region 3 (BH3) domain of synthesis blockage and continuous degradation (Cuthat allows Mule to specifically interact with Mcl-1. conati et al., 2003; Nijhawan et al., 2003). Elimination of Mule expression by RNA interference The degradation of Mcl-1 in HeLa cells can be stabilizes Mcl-1 protein, resulting in an attenuation of blocked by proteasome inhibitors, suggesting a role for the apoptosis induced by DNA-damage agents. Thus, the ubiquitin proteasome pathway in apoptosis up-Mule is a unique BH3-containing E3 ubiquitin ligase stream of Bcl-2 family of proteins (Derouet et al., 2004; apical to Bcl-2 family proteins during DNA damage-Nencioni et al., 2004; Cuconati et al., 2003; Nijhawan et induced apoptosis. al., 2003). In the current report, we demonstrate that Mcl-1 is polyubiquitinated in vivo and in vitro. Using Introduction HeLa cell extracts as a source, we established a cellfree assay for Mcl-1 ubiquitination. Biochemical frac-Apoptosis is a form of cell death orchestrated by chains tionation of HeLa cell extracts allowed us to identify of biochemical reactions. Cells undergoing apoptosis and purify a 482 kDa E3 ubiquitin ligase that mediated show characteristic morphological features such as the polyubiquitination of Mcl-1. This protein turned out condensation of cytoplasmic and nuclear contents, to be a novel homologous to E6-AP carboxyl terminus blebbing of plasma membranes, fragmentation of nu-(HECT)-domain-containing ubiquitin ligase. We named clei, and ultimately breakdown into membrane bound this protein Mule, for Mcl-1 ubiquitin ligase E3. apoptotic bodies that are rapidly phagocytized (Kerr et al., 1972). Results Mitochondria play an important role in regulating apoptosis induced by intracellular damaging signals Ubiquitination of Mcl-1 In Vivo and In Vitro such as DNA damage in mammalian cells (Danial and Proteins targeted for proteasome degradation are usu-Korsmeyer, 2004). Apoptotic stimuli exert their effects ally modified by a polyubiquitin chain (Hershko and on mitochondria to cause the release of proapoptotic Ciechanover, 1998). To test whether Mcl-1 is ubiqfactors like cytochrome c and Smac/Diablo. These facuitinated, we treated HeLa cells expressing Flag-Mcl-1 tors either directly activate caspases, a group of intrawith a proteasome inhibitor MG132 to block the procellular cysteine proteases that execute apoptosis by teasome activity and performed immunoprecipitation cleaving their substrates or releasing caspase inhibition analysis for Flag-Mcl-1 to test for the accumulation of imposed by the inhibitor-of-apoptosis proteins (IAPs; higher-molecular-weight forms of Mcl-1 that could be Du et al., 2000; Liu et al., 1996; Verhagen et al., 2000). ubiquitin modified. As shown in Figure 1A, higher-Mitochondrial response to apoptotic stimuli is regumolecular-weight protein bands recognized by antilated by the pro-and antiapoptotic Bcl-2 family of pro-Mcl-1 antibody were indeed accumulated upon MG132 teins (Gross et al., 1999; Martinou and Green, 2001). treatment (Figure 1A, lanes 3 and 4). To rule out a possi-Antiapoptotic proteins such as Bcl-2, Bcl-xL, and Mcl-1 bility that these higher-molecular-weight bands were protect mitochondrial integrity, while the proapoptotic from Mcl-1-associated protein rather than Mcl-1 itself, the protein mixture was first heated in the presence of 1% SDS and 5 mM DTT to disassociate protein-protein
Functional Identification of Api5 as a Suppressor of E2F-Dependent Apoptosis In Vivo
PLoS Genetics, 2006
Retinoblastoma protein and E2-promoter binding factor (E2F) family members are important regulators of G1-S phase progression. Deregulated E2F also sensitizes cells to apoptosis, but this aspect of E2F function is poorly understood. Studies of E2F-induced apoptosis have mostly been carried out in tissue culture cells, and the analysis of the factors that are important for this process has been restricted to the testing of a few candidate genes. Using Drosophila as a model system, we have generated tools that allow genetic modifiers of E2F-dependent apoptosis to be identified in vivo and developed assays that allow effects on E2F-induced apoptosis to be studied in cultured cells. Genetic interactions show that dE2F1-dependent apoptosis in vivo involves dArk/Apaf1 apoptosome-dependent activation of both initiator and effector caspases and is sensitive to levels of Drosophila inhibitor of apoptosis-1 (dIAP1). Using these approaches, we report the surprising finding that apoptosis inhibitor-5/antiapoptosis clone-11 (Api5/Aac11) is a critical determinant of dE2F1-induced apoptosis in vivo and in vitro. This functional interaction occurs in multiple tissues, is specific to E2F-induced apoptosis, and is conserved from flies to humans. Interestingly, Api5/Aac11 acts downstream of E2F and suppresses E2F-dependent apoptosis without generally blocking E2F-dependent transcription. Api5/Aac11 expression is often upregulated in tumor cells, particularly in metastatic cells. We find that depletion of Api5 is tumor cell lethal. The strong genetic interaction between E2F and Api5/Aac11 suggests that elevated levels of Api5 may be selected during tumorigenesis to allow cells with deregulated E2F activity to survive under suboptimal conditions. Therefore, inhibition of Api5 function might offer a possible mechanism for antitumor exploitation. Citation: Morris EJ, Michaud WA, Ji JY, Moon NS, Rocco JW, et al. (2006) Functional identification of Api5 as a suppressor of E2F-dependent apoptosis in vivo. PLoS Genet 2(11): e196.
This study focuses on determining whether and how the novel PI3 kinase inhibitor NVP-BKM120 (BKM120) induces apoptosis and enhances TRAIL-induced apoptosis in human lung cancer cells. We found that BKM120 reduced Mcl-1 levels across the tested cell lines along with induction of apoptosis and enhancement of TRAIL-induced apoptosis. Enforced expression of ectopic Mcl-1 significantly attenuated the effects of BKM120 alone or in combination with TRAIL on induction of apoptosis. Thus Mcl-1 downregulation contributes to BKM120-induced apoptosis or enhancement of TRAIL-induced apoptosis. Moreover, we have demonstrated that BMK120 decreases Mcl-1 levels through facilitating its degradation involving a GSK3/FBXW7-dependent mechanism.
Apigenin Induces Apoptosis in Human Leukemia Cells and Exhibits Anti-Leukemic Activity In Vivo
2012
In this study, we investigated the functional role of Akt and c-jun-NH 2-kinase (JNK) signaling cascades in apigenin-induced apoptosis in U937 human leukemia cells and anti-leukemic activity of apigenin in vivo. Apigenin induced apoptosis by inactivation of Akt with a concomitant activation of JNK, Mcl-1 and Bcl-2 downregulation, cytochrome c release from mitochondria, and activation of caspases. Constitutively active myristolated Akt prevented apigenin-induced JNK, caspase activation, and apoptosis. Conversely, LY294002 and a dominant-negative construct of Akt potentiated apigenin-induced apoptosis in leukemia cells. Interruption of the JNK pathway showed marked reduction in apigenin-induced caspase activation and apoptosis in leukemia cells. Furthermore, in vivo administration of apigenin resulted in attenuation of tumor growth in U937 xenografts accompanied by inactivation of Akt and activation of JNK. Attenuation of tumor growth in U937 xenografts by apigenin raises the possibility that apigenin may have clinical implications and can be further tested for incorporating in leukemia treatment regimens. Mol Cancer Ther; 11(1); 132-42. Ó2011 AACR.
Ubiquitin-Independent Degradation of Antiapoptotic MCL-1
Molecular and Cellular Biology, 2010
Antiapoptotic myeloid cell leukemia 1 (MCL-1) is an essential modulator of survival during the development and maintenance of a variety of cell lineages. Its turnover, believed to be mediated by the ubiquitin-proteasome system, facilitates apoptosis induction in response to cellular stress. To investigate the contribution of ubiquitinylation in regulating murine MCL-1 turnover, we generated an MCL-1 mutant lacking the lysine residues required for ubiquitinylation (MCL-1 KR ). Here, we demonstrate that despite failing to be ubiquitinylated, the MCL-1 KR protein is eliminated at a rate similar to that of wild-type MCL-1 under basal and stressed conditions. Moreover, the degradation of wild-type MCL-1 is not affected when ubiquitin-activating enzyme E1 activity is blocked. Likewise, both wild-type and MCL-1 KR proteins are similarly degraded when expressed in primary lymphocytes. Supporting these findings, unmodified, in vitro-translated MCL-1 can be degraded in a cell-free system by the 20S proteasome. Taken together, these data demonstrate that MCL-1 degradation can occur independently of ubiquitinylation.
Apigenin-induced-apoptosis is mediated by the activation of PKCδ and caspases in leukemia cells
Biochemical Pharmacology, 2006
Apigenin, a flavone abundantly found in fruits and vegetables, exhibits antiproliferative, anti-inflammatory, and antimetastatic activities through poorly defined mechanisms. In the present study, the treatment of different cell lines with apigenin resulted in selective antiproliferative and apoptotic effect in monocytic and lymphocytic leukemias. Apigenin-induced-apoptosis was mediated by the activation of caspase-9 and caspase-3. Apigenin was found intracellularly and localized to the mitochondria. Treatment of monocytic cells with apigenin was accompanied by an increase in reactive oxygen species (ROS) and phosphorylation of the MAPKs, p38 and ERK. However, the inhibition of ROS, p38 or ERK failed to block apoptosis, suggesting that these cellular responses induced by apigenin are not essential for the induction of apoptosis. In addition, apigenin induced the activation of PKCδ. Pharmacological inhibition of PKCδ, the expression of dominant-negative PKCδ and silencing of PKCδ in leukemia cells showed that apigenin-induced-apoptosis requires PKCδ activity. Together, these results indicate that this flavonoid provides selective activity to promote caspase-dependent-apoptosis of leukemia cells and uncover an essential role of PKCδ during the induction of apoptosis by apigenin.
Carcinogenesis, 2008
Inappropriate activation of phosphatidylinositol 3-kinase-Akt signaling contributes to the development of several human malignancies. Modulation of Akt activity is a strategy that may be valuable in chemopreventive and chemotherapeutic regimens. We have previously demonstrated that apigenin, a plant flavone, causes decreased survival in human prostate cancer cells. However, the molecular mechanism underlying this observation remains elusive. In the present study, we investigated the mechanisms of apigenin action on human prostate cancer PC-3 cells, which possess constitutively active Akt. Treatment of PC-3 cells with apigenin (5-40 mM) resulted in significant dose-and time-dependent decrease in Akt phosphorylation at Serine473. Apigenin-mediated dephosphorylation of Akt resulted in inhibition of its kinase activity, which was confirmed by reduced phosphorylation of proapoptotic proteins BAD and glycogen synthase kinase-3, essential downstream targets of Akt. Hypophosphorylation of BAD resulted in reduced interaction with 14-3-3b protein after 20 mM apigenin exposure to PC-3 cells for 24 h. Inactivation of Akt seems to be associated with downregulation of insulin-like growth factor receptor 1 protein level and inhibition of its autophosphorylation upon apigenin treatment. Exposure to apigenin significantly induced caspase-9 activity and decreased the survival of PC-3 cells in a dose-dependent manner. Furthermore, Serine473 phosphorylation of ectopically expressed Akt in DU145 cells was significantly reduced upon 20 mM apigenin treatment. In vivo, apigenin intake through gavage resulted in inactivation of Akt and induction of apoptosis in PC-3 tumors. These results suggest that Akt inactivation and dephosphorylation of BAD is a critical event, at least in part, in apigenin-induced decreased cell survival and apoptosis.