Long Non-Coding RNA KCNQ1OT1 Regulates Protein Kinase CK2 Via miR-760 in Senescence and Calorie Restriction.

Long non-coding RNAs (lncRNAs) play important biological roles. Here, the roles of the lncRNA KCNQ1OT1 in cellular senescence and calorie restriction were determined. KCNQ1OT1 knockdown mediated various senescence markers (increased senescence-associated ß-galactosidase staining, the p53-p21Cip1/WAF1 pathway, H3K9 trimethylation, and expression of the senescence-associated secretory phenotype) and reactive oxygen species generation via CK2α downregulation in human cancer HCT116 and MCF-7 cells. Additionally, KCNQ1OT1 was downregulated during replicative senescence, and its silencing induced senescence in human lung fibroblast IMR-90 cells. Additionally, an miR-760 mimic suppressed KCNQ1OT1-mediated CK2α upregulation, indicating that KCNQ1OT1 upregulated CK2α by sponging miR-760. Finally, the KCNQ1OT1-miR-760 axis was involved in both lipopolysaccharide-mediated CK2α reduction and calorie restriction (CR)-mediated CK2α induction in these cells. Therefore, for the first time, this study demonstrates that the KCNQ1OT1-miR-760-CK2α pathway plays essential roles in senescence and CR, thereby suggesting that KCNQ1OT1 is a novel therapeutic target for an alternative treatment that mimics the effects of anti-aging and CR.

Downregulation of JMJD2a and LSD1 is involved in CK2 inhibition-mediated cellular senescence through the p53-SUV39h1 pathway.

Lysine methylation is one of the most important histone modifications that modulate chromatin structure. In the present study, the roles of the histone lysine demethylases JMJD2a and LSD1 in CK2 downregulation-mediated senescence were investigated. The ectopic expression of JMJD2a and LSD1 suppressed the induction of senescence-associated ß-galactosidase activity and heterochromatin foci formation as well as the reduction of colony-forming and cell migration ability mediated by CK2 knockdown. CK2 downregulation inhibited JMJD2a and LSD1 expression by activating the mammalian target of rapamycin (mTOR)-ribosomal p70 S6 kinase (p70S6K) pathway. In addition, the downregulation of JMJD2a and LSD1 was involved in activating the p53-p21Cip1/WAF1-SUV39h1-trimethylation of the histone H3 Lys9 (H3K9me3) pathway in CK2-downregulated cells. Further, CK2 downregulation-mediated JMJD2a and LSD1 reduction was found to stimulate the dimethylation of Lys370 on p53 (p53K370me2) and nuclear import of SUV39h1. Therefore, this study indicated that CK2 downregulation reduces JMJD2a and LSD1 expression by activating mTOR, resulting in H3K9me3 induction by increasing the p53K370me2-dependent nuclear import of SUV39h1. These results suggest that CK2 is a potential therapeutic target for age-related diseases. [BMB Reports 2022;55(2): 92-97].

Inhibition of cathepsin K sensitizes oxaliplatin-induced apoptotic cell death by Bax upregulation through OTUB1-mediated p53 stabilization in vitro and in vivo.

Cathepsin K is highly expressed in various types of cancers. However, the effect of cathepsin K inhibition in cancer cells is not well characterized. Here, cathepsin K inhibitor (odanacatib; ODN) and knockdown of cathepsin K (siRNA) enhanced oxaliplatin-induced apoptosis in multiple cancer cells through Bax upregulation. Bax knockdown significantly inhibited the combined ODN and oxaliplatin treatment-induced apoptotic cell death. Stabilization of p53 by ODN played a critical role in upregulating Bax expression at the transcriptional level. Casein kinase 2 (CK2)-dependent phosphorylation of OTUB1 at Ser16 played a critical role in ODN- and cathepsin K siRNA-mediated p53 stabilization. Interestingly, ODN-induced p53 and Bax upregulation were modulated by the production of mitochondrial reactive oxygen species (ROS). Mitochondrial ROS scavengers prevented OTUB1-mediated p53 stabilization and Bax upregulation by ODN. These in vitro results were confirmed by in mouse xenograft model, combined treatment with ODN and oxaliplatin significantly reduced tumor size and induced Bax upregulation. Furthermore, human renal clear carcinoma (RCC) tissues revealed a strong correlation between phosphorylation of OTUB1(Ser16) and p53/Bax expression. Our results demonstrate that cathepsin K inhibition enhances oxaliplatin-induced apoptosis by increasing OTUB1 phosphorylation via CK2 activation, thereby promoting p53 stabilization, and hence upregulating Bax.

Protein Kinase CK2 Is Upregulated by Calorie Restriction and Induces Autophagy.

Calorie restriction (CR) and the activation of autophagy extend healthspan by delaying the onset of age-associated diseases in most living organisms. Because protein kinase CK2 (CK2) downregulation induces cellular senescence and nematode aging, we investigated CK2's role in CR and autophagy. This study indicated that CR upregulated CK2's expression, thereby causing SIRT1 and AMP-activated protein kinase (AMPK) activation. CK2α overexpression, including antisense inhibitors of miR-186, miR-216b, miR-337-3p, and miR-760, stimulated autophagy initiation and nucleation markers (increase in ATG5, ATG7, LC3BII, beclin-1, and Ulk1, and decrease in SQSTM1/p62). The SIRT1 deacetylase, AKT, mammalian target of rapamycin (mTOR), AMPK, and forkhead homeobox type O (FoxO) 3a were involved in CK2-mediated autophagy. The treatment with the AKT inhibitor triciribine, the AMPK activator AICAR, or the SIRT1 activator resveratrol rescued a reduction in the expression of lgg-1 (the Caenorhabditis elegans ortholog of LC3B), bec-1 (the C. elegans ortholog of beclin-1), and unc-51 (the C. elegans ortholog of Ulk1), mediated by kin-10 (the C. elegans ortholog of CK2ß) knockdown in nematodes. Thus, this study indicated that CK2 acted as a positive regulator in CR and autophagy, thereby suggesting that these four miRs' antisense inhibitors can be used as CR mimetics or autophagy inducers.

CK2 Down-Regulation Increases the Expression of Senescence-Associated Secretory Phenotype Factors through NF-κB Activation.

Senescent cells secrete pro-inflammatory factors, and a hallmark feature of senescence is senescence-associated secretory phenotype (SASP). The aim of this study is to investigate the protein kinase CK2 (CK2) effects on SASP factors expression in cellular senescence and organism aging. Here CK2 down-regulation induced the expression of SASP factors, including interleukin (IL)-1ß, IL-6, and matrix metalloproteinase (MMP) 3, through the activation of nuclear factor-κB (NF-κB) signaling in MCF-7 and HCT116 cells. CK2 down-regulation-mediated SIRT1 inactivation promoted the degradation of inhibitors of NF-κB (IκB) by activating the AKT-IκB kinase (IKK) axis and increased the acetylation of lysine 310 on RelA/p65, an important site for the activity of NF-κB. kin-10 (the ortholog of CK2ß) knockdown increased zmp-1, -2, and -3 (the orthologs of MMP) expression in nematodes, but AKT inhibitor triciribine and SIRT activator resveratrol significantly abrogated the increased expression of these genes. Finally, antisense inhibitors of miR-186, miR-216b, miR-337-3p, and miR-760 suppressed CK2α down-regulation, activation of the AKT-IKK-NF-κB axis, RelA/p65 acetylation, and expression of SASP genes in cells treated with lipopolysaccharide. Therefore, this study indicated that CK2 down-regulation induces the expression of SASP factors through NF-κB activation, which is mediated by both activation of the SIRT1-AKT-IKK axis and RelA/p65 acetylation, suggesting that the mixture of the four miRNA inhibitors can be used as anti-inflammatory agents.

4-O-methylascochlorin activates autophagy by activating AMPK and suppressing c-Myc in glioblastoma.

A prior study identified that 4-O-methylascochlorin (MAC), a methylated derivative of ascochlorin (ASC) from the fungus Ascochyta viciae, activates autophagy in leukemia cells by suppressing c-Myc phosphorylation. However, the effects of MAC on autophagy in other cancer cells remain unknown. In the present study, we demonstrated that MAC activated autophagy in human glioblastoma. MAC increased expression of autophagy-related proteins, such as LC3-II and Beclin-1. Moreover, MAC stimulated AMP-activated protein kinase (AMPK) phosphorylation and suppressed phosphorylation of the mTOR, p70S6K, and 4EBP1. The well-known AMPK activator metformin increased LC3-II levels, which were augmented by MAC cotreatment. AMPK knockdown decreased LC3-II levels and inhibited MAC activation of autophagy. Furthermore, MAC suppression of c-Myc expression activated autophagy. Treatment with the c-MYC inhibitor, 10058-FA, induced autophagy, as did c-Myc small interfering RNA knockdown. These effects were augmented by MAC cotreatment. Taken together, these findings indicated that MAC induces autophagy in human glioblastoma by activating AMPK signaling and inhibiting c-Myc protein expression in human glioblastoma.

Protein kinase CK2 activates Nrf2 via autophagic degradation of Keap1 and activation of AMPK in human cancer cells.

Protein kinase CK2 downregulation induces premature senescence in various human cell types via activation of the reactive oxygen species (ROS)-p53-p21Cip1/WAF1 pathway. The transcription factor "nuclear factor erythroid 2-related factor 2" (Nrf2) plays an important role in maintaining intracellular redox homeostasis. In this study, Nrf2 overexpression attenuated CK2 downregulation- induced ROS production and senescence markers including SA-ß-gal staining and activation of p53-p21Cip1/WAF1 in human breast (MCF-7) and colon (HCT116) cancer cells. CK2 downregulation reduced the transcription of Nrf2 target genes, such as glutathione S-transferase, glutathione peroxidase 2, and glutathione reductase 1. Furthermore, CK2 downregulation destabilized Nrf2 protein via inhibiting autophagic degradation of Kelch-like ECHassociated protein 1 (Keap1). Finally, CK2 downregulation decreased the nuclear import of Nrf2 by deactivating AMP-activated protein kinase (AMPK). Collectively, our data suggest that both Keap1 stabilization and AMPK inactivation are associated with decreased activity of Nrf2 in CK2 downregulation-induced cellular senescence. [BMB Reports 2020; 53(5): 272-277].

Dephosphorylation of p53 Ser 392 Enhances Trimethylation of Histone H3 Lys 9 via SUV39h1 Stabilization in CK2 Downregulation-Mediated Senescence.

Cellular senescence is an irreversible form of cell cycle arrest. Senescent cells have a unique gene expression profile that is frequently accompanied by senescence-associated heterochromatic foci (SAHFs). Protein kinase CK2 (CK2) downregulation can induce trimethylation of histone H3 Lys 9 (H3K9me3) and SAHFs formation by activating SUV39h1. Here, we present evidence that the PI3K-AKTmTOR-reactive oxygen species-p53 pathway is necessary for CK2 downregulation-mediated H3K9me3 and SAHFs formation. CK2 downregulation promotes SUV39h1 stability by inhibiting its proteasomal degradation in a p53dependent manner. Moreover, the dephosphorylation status of Ser 392 on p53, a possible CK2 target site, enhances the nuclear import and subsequent stabilization of SUV39h1 by inhibiting the interactions between p53, MDM2, and SUV39h1. Furthermore, p21Cip1/WAF1 is required for CK2 downregulation-mediated H3K9me3, and dephosphorylation of Ser 392 on p53 is important for efficient transcription of p21Cip1/WAF1. Taken together, these results suggest that CK2 downregulation induces dephosphorylation of Ser 392 on p53, which subsequently increases the stability of SUV39h1 and the expression of p21Cip1/WAF1, leading to H3K9me3 and SAHFs formation.

Defect of SIRT1-FoxO3a axis is associated with the production of reactive oxygen species during protein kinase CK2 downregulation-mediated cellular senescence and nematode aging.

We investigated whether SIRT1 is associated with reactive oxygen species (ROS) accumulation during CK2 downregulationmediated senescence. SIRT1 overexpression suppressed ROS accumulation, reduced transcription of FoxO3a target genes, and nuclear export and acetylation of FoxO3a, which were induced by CK2 downregulation in HCT116 and MCF-7 cells. Conversely, overexpression of a dominant-negative mutant SIRT1 (H363Y) counteracted decreased ROS levels, increased transcriptional activity of FoxO3a, and increased nuclear import and decreased acetylation of FoxO3a, which were induced by CK2 upregulation. CK2 downregulation destabilized SIRT1 protein via an ubiquitin-proteasome pathway in human cells, whereas CK2 overexpression reduced ubiquitination of SIRT1. Finally, the SIRT1 activator resveratrol attenuated the accumulation of ROS and lipofuscin as well as lifespan shortening, and reduced expression of the DAF-16 target gene sod-3, which were induced by CK2 downregulation in nematodes. Altogether, this study demonstrates that inactivation of the SIRT1-FoxO3a axis, at least in part, is involved in ROS generation during CK2 downregulationmediated cellular senescence and nematode aging. [BMB Reports 2019; 52(4): 265-270].

CK2 downregulation induces senescence-associated heterochromatic foci formation through activating SUV39h1 and inactivating G9a.

Cellular senescence is an irreversible form of cell cycle arrest and senescent cells have a unique gene expression profile that is frequently accompanied by senescence-associated heterochromatic foci (SAHF). Here, we present evidence that CK2 downregulation induces trimethylation of histone H3 Lys 9 (H3K9me3), selective binding of HP1γ to H3K9me3, formation of SAHF, and reduction of cyclin D1 expression in HCT116 and MCF-7 cells. CK2 downregulation-mediated H3K9me3 is associated with induction of H3K9 trimethylase SUV39h1 as well as reduction of H3K9 dimethylase G9a and GLP in cells. In addition, Pharmacological inhibition of SUV39h1 and G9a overexpression significantly attenuated induction of senescence-associated ß-galactosidase (SA-ß-gal) activity, H3K9me3 and SAHF formation in CK2-downregulated cells. Moreover, CK2 downregulation induced H3K9me3 in nematodes. Taken together, these results demonstrate that CK2 downregulation leads to H3K9me3 and SAHF formation by increasing SUV39h1 and decreasing G9a.

Mitochondrial dysfunction induces the invasive phenotype, and cell migration and invasion, through the induction of AKT and AMPK pathways in lung cancer cells.

Mitochondria are well known for their important roles in oxidative phosphorylation, amino acid metabolism, fatty acid oxidation and ion homeostasis. Although the effects of mitochondrial dysfunction on tumorigenesis in various cancer cells have been reported, the correlation between mitochondrial dysfunction and epithelial­to­mesenchymal transition (EMT) in lung cancer development and metastasis has not been well elucidated. In the present study, the effects of mitochondrial dysfunction on EMT and migration in lung cancer cells were investigated using inhibitors of mitochondrial respiration, oligomycin A and antimycin A. Oligomycin A and antimycin A induced distinct mesenchymal­like morphological features in H23, H1793 and A549 lung cancer cells. In addition, they decreased the expression levels of the epithelial marker protein E­cadherin, but increased the expression levels of the mesenchymal marker proteins Vimentin, Snail and Slug. The results of immunofluorescence staining indicated that oligomycin A and antimycin A downregulated cortical E­cadherin expression and upregulated the expression of Vimentin. In addition, oligomycin A and antimycin A increased the migration and invasion of A549 lung cancer cells, and promoted the expression levels of phosphorylated (p)­protein kinase B (AKT) and p­AMP­activated protein kinase (AMPK). Notably, the production of reactive oxygen species by oligomycin A and antimycin A did not affect the expression of EMT protein markers. Conversely, treatment with the AKT inhibitor wortmannin and the AMPK inhibitor Compound C upregulated E­cadherin and downregulated Vimentin expression. These results suggested that oligomycin A and antimycin A may induce migration and invasion of lung cancer cells by inducing EMT via the upregulation of p­AKT and p­AMPK expression.

Role of phospholipase D in the lifespan of Caenorhabditis elegans.

We have previously shown that phospholipase D (PLD) downregulation accelerates cellular senescence, which is widely believed to play an important role in aging, by stimulating reactive oxygen species (ROS) accumulation in human cells. In this study, we examined the role of PLD in aging using the nematode Caenorhabditis elegans. The mRNA level of pld-1 was found to be inversely correlated with aging. RNAi-mediated knockdown of pld-1 expression in nematodes enhanced ROS and lipofuscin accumulation and decreased lifespan, motility, and resistance to stress compared to that in nematodes treated with control RNAi. Pld-1 knockdown repressed the long lifespan of age-1 and akt-1 mutants but did not further reduce the short lifespan of daf-16 mutants, suggesting that PLD functions between AKT-1 and DAF-16. The ROS scavenger N-acetyl-L-cysteine, a PLD effector phosphatidic acid and a possible CK2 activator spermidine attenuated the lifespan shortening and age-related biomarkers triggered by pld-1 knockdown. Pld-1 RNAi downregulated the expression of DAF-16 target genes such as sod-3, dod-11, and mtl-1 in nematodes. In human cells, furthermore, PLD2 downregulation decreased the transcription of FoxO3a target genes (Cu/ZnSOD, MnSOD, catalase, thioredoxin-2, and peroxiredoxin-5), whereas ectopic PLD2 expression elevated the mRNA levels of these antioxidant genes. Taken together, these results indicated that PLD downregulation shortens longevity and induces age-related biomarkers through ROS accumulation by inhibiting the DAF-16/FoxO3a pathway in nematodes.

Protein kinase C downregulation induces senescence via FoxO3a inhibition in HCT116 and HEK293 cells.

We investigated the impact of protein kinase C (PKC) on cellular senescence. The PKC activity and expression of conventional PKC (cPKC) and atypical PKC (aPKC) isoforms decreased during replicative senescence in IMR-90 cells. Forced inhibition of cPKC or aPKC induced the activation of senescence markers, including senescence-associated ß-galactosidase activity and reactive oxygen species (ROS)-p53-p21Cip1/WAF1 axis in HCT116 and HEK293 cells. PKC inhibition triggered the nuclear exportation of FoxO3a via stimulation of AKT-mediated phosphorylation of FoxO3a, and thereby decreased the transcription of FoxO3a target genes. Conversely, ectopic expression of the PKC isoforms led to stimulation of the nuclear import of FoxO3a and expression of the FoxO3a target genes. Ectopic FoxO3a expression attenuated ROS accumulation and senescent phenotypes induced by PKC inhibition. Therefore, this study suggests for the first time that downregulation of PKC induces senescence through the AKT-FoxO3a-ROS-p53-p21Cip1/WAF1 pathway in HCT116 and HEK293 cells.

Downregulation of protein kinase CK2 activity induces age-related biomarkers in C. elegans.

Studies show that a decrease in protein kinase CK2 (CK2) activity is associated with cellular senescence. However, the role of CK2 in organism aging is still poorly understood. Here, we investigated whether protein kinase CK2 (CK2) modulated longevity in Caenorhabditis elegans. CK2 activity decreased with advancing age in the worms. Knockdown of kin-10 (the ortholog of CK2ß) led to a short lifespan phenotype and induced age-related biomarkers, including retardation of locomotion, decreased pharyngeal pumping rate, increased lipofuscin accumulation, and reduced resistance to heat and oxidative stress. The long lifespan of age-1 and akt-1 mutants was significantly suppressed by kin-10 RNAi, suggesting that CK2 acts downstream of AGE-1 and AKT-1. Kin-10 knockdown did not further shorten the short lifespan of daf-16 mutant worms but either decreased or increased the transcriptional activity of DAF-16 depending on the promoters of the target genes, indicating that CK2 is an upstream regulator of DAF-16 in C. elegans. Kin-10 knockdown increased production of reactive oxygen species (ROS) in the worms. Finally, the ROS scavenger N-acetyl-L-cysteine significantly counteracts the lifespan shortening and lipofuscin accumulation induced by kin-10 knockdown. Therefore, the present results suggest that age-dependent CK2 downregulation reduces longevity by associating with both ROS generation and the AGE-1-AKT-1-DAF-16 pathway in C. elegans.

Naringenin ameliorates kainic acid-induced morphological alterations in the dentate gyrus in a mouse model of temporal lobe epilepsy.

Granule cell dispersion (GCD) in the dentate gyrus (DG) of the hippocampus is a morphological alteration characteristic of temporal lobe epilepsy. Recently, we reported that treatment with naringin, a flavonoid found in grapefruit and citrus fruits, reduced spontaneous recurrent seizures by inhibiting kainic acid (KA)-induced GCD and neuronal cell death in mouse hippocampus, suggesting that naringin might have beneficial effects for preventing epileptic events in the adult brain. However, it is still unclear whether the beneficial effects of naringin treatment are mediated by the metabolism of naringin into naringenin in the KA-treated hippocampus. To investigate this possibility, we evaluated whether intraperitoneal injections of naringenin could mimic naringin-induced effects against GCD caused by intrahippocampal KA injections in mice. Our results showed that treatment with naringenin delayed the onset of KA-induced seizures and attenuated KA-induced GCD by inhibiting activation of the mammalian target of rapamycin complex 1 in both neurons and reactive astrocytes in the DG. In addition, its administration attenuated the production of proinflammatory cytokines such as tumor necrosis tumor necrosis factor-α (TNFα) and interleukin-1ß (IL-1ß) from microglial activation in the DG following KA treatment. These results suggest that naringenin may be an active metabolite of naringin and help prevent the progression of epileptic insults in the hippocampus in vivo; therefore, naringenin may be a beneficial metabolite of naringin for the treatment of epilepsy.

Inactivation of the FoxO3a transcription factor is associated with the production of reactive oxygen species during protein kinase CK2 downregulation-mediated senescence in human colon cancer and breast cancer cells.

We previously showed that protein kinase CK2 downregulation mediates senescence through the reactive oxygen species (ROS)-p53-p21(Cip1/WAF1) pathway in various human cells. In the present study, we investigated whether the FoxO3a transcription factor is associated with ROS production during CK2 downregulation-induced senescence in human colon cancer HCT116 and breast cancer MCF-7 cells. FoxO3a overexpression suppressed ROS production and p53 stabilization induced by a CK2α knockdown. CK2α downregulation induced nuclear export of FoxO3a through stimulation of AKT-mediated phosphorylation of FoxO3a and decreased transcription of its target genes (Cu/ZnSOD, MnSOD, and catalase). In contrast, CK2α overexpression inhibited AKT-mediated FoxO3a phosphorylation. This resulted in nuclear accumulation of FoxO3a, and elevated expression of its target genes. Therefore, these data indicate for the first time that CK2 downregulation stimulates ROS generation by inhibiting FoxO3a during premature senescence in human colon and breast cancer cells.

Fermented Acanthopanax koreanum Root Extract Reduces UVB- and H2O2-Induced Senescence in Human Skin Fibroblast Cells.

The present study assessed the effects of an aqueous extract of Acanthopanax koreanum root (AE) and of AE following fermentation by lactic acid bacteria (Lactobacillus plantarum and Bifidobacterium bifidum) (AEF) on human skin fibroblast HS68 cells exposed to ultraviolet B (UVB) irradiation and oxidative stress. AEF effectively antagonized the senescence-associated ß-galactosidase staining and upregulation of p53 and p21(Cip1/WAF1) induced by UVB or H2O2 treatment in HS68 cells. It also exhibited excellent antioxidant activities in radical scavenging assays and reduced the intracellular level of reactive oxygen species induced by UVB or H2O2 treatment. The antioxidant and antisenescent activities of AEF were greater than those of nonfermented A. koreanum extract. AEF significantly repressed the UVB- or H2O2-induced activities of matrix metalloproteinase (MMP)-1 and -3, overexpression of MMP-1, and nuclear factor κB (NF-κB) activation. This repression of NF-κB activation and MMP-1 overexpression was attenuated by a mitogen-activated protein kinase activator, suggesting that this AEF activity was dependent on this signaling pathway. Taken together, these data indicated that AEF-mediated antioxidant and anti-photoaging activities may produce anti-wrinkle effects on human skin.

Suppression of c-Myc induces apoptosis via an AMPK/mTOR-dependent pathway by 4-O-methyl-ascochlorin in leukemia cells.

4-O-Methyl-ascochlorin (MAC) is a methylated derivative of the prenyl-phenol antibiotic ascochlorin, which was isolated from an incomplete fungus, Ascochyta viciae. Although the effects of MAC on apoptosis have been reported, the underlying mechanisms remain unknown. Here, we show that MAC promoted apoptotic cell death and downregulated c-Myc expression in K562 human leukemia cells. The effect of MAC on apoptosis was similar to that of 10058-F4 (a c-Myc inhibitor) or c-Myc siRNA, suggesting that the downregulation of c-Myc expression plays a role in the apoptotic effect of MAC. Further investigation showed that MAC downregulated c-Myc by inhibiting protein synthesis. MAC promoted the phosphorylation of AMP-activated protein kinase (AMPK) and inhibited the phosphorylation of mammalian target of rapamycin (mTOR) and its target proteins, including p70S6 K and 4E-BP-1. Treatment of cells with AICAR (an AMPK activator), rapamycin (an mTOR inhibitor), or mTOR siRNA downregulated c-Myc expression and induced apoptosis to a similar extent to that of MAC. These results suggest that the effect of MAC on apoptosis induction in human leukemia cells is mediated by the suppression of c-Myc protein synthesis via an AMPK/mTOR-dependent mechanism.

Global analysis of phosphoproteome dynamics in embryonic development of zebrafish (Danio rerio).

The zebrafish (Danio rerio) is a popular animal model used for studies on vertebrate development and organogenesis. Recent research has shown a similarity of approximately 70% between the human and zebrafish genomes and about 84% of human disease-causing genes have common ancestry with that of the zebrafish genes. Zebrafish embryos have a number of desirable features, including transparency, a large size, and rapid embryogenesis. Protein phosphorylation is a well-known PTM, which can carry out various biological functions. Recent MS developments have enabled the study of global phosphorylation patterns by using MS-based proteomics coupled with titanium dioxide phosphopeptide enrichment. In the present study, we identified 3500 nonredundant phosphorylation sites on 2166 phosphoproteins and quantified 1564 phosphoproteins in developing embryos of zebrafish. The distribution of Ser/Thr/Tyr phosphorylation sites in zebrafish embryos was found to be 88.9, 10.2, and 0.9%, respectively. A potential kinase motif was predicted using Motif-X analysis, for 80% of the identified phosphorylation sites, with the proline-directed motif appearing most frequently, and 35 phosphorylation sites having the pSF motif. In addition, we created six phosphoprotein clusters based on their dynamic pattern during the development of zebrafish embryos. Here, we report the largest dataset of phosphoproteins in zebrafish embryos and our results can be used for further studies on phosphorylation sites or phosphoprotein dynamics in zebrafish embryos.