Akt2 Kinase Suppresses Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH)-mediated Apoptosis in Ovarian Cancer Cells via Phosphorylating GAPDH at Threonine 237 and Decreasing Its Nuclear Translocation

Department of Experimental Medicine, Fuzhou General Hospital (Dongfang Hospital), 156 North Xi-er Huan Road, Fuzhou City, Fujian Province 350025, China.
Journal of Biological Chemistry (Impact Factor: 4.57). 12/2011; 286(49):42211-20. DOI: 10.1074/jbc.M111.296905
Source: PubMed


Protein kinase B (Akt) plays important roles in regulation of cell growth and survival, but while many aspects of its mechanism of action are known, there are potentially additional regulatory events that remain to be discovered. Here we detected a 36-kDa protein that was co-immunoprecipitated with protein kinase Bβ (Akt2) in OVCAR-3 ovarian cancer cells. The protein was identified to be glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by MALDI-TOF/TOF MS, and the interaction of Akt2 and GAPDH was verified by reverse immunoprecipitation. Our further study showed that Akt2 may suppress GAPDH-mediated apoptosis in ovarian cancer cells. Overexpression of GAPDH increased ovarian cancer cell apoptosis induced by H(2)O(2), which was inhibited by Akt2 overexpression and restored by the PI3K/Akt inhibitor wortmannin or Akt2 siRNA. Akt2 phosphorylated Thr-237 of GAPDH and decreased its nuclear translocation, an essential step for GAPDH-mediated apoptosis. The interaction between Akt2 and GAPDH may be important in ovarian cancer as immunohistochemical analysis of 10 normal and 30 cancerous ovarian tissues revealed that decreased nuclear expression of GAPDH correlated with activation (phosphorylation) of Akt2. In conclusion, our study suggests that activated Akt2 may increase ovarian cancer cell survival via inhibition of GAPDH-induced apoptosis. This effect of Akt2 is partly mediated by its phosphorylation of GAPDH at Thr-237, which results in the inhibition of GAPDH nuclear translocation.

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    • "On the other hand, overexpression of Akt2 increased the response to EGF and enhanced cell viability. Supportably, other authors indicated that activated Akt2 might increase ovarian cancer cell survival via inhibition of apoptosis 31. Frequent activation of Akt2 is a common occurrence in human ovarian cancer 32 (particularly in undifferentiated tumors) and is often followed by a poor prognosis 33. "
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    ABSTRACT: Objective: Overexpression of the epidermal growth factor receptor (EGFR) is associated with the malignant phenotype in many cancers including ovarian cancer, which leads to increased cell proliferation and survival. In spite of emerging EGFR inhibitors as a potentially useful agent, they are largely ineffective in patients with advanced or recurrent ovarian cancers. Since Akt as a key downstream factor of EGFR is highly activated in some high grade serous ovarian tumors, the augmented Akt activation may attribute to irregular EGFR-mediated signaling observed in ovarian cancer. Here we investigated the differential effect of Akt on the EGF-induced cell viability in a panel of ovarian cancer cell lines. Methods: Cellular viability assay and western blot analysis were used to measure cell viability and expression levels of proteins, respectively. Knockdown of Akt was achieved with siRNA and stable transfection of expression vectors was performed. Results: Cellular viability increased in OVCAR-3 ovarian cancer cells exposed to EGF, but little to no difference was observed in the 5 other ovarian cancer cells including SKOV-3 cells despite of the expression of EGFR. In OVCAR-3 cells, EGF activated Erk and Akt, but an Erk inhibitor had no impact on cellular viability. On the other hand, the EGFR and PI3K inhibitors decreased EGF-induced cellular viability, indicating the involvement of Akt signaling. Although EGF activated Erk in SKOV-3 cells, the Akt activation was very weak as compared to OVCAR-3 cells. Furthermore, we observed a different expression of Akt isoforms: Akt1 was constitutively expressed in all tested ovarian cancer cells, while Akt3 was little expressed. Interestingly, Akt2 was highly expressed in OVCAR-3 cells. Knockdown of Akt2 blocked EGF-induced OVCAR-3 cell viability whereas knockdown for Akt1 and Erk1/2 had no significant effect. Stable transfection of Akt2 into SKOV-3 cells phosphorylated more Akt and enhanced cell viability in response to EGF. Conclusions: Akt2-dependent signaling appears to play an important role in EGFR-mediated cellular viability in ovarian cancer and targeting specific Akt isoform may provide a potential therapeutic approach for EGFR-expressing ovarian cancers.
    Preview · Article · Sep 2014 · Journal of Cancer
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    • "Cell culture and transfection with siRNA were performed in accord with the methods described by us previously [33]. AGS or MKN-45 cells (AGS, American Type Culture Collection, Manassas, VA; MKN-45, Health Service Research Resources Bank, Ibaraki-shi, Osaka, Japan) were grown in F12 or DMEM medium (Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS) at 37°C in an incubator containing 5% CO2. "
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    ABSTRACT: Interleukin-1beta (IL-1beta) has been implicated in the progression of gastric adenocarcinoma (GA); however, the molecular mechanisms of action of IL-1beta in GA are poorly characterized. P38 and JNK are the major MAPK family members that regulate IL-1beta signaling pathways. Here, we investigated the role of both p38 and JNK in IL-1beta-induced GA cell migration, invasion and metastatic potential. The effects of IL-1beta-induced p38 and JNK activation in GA cells were determined using in vitro Transwell migration and invasion assays of MKN-45 and AGS cells, or an in vivo metastasis assay in nude mice. The IL-1beta-induced p38 signaling pathway was further characterized in GA cells. Activation of the IL-1beta/p38 signaling pathway was also assessed in human primary GA tissues by immunohistochemistry. IL-1beta-induced activation of p38 increased GA cell migration and invasion in vitro and promoted the metastatic potential of GA cells in vivo; these effects were attenuated by p38 siRNA or the p38 inhibitor SB202190. MMP2 or MMP9 siRNAs and the MMP2/9 inhibitor BiPS also inhibited IL-1beta-induced GA cell migration and invasion in vitro. IL-1beta-induced p38 activation significantly increased MMP2 and MMP9 mRNA and protein expression and activity. Luciferase reporter assays demonstrated that the activator protein-1 (AP-1) and the AP-1 binding sites of the MMP9 promoter (-670/MMP9) were activated by IL-1beta-induced p38 activation. Phospho-p38 was significantly upregulated in human GA tissues (compared to matched non-neoplastic tissues), and significantly associated with lymph node metastasis, and invasion beyond the serosa. Expression of phospho-p38 significantly correlated with IL-1beta, MMP2, MMP9, and c-fos expression in both human GA tissues and GA cell metastases in the lungs of nude mice. IL-1beta was also capable of activating JNK in GA cells, but activation of JNK was not associated with GA cell migration and invasion. Therefore, IL-1beta-induced the migration and invasion in GA cells was regulated by p38, but not by JNK. IL-1beta-induced p38 activation and the IL-1beta/p38/AP-1(c-fos)/MMP2 & MMP9 pathway play an important role in metastasis in GA; this pathway may provide a novel therapeutic target for GA.
    Full-text · Article · Jan 2014 · Molecular Cancer
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    • "In addition, the formation of COPI transport vesicles is known to be mediated by the interaction of GAPDH with RAB2 [75]. Phosphorylation of GAPDH occurs through the Src/PI3K/AKT pathway, often downstream of receptor tyrosine kinase activation [76]. We found a single receptor tyrosine kinase enriched in iridophores, ltk, which we have previously shown to be required for iridophore development [27]. "
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    ABSTRACT: In order to facilitate understanding of pigment cell biology, we developed a method to concomitantly purify melanocytes, iridophores, and retinal pigmented epithelium from zebrafish, and analyzed their transcriptomes. Comparing expression data from these cell types and whole embryos allowed us to reveal gene expression co-enrichment in melanocytes and retinal pigmented epithelium, as well as in melanocytes and iridophores. We found 214 genes co-enriched in melanocytes and retinal pigmented epithelium, indicating the shared functions of melanin-producing cells. We found 62 genes significantly co-enriched in melanocytes and iridophores, illustrative of their shared developmental origins from the neural crest. This is also the first analysis of the iridophore transcriptome. Gene expression analysis for iridophores revealed extensive enrichment of specific enzymes to coordinate production of their guanine-based reflective pigment. We speculate the coordinated upregulation of specific enzymes from several metabolic pathways recycles the rate-limiting substrate for purine synthesis, phosphoribosyl pyrophosphate, thus constituting a guanine cycle. The purification procedure and expression analysis described here, along with the accompanying transcriptome-wide expression data, provide the first mRNA sequencing data for multiple purified zebrafish pigment cell types, and will be a useful resource for further studies of pigment cell biology.
    Preview · Article · Jul 2013 · PLoS ONE
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