Extensive Crosstalk between O-GlcNAcylation and Phosphorylation Regulates Akt Signaling

Division of Antitumor Pharmacology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China.
PLoS ONE (Impact Factor: 3.23). 05/2012; 7(5):e37427. DOI: 10.1371/journal.pone.0037427
Source: PubMed


O-linked N-acetylglucosamine glycosylations (O-GlcNAc) and O-linked phosphorylations (O-phosphate), as two important types of post-translational modifications, often occur on the same protein and bear a reciprocal relationship. In addition to the well documented phosphorylations that control Akt activity, Akt also undergoes O-GlcNAcylation, but the interplay between these two modifications and the biological significance remain unclear, largely due to the technique challenges. Here, we applied a two-step analytic approach composed of the O-GlcNAc immunoenrichment and subsequent O-phosphate immunodetection. Such an easy method enabled us to visualize endogenous glycosylated and phosphorylated Akt subpopulations in parallel and observed the inhibitory effect of Akt O-GlcNAcylations on its phosphorylation. Further studies utilizing mass spectrometry and mutagenesis approaches showed that O-GlcNAcylations at Thr 305 and Thr 312 inhibited Akt phosphorylation at Thr 308 via disrupting the interaction between Akt and PDK1. The impaired Akt activation in turn resulted in the compromised biological functions of Akt, as evidenced by suppressed cell proliferation and migration capabilities. Together, this study revealed an extensive crosstalk between O-GlcNAcylations and phosphorylations of Akt and demonstrated O-GlcNAcylation as a new regulatory modification for Akt signaling.

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Available from: Cheng Luo, Oct 05, 2015
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    • "Subsequent study from Soesanto and colleagues reports that Akt itself can undergo O-GlcNAcylation [73]. Geng’s and Gong’s groups later demonstrated that O-GlcNAcylation of Akt inhibits its Thr 308 phosphorylation in various cell types [74, 75]. Geng’s group further mapped the O-GlcNAcylation sites of Akt and illustrated that O-GlcNAcylation occurs at Thr305 and Thr312. "
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    ABSTRACT: Akt regulates critical cellular processes including cell survival and proliferation, glucose metabolism, cell migration, cancer progression and metastasis through phosphorylation of a variety of downstream targets. The Akt pathway is one of the most prevalently hyperactivated signaling pathways in human cancer, thus, research deciphering molecular mechanisms which underlie the aberrant Akt activation has received enormous attention. The PI3K-dependent Akt serine/threonine phosphorylation by PDK1 and mTORC2 has long been thought to be the primary mechanism accounting for Akt activation. However, this regulation alone does not sufficiently explain how Akt hyperactivation can occur in tumors with normal levels of PI3K/PTEN activity. Mounting evidence demonstrates that aberrant Akt activation can be attributed to other posttranslational modifications, which include tyrosine phosphorylation, O-GlcNAcylation, as well as lysine modifications: ubiquitination, SUMOylation and acetylation. Among them, K63-linked ubiquitination has been shown to be a critical step for Akt signal activation by facilitating its membrane recruitment. Deficiency of E3 ligases responsible for growth factor-induced Akt activation leads to tumor suppression. Therefore, a comprehensive understanding of posttranslational modifications in Akt regulation will offer novel strategies for cancer therapy.
    Cell and Bioscience 10/2014; 4(1):59. DOI:10.1186/2045-3701-4-59 · 3.63 Impact Factor
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    • "However, the relationship between O-GlcNAcylation and phosphorylation of Akt in cancer cells is not fully elucidated. Wang et al. showed that O-GlcNAcylations at Thr305 and Thr312 inhibited Akt phosphorylation at Thr308 via disrupting the interaction between Akt and PDK1 in MCF-7 cells (51). The impaired Akt activation affected functions of Akt, as evidenced by suppressed cell proliferation and migration capabilities. "
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    ABSTRACT: Although cancer metabolism has received considerable attention over the past decade, our knowledge on its specifics is still fragmentary. Altered cellular metabolism is one of the most important hallmarks of cancer. Cancer cells exhibit aberrant glucose metabolism characterized by aerobic glycolysis, a phenomenon known as Warburg effect. Accelerated glucose uptake and glycolysis are main characteristics of cancer cells that allow them for intensive growth and proliferation. Accumulating evidence suggests that O-GlcNAc transferase (OGT), an enzyme responsible for modification of proteins with N-acetylglucosamine, may act as a nutrient sensor that links hexosamine biosynthesis pathway to oncogenic signaling and regulation of factors involved in glucose and lipid metabolism. Recent studies suggest that metabolic reprograming in cancer is connected to changes at the epigenetic level. O-GlcNAcylation seems to play an important role in the regulation of the epigenome in response to cellular metabolic status. Through histone modifications and assembly of gene transcription complexes, OGT can impact on expression of genes important for cellular metabolism. This paper reviews recent findings related to O-GlcNAc-dependent regulation of signaling pathways, transcription factors, enzymes, and epigenetic changes involved in metabolic reprograming of cancer.
    Frontiers in Endocrinology 09/2014; 5:145. DOI:10.3389/fendo.2014.00145
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    • "Further research into this aspect of PKB phosphorylation is required. It is notable that Thr312 is known to be O-GlcNAcylated36, implying that Thr312 is accessible to modifiers. In addition, Chen et al reported that Tyr315, which is also located on the activation loop, was phosphorylated and that this Tyr-phosphorylation elevated PKB activity in mammalian cells37. "
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    ABSTRACT: Protein kinase B (PKB) also known as Akt is involved in many signal transduction pathways. As alterations of the PKB pathway are found in a number of human malignancies, PKB is considered an important drug target for cancer therapy. However, production of sufficient amounts of active PKB for biochemical and structural studies is very costly because of the necessity of using a higher organism expression system to obtain phosphorylated PKB. Here, we report efficient production of active PKBα using the BmNPV bacmid expression system with silkworm larvae. Following direct injection of bacmid DNA, recombinant PKBα protein was highly expressed in the fat bodies of larvae, and could be purified using a GST-tag and then cleaved. A final yield of approximately 1 mg PKBα/20 larvae was recorded. Kinase assays showed that the recombinant PKBα possessed high phosphorylation activity. We further confirmed phosphorylation on the activation loop by mass spectrometric analysis. Our results indicate that the silkworm expression system is of value for preparation of active-form PKBα with phosphorylation on the activation loop. This efficient production of the active protein will facilitate further biochemical and structural studies and stimulate subsequent drug development.
    Scientific Reports 08/2014; 4:6016. DOI:10.1038/srep06016 · 5.58 Impact Factor
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