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Updated: Jan 23, 2026
Oligopeptide Competition Assay for Phosphorylation Site Determination
Published on: May 18, 2017
Phosphorylation of TET2 by AMPK is indispensable in myogenic differentiation
Ting Zhang1,2, Xiaowen Guan1,2, Un Lam Choi1,2
1Faculty of Health Sciences, University of Macau, Avenida da Universidade, Taipa, Macau.
Background:
TET-mediated oxidation of 5-mC participates in both passive and active DNA demethylation, which exerts a significant influence on diverse biological processes. Mass spectrometry has identified multiple phosphorylation sites of TET2. However, the functions of these phosphosites and their corresponding kinases are mostly unknown.
Results:
Here, we showed that AMP-activated protein kinase (AMPK) phosphorylates murine TET2 at the serine residue 97 (S97), and the phosphorylation enhances TET2 stability through promoting its binding to 14-3-3β. AMPK ablation resulted in decreased global 5-hmC levels at the myotube stages, severe differentiation defects of C2C12 cells and significantly, total loss of expression of Pax7. Genome-wide analyses revealed increased DNA methylation at genic and enhancer regions of AMPK-null myoblasts and myotubes. Using CRISPR/Cas9 technology, we showed that a novel enhancer, which is hypermethylated in AMPK-null cells, regulates Pax7 expression. The phospho-mimicking mutant, TET2-S97E, could partly rescue the differentiation defect in AMPK-ablated C2C12 cells.
Conclusions:
Together, our data demonstrated that AMPK is a critical regulator of myogenesis, partly through phosphorylating TET2.
Insights
AMP-activated protein kinase (AMPK) phosphorylates TET2, enhancing its stability and regulating muscle cell differentiation. This discovery reveals a key mechanism in myogenesis involving DNA demethylation and gene expression.
Area of Science:
- Epigenetics and Gene Regulation
- Molecular Biology
- Cellular Differentiation
Background:
- TET-mediated oxidation of 5-methylcytosine (5-mC) is crucial for DNA demethylation, impacting various biological processes.
- While multiple TET2 phosphorylation sites are known, their functions and associated kinases remain largely uncharacterized.
Purpose of the Study:
- To investigate the role of AMP-activated protein kinase (AMPK) in regulating TET2 function.
- To elucidate the impact of TET2 phosphorylation by AMPK on myogenesis and gene expression.
Main Methods:
- Phosphorylation site identification and functional analysis of TET2.
- AMPK knockout/ablation in C2C12 myoblasts and myotubes.
- Global 5-hydroxymethylcytosine (5-hmC) level assessment.
- Genome-wide DNA methylation analysis.
- CRISPR/Cas9 gene editing to investigate enhancer function.
- Analysis of Pax7 expression and myoblast differentiation.
Main Results:
- AMPK phosphorylates murine TET2 at serine 97 (S97), enhancing its stability via 14-3-3β binding.
- AMPK ablation in C2C12 cells led to reduced 5-hmC, impaired differentiation, and loss of Pax7 expression.
- AMPK-null cells exhibited increased DNA methylation at genic and enhancer regions, including a novel enhancer regulating Pax7.
- A phospho-mimicking TET2 mutant (TET2-S97E) partially rescued differentiation defects in AMPK-ablated cells.
Conclusions:
- AMP-activated protein kinase (AMPK) is a critical regulator of myogenesis.
- AMPK phosphorylates TET2, influencing its stability and downstream effects on DNA methylation and gene expression.
- The AMPK-TET2 pathway plays a significant role in muscle cell differentiation and Pax7 regulation.
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