Biochemical and Biophysical Research Communications
AMP-activated protein kinase (AMPK) cross-talks with canonical Wnt signaling via phosphorylation of β-catenin at Ser 552
Introduction
AMP-activated protein kinase (AMPK) is a key player in the development of obesity and type 2 diabetes (T2D) [1], [2]. It is a serine-threonine kinase consisting of a catalytic subunit (α) and two regulatory subunits (β and γ). AMPK regulates energy metabolism in cells. Its activation is associated with phosphorylation of AMPKα subunit at Thr 172. Protein phosphatase 2C (PP2C) dephosphorylates the Thr 172 phosphorylation of AMPKα subunit, inactivating AMPK [3]. An accumulating body of evidences suggests the involvement of AMPK in mesenchymal stem cell (MSC) differentiation [4], [5], [6]. In 3T3-L1 cells, activation of AMPK inhibits adipogenesis [6], [7]. Activation of AMPK promotes myogenesis [8]. Our previous studies found that enhanced adipogenesis from MSC was associated with down-regulation of AMPK activity [9], [10], [11]. However, mechanisms linking AMPK to MSC remain undefined.
Canonical Wnt/β-catenin signaling pathway is required for early embryonic myogenesis [12]. Activation of the Wnt signaling pathway enhances myogenesis and inhibits adipogenesis in cultured MSCs [13] while down-regulation promoting adipogenesis [14], [15], [16]. Wnt/β-catenin signaling suppresses MSC commitment to the adipogenic lineage and terminal differentiation by blocking induction of peroxisome proliferator-activated receptor (PPAR) γ, an effect mediated by β-catenin [17]. Ser 552 of β-catenin is previously identified as a phosphorylation site by protein kinase A (PKA) [18], [19] and protein kinase B (Akt) [20]. We used GPS 2.0 phosphorylation prediction system [21] and this site was also predicted to be a site for AMPK phosphorylation. We hypothesized that AMPK cross-talks with canonical Wnt signaling through phosphorylation of β-catenin to regulate adipogenesis and MSC differentiation. Our results show that AMPK phosphorylates β-catenin at Ser 552, which enhances β-catenin/TCF mediated transcription, linking AMPK to Wnt/β-catenin signaling pathway to regulate adipogenesis.
Section snippets
Cell culture
Mouse C3H10T1/2 cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 1% antibiotics at 37 °C. Cell differentiation was induced as described previously [22], [23], [24]. Briefly, C3H10T1/2 cells were treated with 10 μM 5′-azacytidine for 3 days, and seeded at 30% confluence in 24-well plates. Cells were grown in adipogenic medium (1 μg/ml insulin, 0.1 μg/ml dexamethazone, 27.8 μg/ml isobutylmethylaxanthine and 10 μM troglitazone) for 3 days, repeated once.
AMPK phosphorylates β-catenin at Ser 552
To determine whether the Ser 552 of β-catenin could serve as a substrate for AMPK, we mutated Ser 552 to Ala and performed in vitro kinase assays using WT β-catenin or β-catenin S552A. β-Catenin was purified by immunoprecipitation, and then incubated with or without pure AMPK. AMPK catalyzed the phosphorylation of WT β-catenin, and the mutation at Ser 552 to Ala abolished this effect (Fig. 1A). The weak band of phosphorylation detected in the S552R β-catenin sample should be due to nonspecific
Discussion
Obesity and T2D are closely linked metabolic complications, both of which are increasing at alarming rates [38]. AMPK has a key role in the development of obesity and T2D [39]. Activation of AMPK inhibits adipogenesis [4], [5]. Activation of AMPK promotes myogenesis [8]. However, the mechanisms linking AMPK to MSC differentiation remains unclear. Wnt/β-catenin signaling pathway regulates morphogenesis during early developmental stages. Activation of Wnt signaling stabilizes β-catenin, which
Acknowledgments
The work was supported by NIH INBREP20RR016474 and 1R03HD057506, and USDA2008-35206-18826.
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