AMP-activated protein kinase (AMPK) cross-talks with canonical Wnt signaling via phosphorylation of β-catenin at Ser 552

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Abstract

AMP-activated protein kinase (AMPK) is a key regulator of energy metabolism; its activity is regulated by a plethora of physiological conditions, exercises and many anti-diabetic drugs. Recent studies show that AMPK involves in cell differentiation but the underlying mechanism remains undefined. Wingless Int-1 (Wnt)/β-catenin signaling pathway regulates the differentiation of mesenchymal stem cells through enhancing β-catenin/T-cell transcription factor 1 (TCF) mediated transcription. The objective of this study was to determine whether AMPK cross-talks with Wnt/β-catenin signaling through phosphorylation of β-catenin. C3H10T1/2 mesenchymal cells were used. Chemical inhibition of AMPK and the expression of a dominant negative AMPK decreased phosphorylation of β-catenin at Ser 552. The β-catenin/TCF mediated transcription was correlated with AMPK activity. In vitro, pure AMPK phosphorylated β-catenin at Ser 552 and the mutation of Ser 552 to Ala prevented such phosphorylation, which was further confirmed using [γ-32P]ATP autoradiography. In conclusion, AMPK phosphorylates β-catenin at Ser 552, which stabilizes β-catenin, enhances β-catenin/TCF mediated transcription, expanding AMPK from regulation of energy metabolism to cell differentiation and development via cross-talking with the Wnt/β-catenin signaling pathway.

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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