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Ankrd2/ARPP is a novel Akt2 specific substrate and regulates myogenic differentiation upon cellular exposure to H(2)O(2).

ABSTRACT: Activation of Akt-mediated signaling pathways is crucial for survival, differentiation, and regeneration of muscle cells. A proteomic-based search for novel substrates of Akt was therefore undertaken in C(2)C(12) murine muscle cells exploiting protein characterization databases in combination with an anti-phospho-Akt substrate antibody. A Scansite database search predicted Ankrd2 (Ankyrin repeat domain protein 2, also known as ARPP) as a novel substrate of Akt. In vitro and in vivo studies confirmed that Akt phosphorylates Ankrd2 at Ser-99. Moreover, by kinase assay with recombinant Akt1 and Akt2, as well as by single-isoform silencing, we demonstrated that Ankrd2 is a specific substrate of Akt2. Ankrd2 is typically found in skeletal muscle cells, where it mediates the transcriptional response to stress conditions. In an attempt to investigate the physiological implications of Ankrd2 phosphorylation by Akt2, we found that oxidative stress induced by H(2)O(2) triggers this phosphorylation. Moreover, the forced expression of a phosphorylation-defective mutant form of Ankrd2 in C(2)C(12) myoblasts promoted a faster differentiation program, implicating Akt-dependent phosphorylation at Ser-99 in the negative regulation of myogenesis in response to stress conditions.

SUBMITTER: Cenni V

PROVIDER: S-EPMC3154889 | biostudies-literature | 2011 Aug

REPOSITORIES: biostudies-literature

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Ankrd2/ARPP is a novel Akt2 specific substrate and regulates myogenic differentiation upon cellular exposure to H(2)O(2).

Cenni Vittoria V Bavelloni Alberto A Beretti Francesca F Tagliavini Francesca F Manzoli Lucia L Lattanzi Giovanna G Maraldi Nadir M NM Cocco Lucio L Marmiroli Sandra S

Molecular biology of the cell 20110707 16

Activation of Akt-mediated signaling pathways is crucial for survival, differentiation, and regeneration of muscle cells. A proteomic-based search for novel substrates of Akt was therefore undertaken in C(2)C(12) murine muscle cells exploiting protein characterization databases in combination with an anti-phospho-Akt substrate antibody. A Scansite database search predicted Ankrd2 (Ankyrin repeat domain protein 2, also known as ARPP) as a novel substrate of Akt. In vitro and in vivo studies confi ...[more]

PMID: 21737686

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Project description:BackgroundSarcopenia, the age-related decline in skeletal muscle mass and function, diminishes life quality in elderly people. Improving the capacity of skeletal muscle differentiation is expected to counteract sarcopenia. However, the mechanisms underlying skeletal muscle differentiation are complex, and effective therapeutic targets are largely unknown.MethodsThe human Gene Expression Omnibus database, aged mice and primary skeletal muscle cells were used to assess the expression level of pyruvate dehydrogenase B (PDHB) in human and mouse aged state. d-Galactose (d-gal)-induced sarcopenia mouse model and two classic cell models (C2C12 and HSkMC) were used to assess the myogenic effect of PDHB and the underlying mechanisms via immunocytochemistry, western blotting, quantitative real-time polymerase chain reaction, RNA interference or overexpression, dual-luciferase reporter assay, RNA sequencing and untargeted metabolomics.ResultsWe identified that a novel target PDHB promoted myogenic differentiation. PDHB expression decreased in aged mouse muscle relative to the young state (-50% of mRNA level, P < 0.01) and increased during mouse and primary human muscle cell differentiation (+3.97-fold, P < 0.001 and +3.79-fold, P < 0.001). Knockdown or overexpression of PDHB modulated the expression of genes related to muscle differentiation, namely, myogenic factor 5 (Myf5) (-46%, P < 0.01 and -27%, P < 0.05; +1.8-fold, P < 0.01), myogenic differentiation (MyoD) (-55%, P < 0.001 and -34%, P < 0.01; +2.27-fold, P < 0.001), myogenin (MyoG) (-60%, P < 0.001 and -70%, P < 0.001; +5.46-fold, P < 0.001) and myosin heavy chain (MyHC) (-70%, P < 0.001 and -69%, P < 0.001; +3.44-fold, P < 0.001) in both C2C12 cells and HSkMC. Metabolomic and transcriptomic analyses revealed that PDHB knockdown suppressed pyruvate metabolism (P < 0.001) and up-regulated ariadne RBR E3 ubiquitin protein ligase 2 (Arih2) (+7.23-fold, P < 0.001) in cellular catabolic pathways. The role of forkhead box P1 (FoxP1) (+4.18-fold, P < 0.001)-mediated Arih2 transcription was the key downstream regulator of PDHB in muscle differentiation. PDHB overexpression improved d-gal-induced muscle atrophy in mice, which was characterized by significant increases in grip strength, muscle mass and mean muscle cross-sectional area (1.19-fold to 1.5-fold, P < 0.01, P < 0.05 and P < 0.001).ConclusionsThe comprehensive results show that PDHB plays a sarcoprotective role by suppressing the FoxP1-Arih2 axis and may serve as a therapeutic target in sarcopenia.

Dataset Information

Ankrd2/ARPP is a novel Akt2 specific substrate and regulates myogenic differentiation upon cellular exposure to H(2)O(2).

Publications

Ankrd2/ARPP is a novel Akt2 specific substrate and regulates myogenic differentiation upon cellular exposure to H(2)O(2).

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