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.

FIGURE 1:

Akt phosphorylates Ankrd2. (A) Cycling C₂C₁₂ cells were grown to confluence and allowed to differentiate from d0 to d7 in the presence of differentiating medium. Cell extracts were immunoblotted with the anti–phospho-Akt substrates (anti P-Akt sub) antibody. Anti-Troponin T and -β tubulin antibodies were used as differentiation and equal loading markers, respectively. Asterisks highlight bands significantly more phosphorylated at d1 of differentiation. Line graph demonstrates the average fold change in phosphorylation, determined as the ratio in the change of Akt-phosphorylated Ankrd2 vs. the amount of total Ankrd2 of the lysates of A and indicates that Ankrd2 phosphorylation by Akt peaks at d1. (B) Top, lysates from d1-differentiating C₂C₁₂ myoblasts were focused with a MicroRotofor device and probed with the anti–phospo-Akt substrates antibody. Nitrocellulose was then stripped and incubated with an anti-Ankrd2 specific antibody (bottom panel).

FIGURE 2:

Akt phosphorylates Ankrd2 at Ser-99 in vitro and in HEK 293T cells. (A) In vitro Akt kinase assay. HEK 293T cells, which do not endogenously express Ankrd2, were transfected with flag-tagged WT or S99A Ankrd2 and then recovered in serum-free medium for 24 h. Overexpressed Ankrd2 forms were then immunoprecipitated by an anti-flag antibody. Immunoprecipitates were used as substrates for an in vitro kinase assay with [γ³²]P-ATP and recombinant active Akt1 (+Akt1) or Akt2 (+Akt2) or kinase buffer alone (–). As control, the activity of each Akt isoform was also monitored on myelin basic protein. Reactions were resolved on SDS–PAGE and blotted on nitrocellulose. (B) HEK 293T cells were transfected with flag-Ankrd2, serum starved for 24 h, and stimulated with 1.6 μM insulin for the indicated times. Cell extracts were resolved on SDS–PAGE and subjected to immunoblotting with the indicated antibodies. (C) HEK 293T cells were transfected with flag-Ankrd2 WT or S99A as indicated, serum starved for 24 h, and treated with insulin or vehicle for 30 min. Where indicated before insulin, cells were also pretreated with the inhibitors for Akt (perifosine and inhibitor IV) and p70 S6K (rapamycin). Lysates were resolved on SDS–PAGE and subjected to immunoblotting with the indicated antibodies. Activities of the inhibitors were checked by the use of antibodies directed against anti–-phospho Akt Ser473 (Cell Signaling) and -phospho p70 S6K Ser411 (Santa Cruz Biotechnology). (D) HEK 293T cells were transfected with flag-Ankrd2 WT, serum starved, and treated with insulin. Where indicated, cells were pretreated with Akt1/2 and Akt2 specific inhibitors. Akt1 and Akt2 were separately immunoprecipitated from cell lysates by the use of specific antibodies, and the corresponding immunocomplexes were resolved on SDS–PAGE, blotted, and probed with the indicated antibodies.

FIGURE 3:

Ankrd2 is an Akt2 specific substrate. (A) HEK 293T cells expressing flag-wild-type (wt) Ankrd2 were infected with Akt1 or Akt2 shRNA lentiviral vector or empty vector for 24 h. Cells were then serum starved and stimulated with insulin for 30 min before addition of lysis buffer. Ankrd2 was then immunoprecipitated from total cell lysates by anti-flag antibody, and pellets were resolved on SDS–PAGE and subjected to immunoblot analysis. Western blot directed against Akt –1 and –2 of total lysates confirms Akt specific isoform silencing. (B) HEK 293T cells were cotransfected with flag-Ankrd2 wt and HA-Akt –1, –2, or –3. Cellular extracts were subjected to immunoprecipitation by an anti-flag antibody. Obtained precipitates and corresponding lysates were resolved on SDS–PAGE and immunoblotted with an anti–HA antibody to check the Akt isoform bound to Ankrd2. (C) HEK 293T cells were cotransfected with HA-Akt2 and wt or S99A forms of flag-Ankrd2. Cells were serum starved for 24 h and treated with 1.6 μM insulin for 30 min. Cells extracts were immunoprecipitated with anti-flag antibody and immunoblotted with the indicated antibodies.

FIGURE 4:

H₂O₂ is a physiological activator of Ankrd2 phosphorylation by Akt2 in C₂C₁₂ cells. (A) Whole lysates from D1-differentiated C₂C₁₂ cells exposed to H₂O₂ 450 μM for the indicated times were resolved on a 12% SDS–PAGE and immunoblotted. Akt activation following H₂O₂ exposure was monitored by the anti Phospho-Akt Ser-473 antibody (α-P-Akt). Cell-cycle exit dependent from H₂O₂ exposure was examined by immunoblotting lysates with anti–cyclin D3. Anti-actin revealed equal loadings. (B) Day 1 (D1)-differentiated C₂C₁₂ cells were exposed to H₂O₂ 450 μM for the indicated times. Whole-cell lysates were immunoprecipitated with anti-Ankrd2, -Phospho-Akt substrate, or -Akt1/2 (N terminus) antibodies, as indicated. Immunocomplexes and corresponding lysates were resolved on 12% SDS–PAGE and immunoblotted as indicated. (C) D1-differentiated C₂C₁₂ cells were left untreated or treated with 4 or 6 μM Akt1/2 or Akt2 inhibitors and then exposed to H₂O₂ 450 μM for 15 min. Total cell lysates were divided and immunoprecipitated with anti–Ankrd2 antibody to check Ankrd2 phosphorylation by Akt, anti-Akt1 and -Akt2 antibodies to verify the efficacy of inhibitors.

FIGURE 5:

Oxidative stress induces nuclear translocation of Ankrd2. (A) Day 1 (D1)-differentiating C₂C₁₂ myotubes were transfected with WT or S99A forms of flag-tagged Ankrd2 and exposed to H₂O₂ 450 μM for 3 h. Immunofluorescence was performed using an anti-flag TRITC-conjugated antibody. Cell nuclei were counterstained with DAPI. Bar, 10 μM. (B) Statistical analysis (mean values ± SD of counts performed in three different samples). C₂C₁₂ multinucleated myotube counts were performed after 2 d of serum deprivation of cells not transfected (nt), transfected with empty vector (ev), wild type (wt), or S99A-mutant Ankrd2. (C) D1-differentiating C₂C₁₂ cells were treated with H₂O₂ 450 μM for 15 min or 3 h or were left untreated, then nuclear and cytoplasmic fractions were purified. Endogenous Ankrd2 was next immunoprecipitated by anti–Ankrd2 antibody, run on SDS–PAGE, blotted, and checked for the presence of Phospho Akt substrates, Ankrd2, activated and total Akt. Lamin A/C and β-tubulin antibodies were used to exclude nuclear or cytoplasmic contamination from cytoplasmic and nuclear extracts, respectively.

FIGURE 6:

Failure of phosphorylation of Ankrd2 at Ser-99 promotes myogenic differentiation. C₂C₁₂ cells at 95% confluence were transfected with wild type (wt), S99A mutant flag-Ankrd2, or empty vector and allowed to differentiate in differentiating medium for 1, 2, and 3 d (d1, d2, and d3). Cell lysates were resolved on SDS–PAGE, and the outcome of muscle differentiation was monitored by differentiation and cell cycle specific markers myogenin, cyclin A and D3, and PCNA (Proliferating Cell Nuclear Antigen). The absence of cleaved PARP confirmed cell viability. β-tubulin was shown for equal loading. Akt activity was monitored during differentiation by anti–phospho Akt Ser-473 (anti P-Akt).

FIGURE 7:

Model for inhibition of myogenesis following Ankrd2 phosphorylation by Akt2 induced by stress conditions. During normal physiological conditions, Ankrd2, which is weakly constitutively phosphorylated at Ser-99, might concur to control muscle differentiation rate, ensuring the time needed to allow proper assembly of the whole differentiation machinery. Under stressful conditions that trigger the production of ROS as H₂O₂ Akt2 induces a huge increase in Ankrd2 phosphorylation at Ser-99, inducing its nuclear translocation and resulting in a negative regulation of muscle differentiation pathways.