Comparison of Akt1 sequences in different species.

<p>Analysis was performed by means of Basic Local Alignment Search Tool (BLAST) software of Uniprot. A 29-aminoacid fragment around S129 Akt1 of human sequences was used as reference. Invariable residues at positions between n-3 and n+3 are in bold, while variations are underlined.</p

List of the peptides used in this work, with sequence (one-letter code) and nomenclature adopted.

<p>The parental peptides are indicated as wild type (wt). The CK2 target site Akt1 S129 and its homologous residue Akt2 S131 are underlined. Positions not conserved in the two Akt isoforms (human sequence), and substitutions that swap Akt1/Akt2 positions in the variant peptides, are in bold. The same colors denote substitutions that swap Akt1/Akt2 positions. Green indicates other substitutions. Three N-terminal Arginine residues (R3) were introduced for technical reasons (see Methods).</p

Effect of n+1 T on Akt2 secondary structure.

<p>(A) Predicted secondary structure for Akt1 / Akt2 peptide variants: α-helix is indicated by red underlining. The results from different algorithms (see Methods) were evaluated and considered acceptable only when consensus was at least 90%, with the exception of Akt2TS peptide, where (*) indicates that a consensus of 70% was considered. (B) Docking sampling of the most energetically favorable complexes clusterized by an RMSD < 3Å. (C) Protein-protein docking analysis and Molecular Dynamics simulation. Comparison between the Akt1 peptide (Violet) and Akt2 peptide (orange) docked against CK2α (green, PDB code: 3Q04); in the inset, the detailed interaction of Akt1 and CK2α substrate binding site is magnified.</p

Comparison of Akt2 sequences in different species.

<p>Analysis was performed by means of Basic Local Alignment Search Tool (BLAST) software of Uniprot. A 29-aminoacid fragment around S131 Akt2 of human sequences was used as reference. Invariable residues at positions between n-3 and n+3 are in bold, while variations are underlined. The common name of species with T at n+1 position, as in human, are underlined.</p

Phosphorylation of Akt1 and Akt2 peptide variants by CK2.

<p>(A) and (B): Peptides at the indicated concentrations were incubated with CK2 for 10 min, under conditions described in Materials and Methods. Results are reported as % of the cpm value obtained with Akt1 wt peptide (means of 4 experiments ± SEM). (C): CK2 was incubated with 2 mM Akt2 peptide for 20 or 120 min in a radioactive mixture; incubations were also performed in the absence of peptide, to evaluate possible contribution of CK2 autophosphorylation. Samples were hydrolyzed to obtain free phospho-aminoacids, that were separated by high voltage electrophoresis and detected by digital autoradiography. Migrations of standard phospho-aminoacids, as well as the starting point of the electrophoresis, are indicated.</p

Phosphorylation of Akt1 and Akt2 peptides by PLK3.

<p>(A): Akt1 and Akt2 peptides at the indicated concentrations were incubated with PLK3, under conditions described in Materials and Methods. Results are reported as % of the cpm value obtained with Akt1 peptide at 0.1 mM (means of 3 experiments ± SEM). cpm detected with 1mM Akt2 peptide were in the range 50000-150000, according to the specific radioactivity; for comparison, a negative control, corresponding to activity in the absence of any peptide, is also shown in the graph (2800-9000 cpm). (B): wt and mutant Akt1 and Akt2 peptides were used at 0.6 and 1.2 mM as substrate in PLK3 assays. Results are reported as % of the cpm value obtained with each wt peptides at each concentration (means of 3 experiments ± SEM).</p

Summary of the phosphorylation of Akt peptides by CK2.

<p>Peptides are listed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193479#pone.0193479.t003" target="_blank">Table 3</a>, follow by (+) or (-) accordingly to their level of phosphorylation by CK2 (see text for details). The underlined residues are S129 of Akt1 and S131 of Akt2 of human sequence, respectively.</p

Effect of T132G mutation on the phosphorylation of full length Akt2 by CK2.

<p>(A): 10 μg proteins of total lysate from HEK 293T transfected cells were analyzed as indicated. Akt pS129 indicates development with phospho-S129 specific antibody (Akt1 S129A mutant was used as a negative control), Akt noP S129 indicates development with an antibody recognizing the same epitope but independently on phosphorylation, Akt tot indicates development with a generic Akt antibody, recognizing all Akt isoforms. (B): HA-Akt T132G or HA-Akt wt were immunoprecipitated with anti-HA from HEK 293T transfected cells. Immunocomplexes were incubated in a radioactive phosphorylation mixture, in the absence or in the presence of CK2. Samples were analyzed by SDS/PAGE and blotting, followed by digital autoradiography to detect radioactivity, and WB with HA antibody to ensure similar immunoprecipitation levels. (C): 10 μg proteins of total lysate from HEK 293T cells transfected for HA-Akt2 wt or HA-Akt2 T132G were analyzed by WB with Akt pT308 or HA antibody, as indicated.</p

Experimental procedure of irradiation and microgravity simulation.

<p>Experimental procedure of irradiation and microgravity simulation.</p

Significantly anti-correlated genes involved in the DNA-damage response to IR of human PBL.

<p>Significantly anti-correlated genes of DDR pathway in 2Gy PBL incubated for 24 h in 1 g or in MMG. Fold change (F.C.) is the mean of the expression values obtained from the transformed log2 (irradiated/non-irradiated) PBL (see Table S5 and Table S6). Genes in bold belong to p53-pathway; genes in italic belong to the GO category “Response to DNA damage stimulus”.</p