B Cell Receptor Activation Predominantly Regulates AKT-mTORC1/2 Substrates Functionally Related to RNA Processing

<div><p>Protein kinase B (AKT) phosphorylates numerous substrates on the consensus motif RXRXXpS/T, a docking site for 14-3-3 interactions. To identify novel AKT-induced phosphorylation events following B cell receptor (BCR) activation, we performed proteomics, biochemical and bioinformatics analyses. Phosphorylated consensus motif-specific antibody enrichment, followed by tandem mass spectrometry, identified 446 proteins, containing 186 novel phosphorylation events. Moreover, we found 85 proteins with up regulated phosphorylation, while in 277 it was down regulated following stimulation. Up regulation was mainly in proteins involved in ribosomal and translational regulation, DNA binding and transcription regulation. Conversely, down regulation was preferentially in RNA binding, mRNA splicing and mRNP export proteins. Immunoblotting of two identified RNA regulatory proteins, RBM25 and MEF-2D, confirmed the proteomics data. Consistent with these findings, the AKT-inhibitor (MK-2206) dramatically reduced, while the mTORC-inhibitor PP242 totally blocked phosphorylation on the RXRXXpS/T motif. This demonstrates that this motif, previously suggested as an AKT target sequence, also is a substrate for mTORC1/2. Proteins with PDZ, PH and/or SH3 domains contained the consensus motif, whereas in those with an HMG-box, H15 domains and/or NF-X1-zinc-fingers, the motif was absent. Proteins carrying the consensus motif were found in all eukaryotic clades indicating that they regulate a phylogenetically conserved set of proteins.</p></div

Biological, physical and functional interaction predictions of the AKT-mTORC1/2 substrate (up-regulated) proteins.

<p>An interaction map was predicted to observe putative AKT-mTORC1/2 substrate interactions by using a web-based interface, String 9 tool for up-regulated proteins. Dense clusters representing high interaction between proteins can be seen for up-regulated proteins.</p

The Effect of MK-2206 and PP242 on RXRXXS/T phosphorylations.

<p>(A). Time course of the BCR-induced AKT activation and its effect on substrates in the presence or absence of MK-2206 (2 μM) for 3 h. Namalwa cells were starved and then stimulated with anti-IgM and followed for 1 h (1–60 min). Lysates were resolved on SDS-PAGE and immunoblotted with different phospho-specific antibodies for AKT and AKT-substrates. (B). Phosphorylation status 3 h after treatment of Namalwa cells with either DMSO or different pharmacological inhibitors, MK-2206, (2 μM), Rapamycin (150 nM), PP242 (1 μM), LY294002 (20 μM) or PD98059 (2 μM). The cells were serum-starved then stimulated with anti-IgM (20 μg/ml). Lysates were resolved on 4–12% SDS-PAGE and immunoblotted with different phospho-specific antibodies for pS473-AKT, RXRXXpS/T and pS660-PKC. Anti-pErk 44/42 antibody was used as control.</p

Homology inference and count of 186 AKT-mTORC1/2 substrates in different eukaryotic clades.

<p>The figure displays the number of homologs of 186 AKT-mTORC1/2 substrates present in different eukaryotic species representing Fungi, Protists, Viridiplantae, Invertebrates and Vertebrates. The proteins with homologs in all clades are also displayed in the figure. The species tree and the two circles are colored according to their clade (color mentioned on top left as figure legend). The polar dendrogram showing the species tree drawn using Archaeopteryx. The count between the inner and outer circle represents the number of 186 AKT-mTORC1/2 substrates with homologous proteins in that species. The HGNC names of proteins (33 proteins) with homologs in all major clades and in more than 65 out of 68 species are written outside the outer circle. It can be inferred from presence of homologs of these proteins in all clades that these proteins were present in the last common ancestor of eukaryotes and that these proteins are evolutionarily and functionally important for eukaryotes.</p

Biological, physical and functional interaction predictions of the AKT-mTORC1/2 substrate (down-regulated) proteins.

<p>An interaction map was predicted to observe putative AKT-mTORC1/2 substrate interactions by using a web-based interface, String 9 tool for down-regulated proteins. Dense clusters representing high interaction between proteins can be seen for down-regulated proteins. The down-regulated proteins have more dense clusters and a larger connected graph in comparison with up-regulated proteins in (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160255#pone.0160255.g007" target="_blank">Fig 7</a>).</p

Phosphorylation of RXRXXS/T motif in MEF-2D and RBM25 proteins.

<p>(A). The immunoprecipitates of proteins carrying the putative AKT motif using anti-RXRXXpS/T antibody in Namalwa cells before and after anti-IgM (20 μg/ml) stimulation. In both experiments of the MS/MS, samples were resolved on 4–12% SDS-PAGE and then immunoblotted with the corresponding antibodies. EXP1 and EXP2 represent two independent experiments conducted prior to the proteomics analysis. (B). Total MEF-2D was immunoprecipitated in Namalwa cells. Anti-RXRXXpS/T antibody was used to identify MEF-2D phosphorylation. The pulled-down protein was identified using anti-MEF-2D antibody. (C). Endogenous RBM25 protein was immunoprecipitated in Namalwa cells and probed for the presence of the consensus site phosphorylation by immunoblotting with anti-RXRXXpS/T antibody.</p

Domain mapping of the MS/MS identified proteins.

<p>Table displays the domain architecture of the 446 proteins in the MS data. The domain architecture is shown as total domains in the 446 proteins and also divided into two parts; AKT motif containing proteins and non-motif containing proteins to identify the domain contents of each part. The table displays the highest number of domains in total, in AKT motif-containing proteins and in non-motif containing proteins.</p

P-element insertion point detection:

<p>Schematic figure showing P-element transposon P(BmΔ-w) insertion site. Sequencing results shows P-element insertion site (arrow) within the genomic DNA of <i>Btk29A</i> at 2L: 8277721. GTCGAGCGC repeats can be seen at both end of the P insertion, which is a characteristic mark of transposon insertion. This information was further use to distinguish between mutant and revertant flies. In mutant flies this breakpoint was easily detected in amplicons (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035640#s3" target="_blank">Materials and Methods</a>), whereas the revertant did not show any P insertion.</p

Comparison between Mouse and Drosophila Btk-dependent transcript:

<p><b>A)</b><b><i>Venn-diagram:</i></b> showing the overlap between Btk-dependent Transitional type 1 B-cells from Btk-defective mice <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035640#pone.0035640-Lindvall1" target="_blank">[40]</a> (a total of 147 differentially expressed genes) and the Btk-dependent transcripts found in <i>Btk29A</i>^ficP<i>Drosophila</i> adult head (a total of 744 differentially expressed genes). <b>B) </b><b><i>Thirteen orthologous transcripts found in Btk KO mouse Transitional type 1 B-lymphocytes and in Btk29A defective flies.</i></b> Bar-graph showing the 16 transcripts, corresponding to 13 genes, found to be common in a homology search between the Btk-dependent transcripts found in the <i>Btk KO</i> mouse Transitional type 1 B-cells compared to its healthy control strain <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035640#pone.0035640-Lindvall1" target="_blank">[40]</a> and the current <i>Drosophila Btk29A</i>^ficP study. Out of these 13 genes, 5 genes showed the same regulatory direction i.e. being up- or down-regulated in the respective Btk-defective strain (denoted as *).</p

Inhibition of cell proliferation and reduction of Bcl-x_L by PP242.

<p>(A). Namalwa and A20 cells were treated with DMSO or PP242 (1 μM) and followed for 72h. A cell proliferation test was performed and the absolute number of viable cells counted. The data are presented as mean ± SEM. P < 0.01 versus vehicle using Duncan test. According to Duncan test, different letters mean that there is a significant difference between groups (B). Namalwa cells were treated with DMSO or different inhibitors; MK-2206, (2 μM), Rapamycin (150 nM), PP242 (1 μM) or LY294002 (20 μM) for 3 h. Samples were resolved on 4–12% SDS-PAGE and probed with anti-Bcl-x_L or pS473-AKT antibodies. (C). Cos-7 cells were pretreated with PP242 (1 μM), Rapamycin (150 nM) or Staurosporine (1 μM) for 3 h. western blot analysis of endogenous Bcl-x_L by staining the blot with anti-Bcl-x_L antibody. Staurosporine was used in this experiment as positive control.</p