| Autor: |
Mohammad DK; Department of Laboratory Medicine, Clinical Research Center, Karolinska Institutet, Karolinska Hospital Huddinge, SE-141 86 Huddinge-Stockholm, Sweden.; Department of Biology, College of Science, University of Salahaddin, 44002 Erbil, Kurdistan Region-Iraq., Ali RH; KTH Royal Institute of Technology, Swedish e-Science Research Center, Science for Life Laboratory, School of Computer Science and Communication, SE-171 77 Solna, Sweden., Turunen JJ; Department of Laboratory Medicine, Clinical Research Center, Karolinska Institutet, Karolinska Hospital Huddinge, SE-141 86 Huddinge-Stockholm, Sweden., Nore BF; Department of Laboratory Medicine, Clinical Research Center, Karolinska Institutet, Karolinska Hospital Huddinge, SE-141 86 Huddinge-Stockholm, Sweden.; Department of Biochemistry, School of Medicine, University of Sulaimani, Sulaimaniyah, Kurdistan Region-Iraq., Smith CI; Department of Laboratory Medicine, Clinical Research Center, Karolinska Institutet, Karolinska Hospital Huddinge, SE-141 86 Huddinge-Stockholm, Sweden. |
| Abstrakt: |
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. |
