title: AKT1 and AKT2 induce distinct phosphorylation patterns in HL-1 cardiac myocytes.
abstract: The protein kinase AKT is a central kinase in the heart and has a major impact on growth/hypertrophy, survival/apoptosis, and metabolism. To gain more insight into AKT isoform-specific signaling at the molecular level, we investigated the phosphoproteome of HL-1 cardiomyocytes carrying AKT1 or AKT2 isoform-specific knock down, respectively. We combined stable isotope labeling with high resolution mass spectrometry and identified 377 regulated phosphopeptides. Although AKT1 is expressed at 4-fold higher levels, insulin stimulation mainly activated AKT2, which might in part rely on a preferred interaction of AKT2 with the mammalian target of rapamycin complex 2. In line with this result, the highest number of regulated phosphopeptides was identified in the AKT2 knock down cells. Isoform-specific regulation of AKT targets not previously described could be observed, and specific regulation of indirect target sites allows a deeper insight into affected biological processes. In the myocardial context, we identified many phosphosites supporting a connection of AKT to excitation-contraction coupling. Phosphoproteins identified included L-type calcium channel, ryanodine receptor, junctophilin, histidine-rich calcium binding protein, phospholamban, heat shock protein beta-6, and Ca²⁺/calmodulin-dependent kinase II. In conclusion, AKT isoform-specific knock down combined with quantitative phosphoproteomics provided a powerful strategy to unravel AKT isoform-specific signaling.
authors.affiliation: Department of Cardiovascular Physiology, and ‡Biological and Medical Research Center (BMFZ, CBiBs), Heinrich-Heine-University Düsseldorf , Universitätsstraße 1, Düsseldorf D-40225, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Reinartz
authors.forename: Annika
authors.initials: A
authors.lastname: Raupach
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Kaisers
authors.forename: Axel
authors.initials: A
authors.lastname: Gödecke
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D055785:Gene Knockdown Techniques; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D008969:Molecular Sequence Data; D032383:Myocytes, Cardiac / Q000378:metabolism*; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D007328:Insulin; D010750:Phosphoproteins; D007334:Insulin-Like Growth Factor I; D051057:Proto-Oncogene Proteins c-akt
keywords: AKT; cardiomyocytes; excitation−contraction coupling; insulin signaling; mass spectrometry; phosphoproteomics