AKT1 and AKT2 Induce Distinct Phosphorylation Patterns in HL-1 Cardiac Myocytes

被引:20
作者
Reinartz, Michael [1 ]
Raupach, Annika [1 ]
Kaisers, Wolfgang [2 ]
Goedecke, Axel [1 ]
机构
[1] Univ Dusseldorf, Dept Cardiovasc Physiol, D-40225 Dusseldorf, Germany
[2] Univ Dusseldorf, CBiBs, Biol & Med Res Ctr BMFZ, D-40225 Dusseldorf, Germany
关键词
AKT; phosphoproteomics; cardiomyocytes; insulin signaling; mass spectrometry; excitation-contraction coupling; BINDING-PROTEIN-C; PHOSPHOLAMBAN PHOSPHORYLATION; KINASE; IDENTIFICATION; PATHWAY; GROWTH; SITES; MICE; PHOSPHOPROTEOME; JUNCTOPHILIN-2;
D O I
10.1021/pr500131g
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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 Ca(2+)/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.
引用
收藏
页码:4232 / 4245
页数:14
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