The AKT/EF2 connection.

<p>AKT is indirectly involved in EF2-activation via mTOR, AMPK and EF2K. Our data reveal a direct interaction of AKT2 and EF2, which is dynamically regulated upon angiotensin II, IGF-1 and insulin stimulation.</p

Systematic Analysis Reveals Elongation Factor 2 and α-Enolase as Novel Interaction Partners of AKT2

<div><p>AKT2 is one of the three isoforms of the protein kinase AKT being involved in the modulation of cellular metabolism. Since protein-protein interactions are one possibility to convey specificity in signal transduction, we performed AKT2-protein interaction analysis to elucidate their relevance for AKT2-dependent cellular functions. We identified heat shock protein 90 kDa (HSP90), Cdc37, heat shock protein 70 kDa (HSP70), 78 kDa glucose regulated protein (GRP78), tubulin, GAPDH, α-enolase and elongation factor 2 (EF2) as AKT2-interacting proteins by a combination of tandem affinity purification and mass spectrometry in HEK293T cells. Quantitative MS-analysis using stable isotope labeling by amino acids in cell culture (SILAC) revealed that only HSP90 and Cdc37 interact stably with AKT2, whereas the other proteins interact with low affinity with AKT2. The interactions of AKT2 with α-enolase and EF2 were further analyzed in order to uncover the functional relevance of these newly discovered binding partners. Despite the interaction of AKT2 and α-enolase, which was additionally validated by proximity ligation assay (PLA), no significant impact of AKT on α-enolase activity was detected in activity measurements. AKT stimulation via insulin and/or inhibition with the ATP-competitive inhibitor CCT128930 did not alter enzymatic activity of α-enolase. Interestingly, the direct interaction of AKT2 and EF2 was found to be dynamically regulated in embryonic rat cardiomyocytes. Treatment with the PI3-kinase inhibitor LY294002 before stimulation with several hormones stabilized the complex, whereas stimulation alone led to complex dissociation which was analyzed <i>in situ</i> with PLA. Taken together, these findings point to new aspects of AKT2-mediated signal transduction in protein synthesis and glucose metabolism.</p></div

Dynamic regulation of the AKT2/EF2-interaction in embryonic rat cardiomyocytes.

<p>A/D/G: PLA after inhibition of PI3-Kinase with LY294002 (1 h, 50 µM) and subsequent angiotensin II (A), IGF-1(D) and insulin (G) stimulation B/E/H: PLA after stimulation of the cells with angiotensin II (30 minutes, 100 nM) (B), IGF-1(10 minutes, 1 µg/ml) (E) and insulin (10 minutes, 1,75 µg/ml) (H). C/F/I: Quantitative data of two independent PLA-experiments for each stimulant. The number of signals/nuclei for the LY294002+ stimulant sample was set as 100%.</p

Overview of proteins most frequently detected in six (I-VI) TAPs with AKT2-NTag.

<p>Listed are those proteins, which were identified in three to six experiments.</p><p>x: Protein was not detected.</p><p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066045#pone.0066045.s005" target="_blank">Table S1</a> shows a detailed table with all identified proteins of these six TAPs.</p

α-enolase activity assay.

<p>A: Activity of immuno-captured α-enolase (ENO1) from HEK293T cells after stimulation with insulin and AKT-inhibition with CCT128930. Data represent means ± SD of n = 7 experiments. B: Detection of immuno-captured α-enolase after α-enolase activity assay. After detection of α-enolase blots were incubated with anti-pan AKT antibody. Note that AKT was also detected after isolation of α-enolase by the α-enolase antibody.</p

Results of PLA of AKT and EF2.

<p>A–D: PLA on HEK293T AKT2-NTag cells A/B: Control experiments with one primary antibody. C: Complete PLA. D: Quantitative analysis of two independent experiments. E–L: Results of PLA with AKT2 and AKT1 with EF2 in embryonic rat cardiomyocytes. E–H: PLA AKT2/EF2 E/F: Control experiments with one primary antibody. G: Complete PLA. H: Quantitative analysis. I–L: PLA AKT1/EF2 I/J: Control experiments with one primary antibody. K: Complete PLA. L: Quantitative analysis. The number of signals/nuclei in the PLA-samples was set as 100%. Controls are shown as percentage of corresponding full PLA.</p

Detail of representative MS-spectra for peptide ratios of a stably and a transiently bound protein after MBP and MAP.

<p>A: Light and heavy form of the peptide DAGTIAGLNVMR of HSP90α with a shifted ratio for both approaches. B: Light and heavy form of the peptide SDIDEIVLVGGSTR of GRP78 with a ∼1∶1 ratio after MBP and a shifted ratio after MAP.</p

Characterization of tagged AKT2.

<p>A: Expression of C- and N-terminally tagged AKT in comparison to endogenous AKT. Tagged AKT was stably expressed in HEK293T cells and detected by pan-AKT and anti-HA-tag antibodies. *degradation product of C-terminally tagged AKT at 50 kDa, detected by pan-AKT-antibody as well as HA-Tag antibody. B: Regulation of serine 473 phosphorylation in AKT2-NTag. Stably AKT2-NTag expressing cells and WT cells were serum deprived overnight, treated with LY294002 (1 h, 25 µM) or stimulated with IGF-1 (10 minutes, 1 µg/ml). C: Regulation of AKT substrate GSK3β phosphorylation at serine 9. D: Silver stained SDS-gel of proteins isolated from AKT2-NTag and wild type cells (WT) by TAP.</p

Circulating NOS3 Modulates Left Ventricular Remodeling following Reperfused Myocardial Infarction

<div><p>Purpose</p><p>Nitric oxide (NO) is constitutively produced and released from the endothelium and several blood cell types by the isoform 3 of the NO synthase (NOS3). We have shown that NO protects against myocardial ischemia/reperfusion (I/R) injury and that depletion of circulating NOS3 increases within 24h of ischemia/reperfusion the size of myocardial infarction (MI) in chimeric mice devoid of circulating NOS3. In the current study we hypothesized that circulating NOS3 also affects remodeling of the left ventricle following reperfused MI.</p><p>Methods</p><p>To analyze the role of circulating NOS3 we transplanted bone marrow of NOS3^−/− and wild type (WT) mice into WT mice, producing chimerae expressing NOS3 only in vascular endothelium (BC−/EC+) or in both, blood cells and vascular endothelium (BC+/EC+). Both groups underwent 60 min of coronary occlusion in a closed-chest model of reperfused MI. During the 3 weeks post MI, structural and functional LV remodeling was serially assessed (24h, 4d, 1w, 2w and 3w) by echocardiography. At 72 hours post MI, gene expression of several extracellular matrix (ECM) modifying molecules was determined by quantitative RT-PCR analysis. At 3 weeks post MI, hemodynamics were obtained by pressure catheter, scar size and collagen content were quantified post mortem by Gomori’s One-step trichrome staining.</p><p>Results</p><p>Three weeks post MI, LV end-systolic (53.2±5.9μl;***p≤0.001;n = 5) and end-diastolic volumes (82.7±5.6μl;*p<0.05;n = 5) were significantly increased in BC−/EC+, along with decreased LV developed pressure (67.5±1.8mmHg;n = 18;***p≤0.001) and increased scar size/left ventricle (19.5±1.5%;n = 13;**p≤0.01) compared to BC+/EC+ (ESV:35.6±2.2μl; EDV:69.1±2.6μl n = 8; LVDP:83.2±3.2mmHg;n = 24;scar size/LV13.8±0.7%;n = 16). Myocardial scar of BC−/EC+ was characterized by increased total collagen content (20.2±0.8%;n = 13;***p≤0.001) compared to BC+/EC+ (15.9±0.5;n = 16), and increased collagen type I and III subtypes.</p><p>Conclusion</p><p>Circulating NOS3 ameliorates maladaptive left ventricular remodeling following reperfused myocardial infarction.</p></div

Flow chart of the presented study.

<p>In a closed chest model, animals were subjected to reperfused myocardial infarction. After 60 min of ischemia, animals were divided into two different groups: 1) 72 h post MI 2) 3 weeks post MI. Further analysis followed as depicted.</p