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An Optogenetic-Phosphoproteomic Approach Links Akt Activation to Dynamic Temporal Changes in Signaling in Endothelial Cells

ABSTRACT: The serine/threonine kinase Akt is a central node in cell signaling. While aberrant Akt activation underlies the development of a variety of human diseases, how different patterns of Akt-dependent phosphorylation dictates downstream signaling and phenotypic outcomes remains largely enigmatic. Herein, we performed a systems-level analysis that integrates method advances of optogenetics, mass spectrometry-based phosphoproteomics, and bioinformatics to elucidate how different Akt intensity, durability, and pattern of stimulation activated temporal phosphorylation profiles in vascular endothelial cells. Through the analysis of ~35,000 phosphorylation sites across multiple conditions precisely controlled by light stimulation, we identified a series of signaling circuits that define the sequential downstream cascade of Akt activation and interrogated how Akt signaling cross-talks with growth factor signaling in endothelial cells. Furthermore, our results categorized substrates of kinases that are preferably activated by oscillating, transient, and sustained Akt signals. We validated a list of Akt motif-carrying phosphosites that covaried with Akt phosphorylation across experimental conditions as potential Akt substrates and presented the entire dataset a rich resource for future studies on Akt signaling and dynamics.

INSTRUMENT(S): Orbitrap Eclipse, Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Wenxue Li

LAB HEAD: Yansheng Liu

PROVIDER: PXD034957 | Pride | 2023-06-27

REPOSITORIES: Pride

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Project description:Most adult patients have a D816V mutation in phosphotransferase domain (PTD), we have described that half of the children carry mutations in extracellular domain (ECD). KIT-ECD versus IT-PTD-mutants were introduced into rodent Ba/F3, EML, Rat2 and human TF1 cells to investigate their biological effect. ECD- and PTD-mutants also displayed distinct whole-genome transcriptional profiles in EML cells. We observed differences in their signaling properties: they both activated STAT pathways, whereas AKT pathway was only activated by ECD-mutants. Consistently, AKT inhibitor suppressed ECD-mutant-dependent proliferation, clonogenicity and erythroid differentiation. Expression of myristoylated AKT restored erythroid differentiation in EMLPTD cells, suggesting the differential role of AKT in those mutants. Overall, our study implied different pathogenesis of pediatric versus adult mastocytosis, which might explain their diverse phenotypes. Each EML cell line (Del417-419insY mutant and D816V mutant) was cultured with or without 250 ng/mL SCF for 48h. Gene expression profile analysis was performed using whole-genome microarrays. RNA expression profiling of cell lines was done with Affymetrix M430 2.0 mouse oligonucleotide microarrays containing 45.101 probe sets, representing 21.408 transcripts and variants including 17.482 well-characterized mouse genes. Preparation of cRNA, hybridizations, washes, detection and quantification were done as recommended by the supplier. For each sample, synthesis of the first-strand cDNA was done from 3 μg total RNA by T7-oligo(dT) priming, followed by second-strand cDNA synthesis. After purification, in vitro transcription associated with amplification generated cRNA-containing biotinylated pseudouridine. Biotinylated cRNA was purified, quantified and chemically fragmented (95°C for 35 min), then hybridized to microarrays in 200 μL hybridization buffer at 45°C for 16 h. Automated washes and staining with streptavidin-phycoerythrin were done as recommended. Signal amplification was done by biotinylated antistreptavidin antibody with goat-IgG blocking antibody. Scanning was done with Affymetrix GeneArray scanner and quantification with Affymetrix GCOS software.

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An Optogenetic-Phosphoproteomic Approach Links Akt Activation to Dynamic Temporal Changes in Signaling in Endothelial Cells

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