A subcellular atlas of AKT as a predictive tool of its physiological and pathological roles

VILA, ANTONELLA; COLMAN-LERNER, ALEJANDRO; BLAUSTEIN, MATÍAS

San Miguel de Tucumán

Simposio; Simposio Argentino de Jóvenes Investigadores en Bioinformática; 2022

International Society for Computational Biology Student Council (ISCB-SC)

Introduction: Regulation of protein kinase AKT activity is associated with a diversity of processes, including cell metabolism, proliferation, differentiation and survival, as well as pathological processes such as viral infection and cancer development. AKT is a therapeutic target for cancer treatment and it is known to be regulated through numerous posttranslational modifications (PTMs) as well as to be recruited to different subcellular compartments. However, little is known about how a cell determines which substrates and functions AKT should regulate. Our hypothesis is that the profile of AKT PTMs can determine the subcellular localization of AKT, and vice versa, affecting each other and thus establishing the subset of AKT target substrates and the set of functions that AKT displays in response to each stimulus and each particular cellular context. This is of particular relevance when defining which targets to attack with therapeutic drugs to specifically block the pathology and not to inhibit physiological processes whose dysfunction may generate toxicity or harm in patients.Aim: To develop an atlas of AKT subcellular localizations as a predictive tool of AKT physiological and pathological functions using bioinformatic and experimental tools.Materials and methods: Bioinformatic analysis. In this study, a protein-protein interaction network of AKT and AKT substrates was built, using the STRING database (v 11.5). The list of known AKT substrates was obtained using the PhosphoSitePlus database. Spatial analysis of functional enrichment (SAFE) was used to identify and color network regions enriched for similar Gene Ontology (GO) biological process terms, GO cellular component terms, diseases (DisGeNET), and drugs (DGIdb).Fluorescence microscopy analysis. HeLa Kyoto cells were co-transfected with AKT reporters and nuclear speckle markers coupled to fluorescent proteins. An immunofluorescence staining was performed using the phospho-AKT-substrate (RXRXXS*/T*) antibody to evaluate the localization pattern of phosphorylated AKT substrates. Images were acquired with an Olympus FluoView1000 confocal microscope.Results: We generated a protein-protein interaction network of AKT and AKT substrates that contains 651 nodes (the three AKT isoforms -AKT1/2/3-, 325 known AKT substrates and 323 direct interactors), and 15923 edges. Using SAFE, we found that de AKT substrates and interactors located within the domain associated with endoplasmic reticulum and Golgi apparatus of the cellular component map, correspond to the domain associated with proliferation, cell death, autophagy, stress response of the biological process map, and the domain associated with breast cancer of the disease map. This is consistent with recently published work from our laboratory.Interestingly, SAFE analysis revealed a nuclear speckle-enriched domain. However, to date, AKT has not been reported to be recruited to this subcellular compartment. Indeed, fluorescence microscopy experiments showed that both AKT and phospho-AKT-substrates colocalize with nuclear speckles.Conclusions: Using a combination of different bioinformatic tools, we performed an analysis of the AKT interactome, which allows us to explain and even predict the functional and subcellular code of AKT. Our bioinformatics analysis allowed us to find a novel subcellular compartment to which AKT is recruited: nuclear speckles.