Phosphoproteomic comparison of Pik3ca and Pten signalling identifies the nucleotidase NT5C as a novel AKT substrate

Abstract

To identify novel effectors and processes regulated by PI3K pathway activation, we performed an unbiased phosphoproteomic screen comparing two common events of PI3K deregulation in cancer: oncogenic Pik3ca mutation (Pik3caH1047R) and deletion of Pten. Using mouse embryonic fibroblast (MEF) models that generate inducible, low-level pathway activation as observed in cancer, we quantified 7566 unique phosphopeptides from 3279 proteins. A number of proteins were found to be differentially-regulated by Pik3caH1047R and Pten loss, suggesting unique roles for these two events in processes such as vesicular trafficking, DNA damage repair and RNA splicing. We also identified novel PI3K effectors that were commonly-regulated, including putative AKT substrates. Validation of one of these hits, confirmed NT5C (5′,3′-Nucleotidase, Cytosolic) as a novel AKT substrate, with an unexpected role in actin cytoskeleton regulation via an interaction with the ARP2/3 complex. This study has produced a comprehensive data resource and identified a new link between PI3K pathway activation and actin regulation.

NODAL/TGFβ signalling mediates the self-sustained stemness induced by PIK3CAH1047R homozygosity in pluripotent stem cells

“…High-depth transcriptomics confirmed that heterozygosity for PIK3CA H1047R fails to induce widespread substantial transcriptional remodeling, whether chronically modelled in CRISPR-edited iPSCs or acutely induced in mouse embryonic fibroblasts (MEFs) by Cre expression, despite induction of canonical PI3K pathway activation in both cases (current study and Ref. ( 5, 28, 48 )). Similarly, iPSCs with heterozygous expression of PIK3CA E418K , a “non-hotspot” mutation, were transcriptionally indistinguishable from their isogenic wild-type controls.…”

“…1D ), with only 30 differentially-expressed genes (table S3 ). We also studied previously reported Pik3ca WT/H1047R mouse embryonic fibroblasts (MEFs) 48 h after Cre -mediated Pik3ca H1047R induction ( 28 ). Wild-type and Pik3ca WT/H1047R MEFs were superimposable on an MDS plot ( Fig.…”

“…The derivation and culture of the wild-type and PIK3CA WT/H1047R MEFs used in this study have been reported previously ( 28 ). Cell pellets were collected on dry ice 48 h after induction of heterozygous PIK3CA H1047R expression, without prior starvation.…”

AKT/PKB Signaling: Navigating the Network

“…For example, in prostate cancer cells with PTEN inactivation, AKT2 is exclusively required for cell-autonomous tumor maintenance (Chin et al, 2014), whereas in Pten +/− mice, the AKT1 isoform appears to suppresses prostate tumor development (Chen et al, 2006), while ablation of AKT2 has little impact (Xu et al, 2012). Global phospho-proteomic screening approaches have identified hundreds of novel phosphorylation sites that conform to the AKT consensus motif and have also provided new insights into potential AKT isoform-specific functions and substrates, many that await further validation (Lee et al, 2014; Moniz et al, 2017; Sanidas et al, 2014). Mechanisms that likely contribute to Akt isoform-specificity for downstream substrates include molecular features of individual isoforms, discrete subcellular localization, and relative expression levels in a given setting.…”

The PI3K Pathway in Human Disease

“…Thus, cytoskeletal remodeling driven by the PI3K-Rac axis drives not only cell motility but also metabolic reprogramming downstream of growth factor receptors. The role of PI3K signaling in effecting cytoskeletal remodeling is further highlighted by the recent identification of NT5C as a novel AKT substrate with a role in cytoskeletal remodeling that is mediated through interaction with ARP2/3 (Moniz et al, 2017). …”