Development of AKT isoform activity peptide arrays - A wide array of extracellular signals “activate PI3K/AKT” across many cell types by engaging receptor tyrosine kinases, nuclear receptors, or G-protein coupled receptors. Despite converging on this common pathway, these stimuli often elicit distinct biological outcomes, suggesting that nuanced interplays between AKT isoforms cell- type-specific cofactors are critical determinants of downstream signaling. the mechanisms underlying these isoform-specific effects remain poorly understood. Notably, no proteome-wide screens have been conducted to identify direct, isoform-selective AKT substrates, leaving a significant gap in our understanding of AKT signaling diversity. This knowledge gap has hindered the development of reliable cellular readouts for the growing number of pan- and isoform-selective AKT inhibitors (AKTi), several of which are already in clinical use. To date, non- redundant—and in some cases, opposing—functions of AKT isoforms have only been revealed through genetic gain- or loss-of-function studies. For example, AKTi therapies have shown some efficacy, though not always durable, in cancers and in genetic syndromes such as Proteus and CLOVES, which involve PI3K or AKT- activating mutations. In contrast, diseases characterized by the of loss PTEN—a natural suppressor of PI3K/AKT signaling—have shown limited responsiveness, likely due to an incomplete understanding of isoform-specific contributions and a lack of robust cellular assays for isoform activity. Currently, among the ~160 known AKT substrates, only five are linked specifically to AKT2, and none to AKT3; all are defined by the canonical RxRxxS*/T* motif shared by all isoforms. Consequently, there are no validated isoform-specific substrates or phosphorylation motifs that can serve as functional readouts for differential AKT activation or inhibitor specificity in cells. To address this, we propose to develop first-in-kind peptide arrays capable of distinguishing AKT isoform activity using either purified AKT isoform enzymes or lysates from diverse human cell types—including normal, cancerous, and genetically altered cells—characterized by either inducible or constitutive PI3K/AKT signaling. Our first aim (SA1) is to perform isoform-specific in vitro kinase (IVK) assays on a near-proteome-wide array of >21,000 full-length human proteins in a cell type- and cell compartment-blind screen, followed by LC-MS/MS analysis to identify phosphorylation sites and substrate motifs. This approach will enable the discovery of novel isoform-specific substrates and potentially non-canonical motifs. In our second aim (SA2), we will design peptide arrays incorporating both shared and isoform-specific phosphorylation sites. These will be used in IVK assays to validate isoform-specific phosphorylation patterns and to evaluate how different cell types, agonists, and AKTi treatments affect phosphorylation profiles. Our long-term goal is to develop a prototype array that enables sensitive, high-throughput assessment of AKT isoform activation and AKTi specificity in cellular contexts. In the long term, optimized versions of these arrays could become powerful tools for researchers investigating how distinct AKT isoforms drive specific biological outcomes or disease progressions.
