Substrate selectivity of semisynthetic Akt with pSer473 or O-GlcNAcSer473

Akt is a Ser/Thr protein kinase that regulates cell growth, metabolism and is considered a therapeutic target for cancer. Regulation of Akt by membrane recruitment and post-translational modifications (PTMs) has been extensively studied. The most well-established mechanism for cellular Akt activation involves phosphorylation on its activation loop on Thr308 by PDK1 and on its C-terminal tail on Ser473 by mTORC2. Other C-terminal tail PTMs have been identified, but their functional impacts have not been well-characterized. We use expressed protein ligation (EPL) as a tool to produce semisynthetic Akt proteins to dissect the enzymatic functions of these PTMs. We performed kinase assays employing human protein microarrays to investigate global substrate specificity of Akt, comparing phosphorylated vs. O-GlcNAcylated Ser473 forms. In this experiments, kinase assays were conducted for each semisynthetic Akt protein containing different modifications at the C-terminal tail: pSer473 and O-GlcNAcSer473. Controls were performed using all reaction components but leaving out Akt to leave out proteins capable of autophosphorylation. All experiments, including the control, were performed twice.