Abstract
Protein kinase B (AKT1) is hyper-activated in diverse human tumors. AKT1 is activated by phosphorylation at two key regulatory sites, Thr308 and Ser473. Active AKT1 phosphorylates many, perhaps hundreds, of downstream cellular targets in the cytosol and nucleus. AKT1 is well-known for phosphorylating proteins that regulate cell survival and apoptosis, however, the full catalog of AKT1 substrates remains unknown. Using peptide arrays, we recently discovered that each phosphorylated form of AKT1 (pAKT1S473, pAKT1T308, and ppAKT1S473,T308) has a distinct substrate specificity, and these data were used to predict potential new AKT1 substrates. To test the high-confidence predictions, we synthesized target peptides representing putative AKT1 substrates. Peptides substrates were synthesized by solid phase synthesis and their purity was confirmed by mass spectrometry. Most of the predicted peptides showed phosphate accepting activity similar to or greater than that observed with a peptide derived from a well-established AKT1 substrate, glycogen synthase kinase 3β (GSK-3β). Among the novel substrates, AKT1 was most active with peptides representing PIP3-binding protein Rab11 family-interacting protein 2 and cysteinyl leukotriene receptor 1, indicating their potential role in AKT1-dependent cellular signaling. The ppAKT1S473,T308 enzyme was highly selective for peptides containing a patch of basic residues at −5, −4, −3 and aromatic residues (Phe/Tyr) at +1 positions from the phosphorylation site. The pAKT1S473 variant preferred more acidic peptides, Ser or Pro at +4, and was agnostic to the residue at −5. The data further support our hypothesis that Ser473 phosphorylation plays a key role in modulating AKT1 substrate selectivity.
Highlights
Protein kinase B (AKT) belongs to the AGC family of serine-threonine kinases (Manning and Toker, 2017)
The AKT isozymes share the same fundamental structure consisting of four distinct domains: an N-terminal regulatory pleckstin homology (PH) domain, an unstructured linker region connecting the PH domain to the serine/threonine-specific kinase domain; and a final C-terminal domain often referred to as the hydrophobic motif, which is responsible for modulating the activity of AKT1 (Franke, 2008; Balasuriya et al, 2020)
Prior to the development of the genetic code expansion system used here, initial attempts at producing recombinant active AKT1 employed the use of phospho-mimetic mutations (Klein et al, 2005)
Summary
Protein kinase B (AKT) belongs to the AGC family of serine-threonine kinases (Manning and Toker, 2017). The AKT isozymes share the same fundamental structure consisting of four distinct domains: an N-terminal regulatory pleckstin homology (PH) domain, an unstructured linker region connecting the PH domain to the serine/threonine-specific kinase domain; and a final C-terminal domain often referred to as the hydrophobic motif, which is responsible for modulating the activity of AKT1 (Franke, 2008; Balasuriya et al, 2020). AKT1 is hyper-phosphorylated and overactive in >50% of human tumors (Agarwal et al, 2013; Spencer et al, 2014; Manning and Toker, 2017). Elevated AKT1 phosphorylation status is linked to poor clinical prognosis (Dai et al, 2005; Suzuki et al, 2010; Antonelli et al, 2012)
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