Figure 1.

A novel route to doubly phosphorylated and active Akt1. A, schematic representation of recombinant Akt1 biosynthesis with pSer-473 genetically encoded in response to the UAG codon and pThr-308 enzymatically phosphorylated in vivo in E. coli. Genetically encoded pSer incorporation requires phosphoseryl-tRNA synthetase (SepRS), a UAG-decoding tRNA^Sep, and the elongation factor mutant (EFSep). B, enzyme activity of differentially phosphorylated Akt1 variants with a GSK-3β substrate peptide. Akt1 quantitatively phosphorylated at both 308 and 473 (ppAkt^S473,T308, blue diamonds) showed maximal activity compared with the unphosphorylated Akt1 (gray circles) and singly phosphorylated Akt1 variants: pAkt^T308 (black cross) and pAkt^S473 (brown diamonds). The reported values represent the mean of triplicate experiments with error bars indicating S.D. Lower-activity variants show above-background kinase activity (inset).