Genetic code expansion and live cell imaging reveal that Thr-308 phosphorylation is irreplaceable and sufficient for Akt1 activity

Abstract

The proto-oncogene Akt/protein kinase B (PKB) is a pivotal signal transducer for growth and survival. Growth factor stimulation leads to Akt phosphorylation at two regulatory sites (Thr-308 and Ser-473), acutely activating Akt signaling. Delineating the exact role of each regulatory site is, however, technically challenging and has remained elusive. Here, we used genetic code expansion to produce site-specifically phosphorylated Akt1 to dissect the contribution of each regulatory site to Akt1 activity. We achieved recombinant production of full-length Akt1 containing site-specific pThr and pSer residues for the first time. Our analysis of Akt1 site-specifically phosphorylated at either or both sites revealed that phosphorylation at both sites increases the apparent catalytic rate 1500-fold relative to unphosphorylated Akt1, an increase attributable primarily to phosphorylation at Thr-308. Live imaging of COS-7 cells confirmed that phosphorylation of Thr-308, but not Ser-473, is required for cellular activation of Akt. We found and in the cell that pThr-308 function cannot be mimicked with acidic residues, nor could unphosphorylated Thr-308 be mimicked by an Ala mutation. An Akt1 variant with pSer-308 achieved only partial enzymatic and cellular signaling activity, revealing a critical interaction between the γ-methyl group of pThr-308 and Cys-310 in the Akt1 active site. Thus, pThr-308 is necessary and sufficient to stimulate Akt signaling in cells, and the common use of phosphomimetics is not appropriate for studying the biology of Akt signaling. Our data also indicate that pThr-308 should be regarded as the primary diagnostic marker of Akt activity.

Phosphorylation-Dependent Inhibition of Akt1

“…We also observed in COS-7 cells that pSer473 is dispensable for Akt1 signaling and phosphorylation of Thr308 alone was sufficient for maximal Akt1 signal prorogation. These results indicated Thr308 phosphorylation status is a superior biomarker for Akt1 activity [11] and called into question the frequent use of Ser473 phosphorylation as a diagnostic marker for Akt1 activity in cancer patients [12][13][14][15].…”

“…Leveraging our ability to produce Akt1 protein in specifically phosphorylated forms (Figure 2), we recently quantified the precise contribution of pThr308 and pSer473 to Akt1 activity in vitro and in mammalian cells [11]. In studies with purified full length Akt1, we found that pAkt1 T308 achieves 30% of the activity of the doubly phosphorylated kinase.…”

“…Full length pAkt1 variants were produced from pCDF-Duet1 plasmids as described previously [11]. The genetic code expansion system for phosphoserine (pSer) is encoded on the pDS-pSer2 plasmid [11,17,18], which contains 5 copies of tRNA Sep [19], phosphoseryl-tRNA synthetase (SepRS9), and elongation factor Tu mutant (EFSep21) [20]. Incorporation of pSer also required site-directed mutagenesis of the Ser473 codon to TAG in the ΔPH-akt1 constructs.…”

“…The PDPK1 gene was purchased from Harvard PlasmidID repository service (plasmid ID: HsCD00001584, Boston, MA, USA) and sub-cloned (KpnI/NdeI) into the second multi-cloning site (MCS) of pCDF-Duet-1. Full length pAkt1 variants were produced from pCDF-Duet1 plasmids as described previously [11]. The genetic code expansion system for phosphoserine (pSer) is encoded on the pDS-pSer2 plasmid [11,17,18], which contains 5 copies of tRNA Sep [19], phosphoseryl-tRNA synthetase (SepRS9), and elongation factor Tu mutant (EFSep21) [20].…”

“…The pDS-pSer2 plasmid [18] was used as before [11] to incorporate pSer in response to a UAG codon at position 473 in ΔPHAkt1 and full length Akt1 variants. To produce both full length and PH domain deficient Akt1 variants containing pSer473 (Table S1), the pCDFDuet-1 Akt1 bearing plasmid was co-transformed with pDS-pSer2 into E. coli Bl21(DE3) and plated on LB agar plates with 25 μg/ml kanamycin and 50 μg/ml streptomycin.…”