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. 2022 Aug 15;11:e80148. doi: 10.7554/eLife.80148

Figure 2. Kinase activities of Akt PH domain basic residue mutations and analysis of R86A PH domain intermolecular binding to the Akt kinase domain.

Figure 2.

(A) Enzymatic activities of full-length Akt mutants possessing pT308 and pS477/pT479 (A1–A4) prepared using a three-piece expressed protein ligation (EPL) strategy. (B) Enzymatic activities of full-length R86A Akt mutant forms with differentially phosphorylated C-tails (A5: pS477/pT479, A7: Non-P, A11: pS473) relative to WT Akt containing pS473 (A10) as a control. These Akt forms were prepared using a two-piece EPL strategy. These kinase assays were performed in buffer containing 250 μM ATP and 20 μM GSK3 peptide as substrates (n≥3, SD shown). (C) MST (microscale thermophoresis) binding experiments using the N-terminally Cy5-labeled kinase domain with pT308 as a target protein and the isolated PH domain (WT or R86A) as a ligand. WT: red, R86A: blue (n=3, SEM shown).

Figure 2—source data 1. Kinase activity assays with full-length Akt mutants having pT308 and pS477/pT479.
Figure 2—source data 2. Kinase activity assays with full-length R86A Akt mutant forms with differentially phosphorylated C-tails.