Akt Kinase Activation Mechanisms Revealed Using Protein Semisynthesis

Abstract

Akt is a critical protein kinase that drives cancer proliferation, modulates metabolism, and is activated by C-terminal phosphorylation. The current structural model for Akt activation by C-terminal phosphorylation has centered on intramolecular interactions between the C-terminal tail and the N lobe of the kinase domain. Here, we employ expressed protein ligation to produce site-specifically phosphorylated forms of purified Akt1 that are well suited for mechanistic analysis. Using biochemical, crystallographic, and cellular approaches, we determine that pSer473-Akt activation is driven by an intramolecular interaction between the C-tail and the pleckstrin homology (PH)-kinase domain linker that relieves PH domain-mediated Akt1 autoinhibition. Moreover, dual phosphorylation at Ser477/Thr479 activates Akt1 through a different allosteric mechanism via an apparent activation loop interaction that reduces autoinhibition by the PH domain and weakens PIP3 affinity. These results provide a new framework for understanding how Akt is controlled in cell signaling and suggest distinct functions for differentially modified Akt forms.

Keywords: X-ray crystal structure; bisubstrate analog; expressed protein ligation; kinase; mass spectrometry; peptide; phosphorylation; photocrosslinking.

Figure 1.. Semisynthesis of site-specifically phosphorylated Akt1 constructs.

A) Semisynthesis strategy for C-terminally phosphorylated Akt1 proteins with highlighted phosphorylations (red balls) that are studied in this work. C-terminally truncated recombinant Akt1 containing PDK1-catalyzed pThr³⁰⁸ and a C-terminal intein generated thioester is ligated to N-Cys synthetic C-tail peptides without or with phosphorylations at residues Ser473, Ser477 and Thr479. B) Representative C-terminal tail synthetic peptide sequences containing distinct phosphorylation states. C) Coomassie stained SDSPAGE of selected purified pThr³⁰⁸ containing Akt1 semisynthetic proteins: M, molecular weight standards, lane 1, unligated C-terminally truncated Akt1 MESNA thioester, lane 2, full-length FL-Akt1-pThr³⁰⁸, lane 3, FL-Akt1-pThr³⁰⁸/pSer⁴⁷³, lane 4, FL-Akt1-pThr³⁰⁸/3p-Ser⁴⁷³,Ser⁴⁷⁷,Thr⁴⁷⁹. D) Western blot analysis of selected purified semisynthetic Akt1 forms: M, MW markers, lane 1, non-pThr³⁰⁸, non-C-terminally phosphorylated full length Akt1, lane 2, FL-Akt1-pThr³⁰⁸, lane 3, FL-Akt1-pSer⁴⁷³, lane 4, FL-Akt1-pThr³⁰⁸/pSer⁴⁷³.

Figure 2.. Enzymatic characterization of semisynthetic Akt constructs by radiometric measurements.

A) Representative steady-state kinetic plots for v/[E] vs [ATP] with 20 mM GSK3 peptide substrate (left), and v/[E] vs [GSK3 peptide] with 20 mM ATP (right). Enzyme concentrations are 2 nM FL-Akt1-pThr³⁰⁸/pSer⁴⁷³; 4 nM FL-Akt1-pThr³⁰⁸/2p-Ser⁴⁷⁷,Thr⁴⁷⁹, and 500 nM non-phosphorylated FL-Akt1. The reactions were carried out at 30⁰C for 10 min. B) Enzymatic parameters for the semisynthetic full length or Δ143 Akt1 proteins are shown ± standard error. The k_cat and k_cat/K_m values were obtained from kinetic plots for v/[E] vs [ATP] (*), (n=2).

Figure 3.. X-ray structural analysis of Δ143-Akt1-pThr³⁰⁸/3p-Ser⁴⁷³,Ser⁴⁷⁷,Thr⁴⁷⁹

A) Overall crystal structure of Δ143-Akt1-pThr³⁰⁸/3p-Ser⁴⁷³,Ser⁴⁷⁷,Thr⁴⁷⁹ complexed with bisubstrate ATP-GSK3 (teal). B) A blow up of the region highlighted with a square box in Figure 3A showing chemical formula (top) and 3D structure (bottom) of bisubstrate ATP-GSK3. C) Structural comparison of the C-tail with S473D (orange, PDB: 4EKK) and pS473 (purple), interactions with Q218 are highlighted. D) Kinase assays of semisynthetic Akt1 proteins with pSer⁴⁷³ and Q218A and basic patch (142-KHR-144) AAA replacements. The kinase assays were carried out as described in Figure 2A and the enzymatic parameters are shown in Figure 2B. E) Alignment of the PH-kinase linkers (aa 135-164) from different human Akt paralogs and from Akt1 from different species with Uniprot IDs in parentheses and position of Arg144 highlighted in the red box.

Figure 4.. Phospho-Ser473 interacts with Arg144 in the PH-kinase linker basic patch.

A) Cellular analysis of the effect of basic patch on the Akt phosphorylation and activity. Akt1/2 knock-out HCT116 cells (Ericson et al., 2010) were transfected with pcDNA3 plasmids expressing wild type (WT) and K142A, H143A, R144A (3A) full-length Akt1s. After serum starvation, the cells were stimulated with growth factors for the time indicated. The cells were lysed and analyzed by western blot with Akt antibodies (left panel) and Foxo1 or Foxo3a antibodies (right panel). N.: non-transfected and stimulated with growth factors for 10 min., (−): transfected with DNA plasmids but not stimulated with growth factors; n=3 forassays. B) Quantification of phosphorylation level of Akt Ser473 (blue for WT Akt1 and orange for 3A-Akt1) and Foxo1 Thr24/Foxo3a Thr32 (pink for WT Akt1 and green for 3A-Akt1) using Image J (n=3, SEM shown, p<0.01) of blots represented by Figure 4A. C) X-ray structure of D122-Akt1-pThr³⁰⁸/3p-Ser⁴⁷³,Ser⁴⁷⁷,Thr⁴⁷⁹complexed with bisubstrate analog and residues Gln218, pSer⁴⁷³ and Arg144 are highlighted. D) Zoom-in of the region highlighted with a square in Figure 4C shows the interaction of pSer⁴⁷³ with Gln218 and Arg144, the pSer⁴⁷³ to Arg144 H-bond is 3.0 Å

Figure 5.. The molecular mechanism of Akt1 activation by C-terminal Ser473-phosphorylation.

A) Cartoon model for Akt1 activation induced by phospho-Ser473. Without C-terminal phosphorylation, Akt1 exists in an inactive conformation with autoinhibition involving a PH domain-kinase domain interaction. Phosphorylation of Ser473 by mTORC2 can activate Akt1 via the interaction of pSer⁴⁷³ with the N-lobe and Arg144 within the PH-kinase linker that relieves autoinhibition by the PH domain.B) Cellular assays for an Akt construct in which a hexa-Gly (6G) segment is inserted into the PH-kinase linker compared with wild type Akt1. Assays were carried out as described in Figure 4A. C) Quantification of the phosphorylation level of Ser473 (blue for WT Akt1 and purple for 6G-Akt) and Foxo1 Thr24/Foxo3a Thr32 (pink for WT Akt1 and yellow for 6G-Akt1) from three replicates (n=3, SEM shown, p<0.01). D) Cartoon depicted shows the model for the effect of a hexa-Gly insertion into the Akt1 PH-linker. The 6 Gly insertion is proposed to enhance linker flexibility stabilizing the intramolecular PH-kinase interaction even after C-terminal phosphorylation of Akt1 on Ser473.

Figure 6.

Dissecting the pSer⁴⁷⁷/pThr⁴⁷⁹ intramolecular interaction in Akt1 using photo-crosslinking. A) Schematic representation for the UV-activatable crosslinking of the Bpa group from the pSer⁴⁷⁷/pThr⁴⁷⁹ C tail with the activation loop. B) MS and C) MS/MS spectra of RPKbioFPQBSYSASGTA (peptide 1) crosslinked to DGATMK (peptide 2) in the activation loop of Akt1. (C) Ions of type b from peptide 1 and peptide 2 are highlighted in blue and green, respectively. Similarly, ions of type y from peptide 1 and peptide 2 are shown in red and yellow. Each product ion is also labeled with its parent sequence (“1” or “2”) followed by the charge state of the ion (i.e. 2+). For example, y₁ (2/1+) corresponds to the y1 ion of peptide 2 singly charged. Specific crosslinked sequences and internal fragments are also indicated (See Figure S7 for the rest of m/z spectrum). −H₂O, water loss; K_bio, biotinylated lysine; B, benzoylphenylalanine.

Figure 7.. The dual phosphorylations at Ser477 and Thr479 activate Akt1 in a mechanism distinct from that of pSer473.

A) Binding assay of PIP3 with FL-Akt1-pThr³⁰⁸, pSer⁴⁷⁷,pThr⁴⁷⁹ (A6) measured with fluorescence anisotropy. Varying concentrations of Akt1 were mixed with 50 nM fluorescein-labeled soluble PIP3. Measurements (n=2) were fit to quadratic binding isotherms and K_d value shown ± SEM. B) Table for the affinity binding (K_d) values of soluble PIP3 with three semisynthetic FL-Akt1 proteins: A1, A2, and A6.C) Cartoon model for how phosphorylation of Ser477 and Thr479 of Akt1 can relieve autoinhibition by interaction with the activation loop and PH domain, partially displacing the PH domain from its interaction with the kinase domain.