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. 2020 Aug 3;9:e59151. doi: 10.7554/eLife.59151

Figure 5. The PH domain of Akt interacts with the kinase domain differently depending on C-tail phosphorylations.

(A) Cartoon representation of secondary structure elements (rectangle for β-strands, zigzag for α-helices) in Akt PH domain. Color coding represents regions with distinct binding modes to the kinase domain. Star indicates that the α-helix is present only when Akt is bound to IP4. (B–C) Combined chemical shift perturbations derived from spectra in Figure 4 and plotted along the PH domain primary sequence for pSer473 (B) and pSer477/pThr479 (C) referenced to non-p-C. Dashed red line corresponds to the standard deviation to the mean, excluding outliers (higher than 3xStDev). Grey bars indicate peaks that disappeared from the spectrum, also indicating strong interaction. Red area highlights the short hinge primarily studied here. Negative bars (−0.05) indicate non-assigned residues, negative bars (−0.025) indicate residues which assignment could not be easily transferred or recovered in the context of full-length Akt. (D) Structure of allosteric drug inhibited Akt (PDB: 3O96, [Wu et al., 2010]) with PH domain as ribbon in the front and kinase domain as surface in the back (N-lobe in light blue, C-lobe in dark blue). Color coding of secondary structure elements in the PH domain corresponds to (A). Allosteric inhibitor MK2206 is displayed in white. Main Akt domains are labeled. (E–F) Same structure representation as in (D) with the most significantly affected residues (chemical shift perturbations higher than the standard deviation) colored in red in the PH domain in case of pSer473 (E) and pSer477/pThr479 (F). Non-affected residues are shown in light orange and non-assigned residues in grey. Representations rotated by 180° are shown. (G) Statistical bar and whisker plots of saturation transfer efficiencies from CST data (Figure 5—figure supplement 3) for each C-tail phospho-state, categorized and color coded according to secondary structure elements as in (A) and (D). A ratio of 0 indicates maximum saturation transfer efficiency (very tight interaction) whereas a ratio of 1 indicate no saturation transfer (no interaction).

Figure 5.

Figure 5—figure supplement 1. The PH domain of Akt is significantly affected in the context of full-length Akt.

Figure 5—figure supplement 1.

(A) 15N-1H HSQC spectra of the isolated PH domain (black) and the PH domain in the context of full-length semisynthetic Akt with pS473 C-tail (red). Select residue-specific assignments are shown, color coded according to (B). (B) Cartoon representation of secondary structure elements (rectangle for β-strands, zigzag for α-helices) in Akt PH domain. Color coding represents regions with distinct binding modes to the kinase domain. Star indicates that the α-helix is present only when Akt is bound to IP4. (C) Combined chemical shift perturbations derived from spectra in (A) and plotted along the PH domain primary sequence. Dashed red line corresponds to the standard deviation to the mean, excluding outliers (higher than 3xStDev). Grey bars indicate peaks that disappeared from the spectrum, also indicating strong interaction. Red area highlights the short hinge primarily studied here. Negative bars (−0.05) indicate non-assigned residues. (D) Structure of allosteric drug-inhibited Akt (PDB: 3O96, [Wu et al., 2010]) with PH domain as carton on top and kinase domain as surface in the back. Color coding of secondary structure elements in the PH domain corresponds to (B). Allosteric inhibitor MK2206 is displayed in white.
Figure 5—figure supplement 2. Akt kinase domain and inositol phosphates compete for interaction with PH domain.

Figure 5—figure supplement 2.

(A–B) 15N-1H HSQC spectra of the isolated PH domain (black) in the absence and in the presence of kinase domain (A, red) or IP6 (B, red). Select residue-specific assignments are shown, color coded according to (C). (C) Cartoon representation of secondary structure elements (rectangle for β-strands, zigzag for α-helices) in Akt PH domain. Color coding represents regions with distinct binding modes to the kinase domain. Star indicates that the α-helix is present only when Akt is bound to IP4. (D–E) Combined chemical shift perturbations derived from spectra in (A–B) and plotted along the PH domain primary sequence for PH domain in the presence of kinase domain (D) or IP6 (E), respectively, referenced to the isolated PH domain. Dashed red line corresponds to the standard deviation to the mean, excluding outliers (higher than 3xStDev). Grey bars indicate peaks that disappeared from the spectrum, also indicating strong interaction. Red area highlights the short hinge primarily studied here. Negative bars indicate non-assigned residues. (F–G) Crystal structures of the isolated PH domain (F, PDB:1UNP) and IP4-bound PH domain (G, PDB: 1UNQ) (Milburn et al., 2003). IP4 is shown in magenta. The short hinge shown in red is folded as a short α-helix. (H) Structure of allosteric drug-inhibited Akt (PDB:3O96, [Wu et al., 2010]) with PH domain as a ribbon cartoon and the kinase domain as a surface representation (purple). The allosteric inhibitor MK2206 is displayed in white. It is evident that IP4 and the kinase domain partially overlap in their interactions with the PH domain.
Figure 5—figure supplement 3. Cross-saturation transfer NMR analysis of the the PH-kinase domain interactions.

Figure 5—figure supplement 3.

(A–C) 15N-1H cross-saturation transfer spectra of the deuterated PH domain in the context of the semisynthetic full-length protonated Akt. Spectra where the kinase domain’s protons have been saturated are shown in red and their unsaturated reference in black for the non-phosphorylated C-tail (A), pSer473 (B) and pSer477/pThr479 (C) forms, respectively. Select residue-specific assignments are shown, color coded according to (E). (D) Structure of allosteric drug-inhibited Akt (PDB:3O96, [Wu et al., 2010]) with the PH domain as a ribbon cartoon on top and the kinase domain as a surface representation in the back. Color coding of secondary structure elements in the PH domain corresponds to (E). Allosteric inhibitor MK2206 is displayed in white. (E) Cartoon representation of secondary structure elements (rectangle for β-strands, zigzag for α-helices) in Akt PH domain. Color coding highlights regions with distinct binding modes to the kinase domain. Star indicates that the α-helix is present only when Akt is bound to IP4. (F–H) Saturation transfer efficiency derived from (A–C) plotted as the ratio of peak intensities of saturated over unsaturated spectra against the PH domain primary sequence for non-p (F), pSer473 (G) and pSer477/pThr479 (H) forms, respectively. A ratio of 0 indicates maximum saturation transfer efficiency, whereas a ratio of 1 indicate no saturation transfer. An indicative dashed red line has been drawn at 0.5. Red area highlights the short hinge primarily studied here. Negative ratios indicate non-assigned residues. Residues that were not present in 70% 2H2O have ratios set to 0.