Regulation of the PI3K pathway through a p85α monomer–homodimer equilibrium
- University of Texas MD Anderson Cancer Center, United States
- King Abdullah University of Science and Technology, Saudi Arabia
Figures
Figure 1 with 5 supplements
Intermolecular interactions contribute to p85α homodimerization and PTEN binding.
(A) Theoretical molecular model of the p85α SH3:PR1 interaction. The Qualitative Model Energy Analysis (QMEAN) Z-score of the model is −0.8 [the QMEAN Z-score ranges from −4 (worse) to +4 (best) …
Figure 1—figure supplement 1
(A–F) Sedimentation equilibrium data for full-length p85α, p85α PR1-SH3-BH-PR2 (residues 1–333), and p85α PR1-BH (residues 79–301).
The data of final equilibrium profiles (black symbols) were fitted (red line) to the monomer–dimer–tertramer model (p85α and p85α PR1-SH3-BH-PR2) or a monomer–dimer model (PR1-BH) with reduced χ2 of …
Figure 1—figure supplement 2
(J–L) Microscale thermophoresis (MST) data on dimerization of p85α (full length, SH3-PR1-BH-PR2 and PR1-BH).
Affinities for individual constructs were determined at Kd p85α = 3.9 ± 0.2 μM, Kd p85α1-333 = 441 ± 30 nM, and Kd p85α PR1-BH = 22.3 ± 1.5 μM. The errors are standard deviation calculated from …
Figure 1—figure supplement 3
(M–N) Isothermal titration calorimetry (ITC) data on p85α SH3:PR1 and p85αΔSH3 interactions.
Kd = 23.3 ± 6 μM, ΔG = −26.5 kJ/mol, ΔH = −4.9 kJ/mol, TΔS = 21.5 kJ/mol and the stoichiometry N = 1.06 ± 0.3 (M) and Kd = 16.8 ± 4.2 μM ΔG = −27.7 kJ/mol, ΔH = −13.4 kJ/mol, TΔS = 14.4 kJ/mol and …
Figure 1—figure supplement 4
(A, B) p85α knockout mouse embryonic fibroblast (MEF) cells (A) or PTEN knockout MEF cells (B) co-transfected with Flag-tagged WT p85α (Flag-WT) and HA-tagged WT p85α or PR mutants for 72 hr were collected for IP with anti-HA and WB.
(C) KLE cells co-transfected with Flag-WT and HA-tagged WT p85α or truncated mutant A360* were collected for IP with anti-HA and WB. LacZ was used as control. Numerical values below the immunoblots …
Figure 1—figure supplement 5
(A) KLE cells co-transfected with HA-tagged WT p85α (or p85β) and Flag-tagged WT p85α (or p85β) for 72 hr were harvested for IP with anti-HA and WB.
(B) The crystallographic BH domain dimers from p85α (PDB id. 1PBW; light and dark red) and p85β (PDB 2XS6; yellow and pale yellow) are superimposed on one of the BH domains. (C) KLE cells …
Figure 2 with 2 supplements
p85α homodimer increases protein stability, lipid phosphatase activity, and membrane association of PTEN.
(A–F) KLE cells transfected with WT p85α or PR1 and PR2 mutants (A, D), SH3 domain mutants (B, E), or BH domain mutants (C, F) for 72 hr were collected for IP with anti-PTEN and WB with …
Figure 2—figure supplement 1
(A) Negative feedback of the phosphatidylinositol 3-kinase (PI3K) pathway mediated by S6K.
(B) The efficiency of siRNAs targeting S6K was confirmed by WB. Non-specific (NS) siRNA was used as control. (C, D) KLE cells co-transfected with WT p85α or PR1+PR2mut and 10 nM siRNA targeting S6K …
Figure 2—figure supplement 2
PTEN in vitro lipid phosphatase activity was determined using a malachite green phosphatase assay with soluble PIP3 as the substrate.
Endogenous PTEN proteins were immunoprecipitated in lysates using anti-PTEN antibody extracted from transfected cells and phosphatase activity was measured in triplicate. The activity was normalized …
Figure 3 with 2 supplements
p85α homodimer competes with E3 ligase WWP2 for PTEN binding.
(A) KLE cells were harvested for IP with anti-PTEN and WB. Normal IgG was used as a negative control. (B) Cells transfected with WT p85α or PR mutants were harvested for IP after 72 hr. (C) Cells …
Figure 3—figure supplement 1
(A, B) KLE cells co-transfected with WWP2 and 10 nM siRNA targeting S6K or NS control for 72 hr were harvested for WB directly (A) or IP with anti-Flag and WB (B).
(C, D) Cells co-transfected with WWP2 and 10 nM siRNA targeting Rictor or NS control for 72 hr were harvested for WB (C) or IP with anti-Flag (D). (E) Cells transfected with WWP2 were treated with …
Figure 3—figure supplement 2
(A, B) HEC1A cells were transfected with 10 nM siRNA targeting WWP2 (A) or p85α (B) for 72 hr.
The efficiency of the siRNAs was confirmed by WB. NS siRNA was used as control. (C, D) Cells transfected with 10 nM siRNA targeting WWP2 (C) or p85α (D) or NS control for 72 hr were harvested for IP …
Figure 4 with 1 supplement
Binding of PTEN to WWP2 and to p85α homodimer is mutually exclusive.
(A–F) Cell lysates from KLE cells transfected with WT p85α (A–C) or combined PR1 and PR2 mutant (PR1+PR2) (D–F) were fractionated using a gel filtration column and the indicated fractions were …
Figure 4—figure supplement 1
(A-B) KLE cells were transfected with WT p85α for 72 hr.
Whole cell lysates were then fractionated using a gel filtration column and the indicated fractions were pooled for IP with anti-p85α antibody. The immunocomplexes were subjected to SDS-PAGE …
Figure 5 with 1 supplement
Oncogenic cancer patient-derived PIK3R1 mutation perturbs p85α homodimerization leading to PI3K pathway activation.
(A) KLE cells co-transfected with Flag-tagged WT p85α (Flag-WT) and HA-tagged WT p85α or patient-derived p85α BH domain mutants were collected for IP with anti-HA and WB. (B, C) Cells transfected …
Figure 5—figure supplement 1
PIK3R1 mutations from The Cancer Genome Atlas (TCGA) data sets across tumor lineages are represented by lollipops (green, missense; red, nonsense, frameshift, or splice; black, in-frame deletion/insertion; purple, different types of mutations at the same site).
The diagram was adopted from the cBioPortal (http://www.cbioportal.org/public-portal/).
Figure 6 with 1 supplement
Molecular model of the p85α homodimer:PTEN complex reveals cancer patient-derived p85α mutant with decreased PTEN binding.
(A) Schematic theoretical molecular working model of the homodimerized p85α:PTEN. This speculative model has been constructed by integrating experimental data, physical constraints, and …
Figure 6—figure supplement 1
(A) KLE cells co-transfected with Flag-tagged WT p85α (Flag-WT) and HA-tagged WT p85α or mutants were harvested for IP with anti-HA and then subjected to WB.
The mutated amino acids are highly conserved residues potentially for small GTPase binding. (B) Cells were co-transfected with HA-tagged p85α in the absence or presence of increasing amounts of PTEN …
Figure 7
Schematic working model of how homodimerized p85α promotes PTEN stabilization and lipid phosphatase activity.
Our data support a p85α homodimer model that includes intermolecular interactions between SH3:PR1 in trans and BH:BH interactions between protomers. Key contact residues at the interfaces are shown …
Appendix figure 1
Superimposition of the crystallographic complex Cdc42GAP (light green) bound to Cdc42 (dark green) (1GRN) onto p85α BH (black) and the crystal structure of Rab5 (yellow; 1R2Q).
The GTP analogue (magenta) and the important catalytic arginine residue (blue) are highlighted.
Appendix figure 2
Model for Rab5 bound to the BH domain. Left and right panel are 90° views.
Cyan: Residues mutated in the GTPase-binding site that did not affect PTEN binding.
Appendix figure 3
(Left) distance constraints used in BUNCH.
(A-C) Examples of dimeric SH3-PR1-BH models obtained by BUNCH. SH3: green; SH3-BH linker: magenta; BH dimer: light and dark gray.
Appendix figure 4
(A) approximate positioning of PTEN with respect to the membrane (green).
C2 residues required for membrane binding are highlighted. Orange spheres represent a bound L(+)-tartrate molecules found in the PTEN crystal structure (1D5R) and are thought to mimic interactions …
Appendix figure 5
(A) PTEN is shown in the crystal lattice of 1D5R.
Gray: C2; orange: phosphatase; magenta: symmetric molecules. Highly conserved regions are shown as blue spheres. (B) Result of Sitehound search (http://scbx.mssm.edu/sitehound/ (Hernandez et al., …
Appendix figure 6
Cluster of ClusPro models that satisfy all constraints.
(A) Complexes viewed from the membrane; (B) side view with respect to the membrane.
