Akt phosphorylates insulin receptor substrate to limit PI3K-mediated PIP3 synthesis
- Charles Perkins Centre, School of Life and Environmental Sciences, University of Sydney, Australia
- Metabolic Research Laboratories, Wellcome Trust-Medical Research Council Institute of Metabolic Science, University of Cambridge, United Kingdom
- Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Australia
- Biomedicine Discovery Institute, Monash University, Australia
- Charles Perkins Centre, School of Mathematics and Statistics, University of Sydney, Australia
- Computational Systems Biology Group, Children's Medical Research Institute, University of Sydney, Australia
- School of Medical Sciences, University of Sydney, Australia
Figures
Figure 1 with 1 supplement
A mechanistic model of insulin signalling predicts a rapid negative feedback mechanism that is not dependent on mammalian target of rapamycin complex 1 (mTORC1).
(A) Schematic depicting model architecture with negative feedback via mTORC1 onto the insulin receptor substrate/phosphoinositide 3-kinase (IRS/PI3K) node (IR, insulin receptor; NFB, negative …
Figure 1—figure supplement 1
A mechanistic model of insulin signalling without negative feedback does not capture signalling kinetics.
(A) Box and whiskers plots (median; interquartile range and min/max) of the local minima from 200 independent calibration runs (repeats) of negative feedback models 1 (mammalian target of rapamycin …
Figure 2 with 2 supplements
Negative feedback is dependent on Akt activity.
(A) Schematic depicting the model architecture including the hypothetical negative feedback loops from phosphoinositide-dependent protein kinase 1 (PDPK1), mammalian target of rapamycin complex 2 …
Figure 2—figure supplement 1
Six hypothetical mechanistic models of insulin signalling predict signalling outcomes.
The hypothetical negative feedback models 4 (A), 5 (B), 6 (C), 7 (D), 8 (E), and 9 (F) were used to predict Akt recruitment, T309 and S474 phosphorylation, and PRAS40 phosphorylation in response to …
Figure 2—figure supplement 2
Akt inhibition increases its phosphorylation and membrane recruitment following insulin stimulation, in diverse cell types.
(A) 3T3-L1 adipocytes were preincubated with vehicle (DMSO) or 10 μM GDC0068 for 5 min, then stimulated with 1 nM insulin for times specified. Lysates were immunoblotted with antibodies as …
Figure 3 with 1 supplement
Akt regulates PI(3,4,5)P3 abundance.
(A) 3T3-L1 adipocytes were treated with 10 μM GDC0068, 10 μM MK2206, 10 μM GSK2334470, or vehicle control for 15 min, followed by 1 nM insulin for 10 min. Lipids were extracted from the cells and …
Figure 3—figure supplement 1
GSK2334470 inhibits phosphoinositide-dependent protein kinase 1 (PDPK1)-dependent signalling outcomes in 3T3-L1 adipocytes.
3T3-L1 adipocytes were incubated with GSK2334470 (PDPK1 inhibitor) at the indicated dose for 15 min followed by 1 nM insulin for 10 min. Lysates were immunoblotted with antibodies as specified, with …
Figure 4
Akt regulates insulin-stimulated production of PI(3,4,5)P3 by phosphoinositide 3-kinase (PI3K).
(A) Model predictions detailing the effect of PI3K inhibition following insulin stimulation, on the plasma membrane dissociation of Akt with and without Akt inhibition. The parameter value of Kf3 …
Figure 5
Akt releases insulin receptor substrate 1/2 (IRS1/2) from the plasma membrane to the cytosol.
(A) 3T3-L1 adipocytes expressing IRS1-eGFP were stimulated with 10 μM GDC0068, 10 μM MK2206, or vehicle control for 10 min, followed by 1 nM insulin. Recruitment was assessed by total internal …
Figure 6 with 4 supplements
Akt-mediated phosphorylation of insulin receptor substrate (IRS) removes it from the plasma membrane to engage negative feedback.
(A) IRS1-eGFP was concurrently mutated at six phosphorylation sites to make up ‘6P IRS1’ – S270A, S307A, S330A, T525A, S527A, and S1101A (human). (B) IRS2-eGFP was concurrently mutated at five …
Figure 6—figure supplement 1
Phosphorylation of a variety of IRS1 residues alter IRS1 and Akt localisation at the plasma membrane.
(A–F) 3T3-L1 adipocytes were co-electroporated with insulin receptor substrate 1 (IRS1)-eGFP (WT or mutant) and TagRFP-T-Akt2. Cells were stimulated with 1 nM insulin and recruitment assessed by …
Figure 6—figure supplement 2
Phosphorylation of a variety of IRS2 residues alter IRS2 and Akt localisation at the plasma membrane.
(A–E) 3T3-L1 adipocytes were co-electroporated with insulin receptor substrate 2 (IRS2)-eGFP (WT or mutant) and TagRFP-T-Akt2. Cells were stimulated with 1 nM insulin and recruitment assessed by …
Figure 6—figure supplement 3
Phosphorylation of IRS2 at S365 and S1149 in vitro and in cells.
(A) Quantification of phosphorylated insulin receptor substrate 2 (IRS2) S365 (S362 in mouse) peptides across three mass spectrometry experiments. (B) Quantification of phosphorylated IRS2 S1149 …
Figure 6—figure supplement 4
Phosphorylation of IRS1 at S270 and S527 only subtly alter IRS1 and Akt localisation at the plamsa membrane.
3T3-L1 adipocytes were co-electroporated with human insulin receptor substrate 1 (IRS1)-eGFP (WT or S270/527A) and TagRFP-T-Akt2. Cells were stimulated with 1 nM insulin and recruitment assessed by …
Figure 7
Akt phosphorylates insulin receptor substrate 1/2 (IRS1/2) to limit phosphoinositide 3-kinase (PI3K)-mediated phosphatidylinositol (3,4,5)-trisphosphate (PIP3) synthesis.
Schematic for a model of Akt-mediated negative feedback. Basal; IRS is plasma membrane (PM) localised due to a pleckstrin homology (PH) domain-mediated interaction with the PM. Insulin (with …
Author response image 1
Models’ simulations of Akt recruitment, T309 and S474 phosphorylation, and PRAS40 phosphorylation in response to 1 and 100 nM insulin (simulation; dotted lines).
This was overlaid with the experimentally observed kinetics for each outcome (experiment; solid lines, mean ± S.E.M). Red boxes highlight inconsistencies between model’s simulations and experimental …
Author response image 2
3T3-L1 adipocytes were electroporated with WT (left) or DelPH (right) IRS1-eGFP and stimulated with 1 nM Insulin.
Recruitment was assessed by TIRFM (three independent experiments, data expressed as mean ± S.E.M, PM; plasma membrane).
Author response image 3
3T3-L1 adipocytes were electroporated with IRS1-eGFP or IRS2-eGFP.
Cells were stimulated with DMSO, 10 µM MK2206 or 10 µM GDC0068 5 min prior to 100 ng/mL EGF (epidermal growth factor) and recruitment assessed by TIRFM (two independent experiments, data expressed …
Author response image 4
3T3-L1 adipocytes were electroporated with TagRFP-T-Akt2.
Cells were stimulated with 100 nM insulin with or without a 1 nM insulin pre-stimulation. Recruitment was assessed by TIRFM (data expressed as mean ± S.E.M, PM; plasma membrane).
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (mouse) | 3T3-L1 | Dr Howard Green, Harvard Medical School | RRID:CVCL_0A20 | |
| Cell line (human) | HEK293E | American Type Culture Collection | RRID:CVCL_6974 | |
| Cell line (human) | HeLa | American Type Culture Collection | RRID:CVCL_0030 | |
| Cell line (human) | HCC1937 | American Type Culture Collection | RRID:CVCL_0290 | |
| Cell line (human) | MCF7 | Associate Prof. Jeff Holst, Centenary Institute | RRID:CVCL_0031 | |
| Antibody | pAkt T309 (rabbit polyclonal) | Cell Signalling Technology | Cat#9275 | WB (1:1000) |
| Antibody | pAkt T309 (rabbit monoclonal) | Cell Signalling Technology | Cat#13038 | WB (1:1000) |
| Antibody | pAkt S474 (mouse monoclonal) | Cell Signalling Technology | Cat#4051 | WB (1:1000) |
| Antibody | pAS160 T642 (rabbit polyclonal) | Cell Signalling Technology | Cat#4288 | WB (1:1000) |
| Antibody | pPRAS40 T246 (rabbit monoclonal) | Cell Signalling Technology | Cat#2997 | WB (1:1000) |
| Antibody | pGSK3ɑ/β S21/29 (rabbit monoclonal) | Cell Signalling Technology | Cat#9327 | WB (1:1000) |
| Antibody | pFOXO1 S256 (rabbit polyclonal) | Cell Signalling Technology | Cat#9461 | WB (1:1000) |
| Antibody | 14-3-3 (rabbit polyclonal) | Santa Cruz | Cat#sc-629 | WB (1:1000) |
| Antibody | pP70S6K T389 (rabbit polyclonal) | Cell Signalling Technology | Cat#9205 | WB (1:1000) |
| Antibody | Akt2 (rabbit monoclonal) | Cell Signalling Technology | Cat#3063 | WB (1:1000) |
| Antibody | Caveolin (mouse monoclonal) | Abcam | Cat#ab17052 | WB (1:1000) |
| Antibody | Pan Akt (rabbit monoclonal) | Cell Signalling Technology | Cat#4685 | WB (1:1000) |
| Antibody | PI3K p85 (rabbit monoclonal) | Cell Signalling Technology | Cat#4257 | WB (1:1000) IF (1:100) |
| Antibody | PI3K p110 (rabbit monoclonal) | Cell Signalling Technology | Cat#4249 | WB (1:1000) |
| Antibody | IRS1 (rabbit polyclonal) | Cell Signalling Technology | Cat#2382 | WB (1:1000) |
| Antibody | Na,K-ATPase (rabbit polyclonal) | Prof. Gus Lienhard | ||
| Antibody | IR (rabbit monoclonal) | Cell Signalling Technology | Cat#3025 | WB (1:1000) |
| Antibody | pTyrosine (rabbit monoclonal mix) | Cell Signalling Technology | Cat#8954 | WB (1:1000) |
| Antibody | ɑ-Tubulin (mouse monoclonal) | Sigma-Aldrich | Cat#T9026 | WB (1:1000) |
| Antibody | IRS1 (rabbit monoclonal) | Cell Signalling Technology | Cat#3407 | IP (2 µL per sample) |
| Antibody | IRS2 (rabbit polyclonal) | Cell Signalling Technology | Cat#3089 | IP (2 µL per sample) |
| Antibody | FLAG (mouse monoclonal) | Sigma-Aldrich | Cat#F1804 | IP (5 µg per sample) |
| Antibody | IgG control (rabbit) | Santa Cruz | Cat#sc-2027 | IP (control) |
| Antibody | Alexa Fluor 555 (goat polyclonal) | Thermo Fisher Scientific | Cat#A21428 | IF(1:200) |
| Peptide, recombinant protein | 3X FLAG Peptide | Sigma-Aldrich | Cat#F4799 | |
| Chemical compound, drug | Insulin | Sigma-Aldrich | Cat# I5500; CAS 11070-73-8 | |
| Chemical compound, drug | Insulin-like growth factor 1 (IGF1) | Miltenyi Biotec | Cat#130-093-886 | |
| Chemical compound, drug | Epidermal growth factor (EGF) | Miltenyi Biotec | Cat#130-097-749 | |
| Chemical compound, drug | GDC0068 | Selleck Chemicals | Cat#S2808; CAS 1001264-89-6 | |
| Chemical compound, drug | MK2206 | MedChemExpress | Cat#HY-10358; CAS 1032350-13-2 | |
| Chemical compound, drug | Wortmannin | Sigma-Aldrich | Cat#W1628; CAS 19545-26-7 | |
| Chemical compound, drug | Rapamycin | LC Laboratories | Cat#R-5000; CAS 53123-88-9 | |
| Chemical compound, drug | Rapalink | Prof. Kevan Shokat (Rodrik-Outmezguine et al., 2016) | ||
| Chemical compound, drug | GSK2334470 | Tocris | Cat# 4143; CAS 1227911-45-6 | |
| Commercial assay or kit | PIP3 ELISA | Echelon Biosciences | Cat#K-2500S | |
| Commercial assay or kit | PI(3,4)P2 ELISA | Echelon Biosciences | Cat#K-4500 | |
| Recombinant DNA reagent | TagRFP-T-Akt2 (plasmid) | Norris et al., 2017 | ||
| Recombinant DNA reagent | TagRFP-T-Akt2 W80A; W80A-T309A; W80A-S474A; W80A-T309A-S474A; W80A-K181A (plasmids) | This paper | See Materials and methods and Supplementary file 3 for DNA sequences | |
| Recombinant DNA reagent | PDPK1-eGFP (plasmid) | This paper | See Materials and methods and Supplementary file 3 for DNA sequences | |
| Recombinant DNA reagent | PDPK1-TagRFP-T (plasmid) | This paper | See Materials and methods and Supplementary file 3 for DNA sequences | |
| Recombinant DNA reagent | PH-Gab2-GFP (plasmid) | Sergio Grinstein – Addgene | Plasmid #35147 | |
| Recombinant DNA reagent | PI3K p110* (plasmid) | Morris Birnbaum | ||
| Recombinant DNA reagent | pMIG FLAG-Akt2 W80A; W80A-T309A (plasmids) | Kearney et al., 2019 | ||
| Recombinant DNA reagent | IRS1-eGFP (plasmid) | This paper | See Materials and methods and Supplementary file 3 for DNA sequences | |
| Recombinant DNA reagent | IRS1-eGFP S270A; S307A; S330A; T525A; S527A; S1101A; S270A-S307A-S330A-T525A-S527A-S1101A (6P); S270A-S527A; DelPH; DelPTB (plasmids) | This paper | See Materials and methods and Supplementary file 3 for DNA sequences | |
| Recombinant DNA reagent | IRS2-eGFP (plasmid) | This paper | See Materials and methods and Supplementary file 3 for DNA sequences | |
| Recombinant DNA reagent | IRS2-eGFP S306A; S346A; S365A; S577A; S1149A; S306A-S346A-S365A-S577A-S1149A (5P); S306A-S577A (plasmids) | This paper | See Materials and methods and Supplementary file 3 for DNA sequences | |
| Recombinant DNA reagent | IRS2-FLAG (plasmid) | This paper | See Materials and methods and Supplementary file 3 for DNA sequences | |
| Sequence-based reagent | DNA Primers | Sigma-Aldrich | See Materials and methods and Supplementary file 3 for DNA sequences | |
| Software, algorithm | MaxQuant | Cox and Mann, 2008; Tyanova et al., 2016 | RRID:SCR_014485 | |
| Software, algorithm | Image Studio | LI-COR Biosciences | RRID:SCR_013715 | |
| Software, algorithm | Graphpad Prism | GraphPad Software Inc. | RRID:SCR_002798 | |
| Software, algorithm | Fiji ImageJ | Schindelin et al., 2012 | RRID:SCR_003070 | |
| Software, algorithm | Tableau Prep | Tableau Software | ||
| Software, algorithm | R | https://www.R-project.org/ | RRID:SCR_001905 | |
| Software, algorithm | MATLAB | MathWorks | RRID:SCR_001622 | |
| Software, algorithm | IQM | IntiQuan |
Additional files
-
Supplementary file 1
Reactions and rate equations for the PI3K/Akt pathway models.
- https://cdn.elifesciences.org/articles/66942/elife-66942-supp1-v1.docx
-
Supplementary file 2
Parameter identifiability analysis of models 4 to 9.
- https://cdn.elifesciences.org/articles/66942/elife-66942-supp2-v1.pdf
-
Supplementary file 3
DNA sequence of plasmids and primers generated in this study.
- https://cdn.elifesciences.org/articles/66942/elife-66942-supp3-v1.docx
-
Transparent reporting form
- https://cdn.elifesciences.org/articles/66942/elife-66942-transrepform-v1.docx
