Paradoxical dominant negative activity of an immunodeficiency-associated activating PIK3R1 variant

Patsy R Tomlinson
Rachel Knox
Olga Perisic
Helen C Su
Gemma V Brierley
Roger L Williams
Robert K Semple author has email address

The University of Cambridge Metabolic Research Laboratories, Wellcome Trust-MRC Institute of Metabolic Science, Cambridge, UK
MRC Metabolic Diseases Unit, Wellcome Trust-MRC Institute of Metabolic Science, Cambridge, UK
The National Institute for Health Research Cambridge Biomedical Research Centre, Cambridge, UK
MRC Laboratory of Molecular Biology, Cambridge, UK
Laboratory of Clinical Immunology & Microbiology, Intramural Research Program, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, USA
Department of Comparative Biomedical Science, The Royal Veterinary College, London, UK
Centre for Cardiovascular Science, University of Edinburgh, Edinburgh, UK
MRC Human Genetics Unit, Institute of Genetics and Cancer, University of Edinburgh, Edinburgh, UK

https://doi.org/10.7554/eLife.94420.2

Open access
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Figures and data

PI3K subunit expression and signalling in primary dermal fibroblasts.
Immunoblotting of AKT, AKT phosphorylated at threonine 308 (T308) or serine 473 (S473), p85α, p110δ and p110α and are shown with and without stimulation by 100 nM insulin (Ins) for 10 minutes. β-actin is shown as a loading control, with different amounts of pooled lysate (Pool) used to demonstrate signal intensity in the linear range. Results from 4 healthy controls (WT; 1-4), 1 patient with Activating p110 Delta Syndrome 2 (APDS2) due to the p85α Δexon11 variant, and 3 patients with PIK3CA-Related Overgrowth Spectrum (PROS) caused by the activating PIK3CA mutations indicated. (A) Immunoblots, with the truncated p85α Δexon11 variant arrowed; (B)-(E) quantification of immunoblot bands from 3 independent experiments shown for phosphoAKT-S473, phosphoAKT-T308, p110δ and p110α respectively. Each point represents data from one of the patient cell lines in the immunoblots. Paired datapoints +/- insulin are shown in (B) and (C), and dotted lines mark means. Asterisks indicate a significant difference. More detailed statistical analysis including 95% confidence intervals for the paired mean differences for these comparisons are shown in Figure 1 figure supplement 2.

Blunted insulin signaling in 3T3-L1 preadipocyte models of APDS2 and SHORT syndrome.
Immunoblotting of Akt, Akt phosphorylated at threonine 308 (T308) or serine 473 (S473), p85α, and p110α and are shown with and without stimulation with 100 nM insulin (Ins) for 10 minutes. Cells were treated with doxycycline (Dox) 1μg/mL for 72 hours prior to insulin stimulation as indicated. (A) One immunoblot representing 3 experiments is shown. (B)-(C) Quantification of immunoblot bands from all 3 independent experiments shown for phosphoAkt-S473 and phosphoAkt-T308 respectively. Paired datapoints +/- insulin are shown, and dotted lines mark means. Asterisks indicate a significant difference. More detailed statistical analysis including 95% confidence intervals for the paired mean differences for these comparisons are shown in Figure 2 figure supplement 2. (D) Staining for neutral lipid with Oil Red O of 3T3-L1 cells at day 10 of adipocyte differentiation. Induction of transgene expression by 1μg/mL doxycycline throughout differentiation is shown. Images of entire plates are shown above, with representative bright field microscopy images below.

SHORT syndrome p85α mutations impair phosphotyrosine-stimulated phosphoinositide 3-kinase activity.
Lipid kinase activity of purified recombinant PI3K complexes generated using baculoviral expression in Sf9 cells was measured using a modified fluorescence polarisation assay. WT p85α or p85α SHORT syndrome mutations, E489K, R649W or Y657X bound to either (A) p110α (B) p110β or (C) p110δ were assayed for basal and bisphosphotyrosine (pY2)-stimulated lipid kinase activity. Dotted lines mark means, and asterisks indicate a significant difference between the bisphosphotyrosine (pY2)-stimulated state for WT and comparator mutant p85α. More detailed statistical analysis including 95% confidence intervals for the paired mean differences for these comparisons are shown in Figure 3 figure supplement 1.

Ability of pathogenic p85α variants to bind p110α, assessed by co-immunoprecipitation.
Results of immunoblotting of anti-p110α immunoprecipitates from 3T3-L1 cells expressing wild-type, APDS2-associated or SHORT syndrome-associated mutant p85α under the control of doxycycline (Dox) are shown. (A) One representative immunoblot of immunoprecipitate, cell lysate prior to immunoprecipitation, and post immunoprecipitation supernatant is shown. (B) Quantification of immunoblot bands from immunoprecipitates from 3 independent experiments, expressed as a percentage relative to the intensity of the band in WT cells without doxycycline exposure. Co-immunoprecipitated p85α is shown normalized to immunoprecipitated p110α from all 3 independent experiments. Datapoints from the same experiment +/- doxycline are connected by lines. No significant differences were found among conditions.

Attenuated insulin-induced association of p110α with Irs1 in the presence of APDS2 and SHORT syndrome mutant p85α.
Results of immunoblotting of anti-Irs1 immunoprecipitates from 3T3-L1 cells expressing wild-type, APDS2-associated or SHORT syndrome-associated mutant p85α under the control of doxycycline (Dox) are shown. Treatment with 100nM insulin (Ins) is indicated. (A) One representative immunoblot of immunoprecipitate, cell lysate prior to immunoprecipitation, and post immunoprecipitation supernatant is shown. Two separate sets of gels, including independent wild-type controls are shown on left and right. (B-C) Quantification of immunoblot bands from immunoprecipitates from 3 independent experiments. Immunoprecipitated p110α is shown normalized to immunoprecipitated Irs1 from all 3 independent experiments in (B), and immunoprecipitated p85α similarly in (C). Datapoints from the same experiment +/- insulin are connected by lines. Asterisks indicate significant differences induced by transgene overexpression (i.e. plus versus minus doxycycline). More detailed statistical analysis including 95% confidence intervals for the paired mean differences for these comparisons are shown in Figure 5 figure supplement 1.

Attenuated insulin-induced association of p110α with Irs2 in the presence of APDS2 and SHORT syndrome mutant p85α.
Results of immunoblotting of anti-Irs2 immunoprecipitates from 3T3-L1 cells expressing wild-type, APDS2-associated or SHORT syndrome-associated mutant p85α under the control of doxycycline (Dox) are shown. Treatment with 100nM insulin (Ins) is indicated. (A) One representative immunoblot of immunoprecipitate, cell lysate prior to immunoprecipitation, and post immunoprecipitation supernatant is shown. Two separate sets of gels, including independent wild-type controls are shown on left and right. (B-C) Quantification of immunoblot bands from immunoprecipitates from 3 independent experiments. Immunoprecipitated p110α is shown normalized to immunoprecipitated Irs2 from all 3 independent experiments in (B), and immunoprecipitated p85α similarly in (C). Datapoints from the same experiment +/- insulin are connected by lines.

Antibodies Used

Plasmids Used

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