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

PIK3R1 encodes three regulatory subunits of class IA phosphoinositide 3-kinase (PI3K), each associating with any of three catalytic subunits, namely p110α, p110β or p110δ. Constitutional PIK3R1 mutations cause diseases with a genotype-phenotype relationship not yet fully explained: heterozygous loss-of-function mutations cause SHORT syndrome, featuring insulin resistance and short stature attributed to reduced p110α function, while heterozygous activating mutations cause immunodeficiency, attributed to p110δ activation and known as APDS2. Surprisingly, APDS2 patients do not show features of p110α hyperactivation, but do commonly have short stature or SHORT syndrome, suggesting p110α hypofunction. We sought to investigate this. In dermal fibroblasts from an APDS2 patient, we found no increased PI3K signalling, with p110δ expression markedly reduced. In preadipocytes, the APDS2 variant was potently dominant negative, associating with Irs1 and Irs2 but failing to heterodimerise with p110α. This attenuation of p110α signalling by a p110δ-activating PIK3R1 variant potentially explains co-incidence of gain-of-function and loss-of-function PIK3R1 phenotypes.


Introduction
PIK3R1 gene products (p85α, p55α and p50α) are essential for class IA phosphoinositide 3kinase (PI3K) signalling.Each of the three protein products can bind any of three catalytic subunits, p110α, β and δ, encoded by PIK3CA, PIK3CB, and PIK3CD respectively.This stabilises and inhibits the catalytic subunits in the basal state and confers responsiveness to upstream stimuli including receptor tyrosine kinase activation, enabled by two phosphotyrosine-binding SH2 domains shared by all PIK3R1 products (Fruman et al, 2017).
PI3K signalling mediates responses to a myriad of cues, including growth factors, hormones such as insulin, and processed antigens, and so recent discovery that genetic perturbation of PIK3R1 in humans disrupts growth, insulin action and immunity is no surprise.Beyond this headline observation lies considerable complexity, however, and important questions about genotype-phenotype correlations in PIK3R1-related disorders are unresolved.Some PIK3R1 mutations reduce basal inhibition of catalytic subunits, usually due to disruption of the inhibitory inter-SH2 domain, and are found in cancers (Philp et al, 2001) and vascular malformations with overgrowth (Cottrell et al, 2021).In both diseases, hyperactivated PI3Kα, composed of heterodimers of PIK3R1 products and p110α, drives pathological growth.Distinct inter-SH2 domain PIK3R1 mutations, mostly causing skipping of exon 11 and deletion of residues 434-475, hyperactivate PI3Kδ in immune cells, causing immunodeficiency (Deau et al, 2014;Lucas et al, 2014b).This phenocopies the immunodeficiency caused by genetic activation of p110δ itself, called Activated PI3K Delta Syndrome 1 (APDS1) (Angulo et al, 2013;Lucas et al, 2014a), and is thus named APDS2.
Despite ubiquitous PIK3R1 expression, features attributable to PI3Kα activation have been described in only a single case of APDS2 to date (Wentink et al, 2017).In that case, the causal variant was a constitutional activating mutation associated with cancer rather than one of the common APDS2 variants.Biochemical studies have suggested that apparently selective p110δ activation occurs because APDS2 mutations derepress p110δ, the .CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.It is made The copyright holder for this preprint this version posted November 2, 2023.; https://doi.org/10.1101/2023.11.02.565250 doi: bioRxiv preprint predominant immune system catalytic subunit, more than p110α, due to differences in the inhibitory contacts between PIK3R1 products and different catalytic subunits (Dornan et al, 2017).
More surprisingly, phenotypic overlap is reported between APDS2 and SHORT syndrome.
SHORT syndrome is caused by loss of PI3Kα function due to disruption of the phosphotyrosine-binding C-terminal SH2 domain (Chudasama et al, 2013;Dyment et al, 2013;Thauvin-Robinet et al, 2013).It features reduced linear growth, insulin resistance, and dysmorphic features (Avila et al, 2016).In recent years, both case reports (Bravo Garcia-Morato et al, 2017;Petrovski et al, 2016;Ramirez et al, 2020;Szczawinska-Poplonyk et al, 2022) and larger series (Elkaim et al, 2016;Jamee et al, 2020;Maccari et al, 2023;Nguyen et al, 2023;Olbrich et al, 2016;Petrovski et al., 2016) have established that many people with APDS2 have overt features of SHORT syndrome, while, more generally, linear growth impairment is common in APDS2, but not in APDS1.These clinical observations are bolstered by the impaired linear growth and increased in utero mortality reported in mice with knock in of the common causal APDS2 mutation in Pik3r1 (Nguyen et al., 2023).All people with SHORT-APDS2 overlap syndromes have well established activating PIK3R1 mutations in the inter-SH2 domain implicated in APDS2, but none have characteristic SHORT syndrome mutations, which are usually in the C-terminal SH2 domain.Conversely, no patient with a classical SHORT syndrome mutation has been shown to have immunodeficiency.There is thus now convincing evidence of a syndrome with features of both gain and loss of PI3K function.The mechanistic basis of this is unexplained.PI3K activity is determined not just by activity of individual PI3K holoenzymes, but also by subunit stoichiometry.Regulatory and catalytic subunits stabilise each other upon binding (Brachmann et al, 2005;Yu et al, 1998), and only regulatory subunit monomers are stable enough to be detected in mammalian cells (Yu et al., 1998).Molar excess of regulatory subunits over catalytic subunits gives rise to free regulatory subunits that compete with holoenzyme for phosphotyrosine binding (Ueki et al, 2002).This has been invoked to explain increased insulin sensitivity on knockout or reduction of p85α alone (Mauvais-Jarvis et al, 2002), of p55α and p50α (Chen et al, 2004) or of all Pik3r1 products (Fruman et al, 2000).
The "free-p85" model has been contested by some reports suggesting no excess p85 (Geering et al, 2007;Zhao et al, 2006), however most observations, including some made recently using in vivo tagging and pulldown of PI3K components (Tsolakos et al, 2018) suggest that monomeric p85α can be seen in vivo in some tissues.
We now address mechanisms underlying human mixed gain-and loss-of function phenotypes observed in association with PIK3R1 mutations using primary cells, cell models and in vitro enzyme assay and suggest that they are explained by competing effects of APDS2 PIK3R1 mutations on PI3K activity and stability.

PI3K subunit expression and signalling in APDS2 fibroblasts
Disorders in the PIK3CA-related overgrowth spectrum (PROS) feature soft tissue overgrowth and are caused by mosaic activating mutations in PIK3CA, encoding the p110α catalytic subunit of PI3Kα (Madsen et al, 2018).In this group, basal hyperactivation of PI3Kα signalling can be easily discerned in dermal fibroblasts from affected areas of the body (Lindhurst et al, 2012).PIK3R1, like PIK3CA, is ubiquitously expressed, yet overgrowth is not reported in APDS2 caused by constitutional activating mutations in PIK3R1.Dermal fibroblasts strongly express PIK3R1, but no studies to date have evaluated whether PI3K activity is increased in APDS2.
We began by assessing dermal fibroblasts cultured from a previously described woman with APDS2 due to the common causal PIK3R1 mutation in the splice donor site that causes skipping of exon 11 and an in-frame deletion in the inter-SH2 domain shared by all PIK3R1 isoforms (Patient A.1 in (Lucas et al., 2014b)).These cells were compared to cells from healthy control volunteers, or from people with PROS.Confirmation of expression of all pathogenic mutations was undertaken by cDNA sequencing prior to further study (Fig

EV1A
).We found that truncated p85α was expressed, but at a much lower level than full Previous studies have suggested that the truncated, APDS2-causal p85α variant exerts a much greater activating effect on p110δ, which has a more restricted tissue expression, including immune cells, than p110α, which is ubiquitous (Dornan et al., 2017).We found that both p110α and p110δ were easily detectable in control dermal fibroblasts, however p110δ was almost absent in APDS2 fibroblasts compared to controls, with lower levels also seen in

Overexpressed PIK3R1 ΔExon11 is potently dominant negative in 3T3-L1 preadipocytes
We next turned to a well-established cellular system allowing conditional overexpression of PK3R1 alleles of interest.We have previously shown that overexpression of two SHORT syndrome PIK3R1 variants -R649W and Y657X -impair insulin signalling and adipocyte differentiation of murine 3T3-L1 preadipocytes, consistent with impaired PI3Kα function and with the lipodystrophy and insulin resistance seen in SHORT syndrome (Huang-Doran et al, 2016).To assess the effect of the APDS2 ΔEx11 in the same, non-immunological, cell context, we used lentiviral vectors, as previously described (Huang-Doran et al., 2016), to generate clonal 3T3-L1 cell lines allowing conditional, tuneable overexpression of PIK3R1 variants in response to doxycycline.For signalling studies, we also generated cells  et al., 2016).
In undifferentiated cells, we first confirmed doxycycline-dependent overexpression of p85α To assess whether the inhibitory effect of ΔEx11 p85α might be an artefact caused by strong overexpression, doxycycline titration was used to assess whether a low level of overexpression might unmask signalling hyperactivation.However, no such hyperactivation was seen, and instead, a graded diminution of insulin-induced AKT phosphorylation was observed from the lowest to highest levels of p85α ΔEx11 overexpression (Fig EV4).

Effect of PIK3R1 mutations on Phosphoinositide 3-kinase activity in vitro
Given this evidence that APDS2-associated PIK3R1 ΔEx11 potently inhibits PI3Kα when overexpressed in 3T3-L1 preadipocytes, we next sought to investigate the biochemical basis of this observation.First, we assessed the effect of disease-causing PIK3R1 mutations on basal and phosphotyrosine-stimulated activity of purified PI3Kα, β and δ holoenzyme in a previously described reconstituted in vitro system (Burke et al, 2012).As well as the APDS2 ΔEx11 mutation, we selected three SHORT syndrome-associated mutations to study.These were the most common causal mutation, R649W, which abolishes phosphotyrosine binding by the C-terminal SH2 (cSH2) domain (Chudasama et al., 2013), Y657X, which truncates the cSH2 domain (Huang-Doran et al., 2016;Kwok et al, 2020), and E489K which, atypically, lies in the inter-SH2 domain where most cancer, overgrowth and APDS2associated mutations lie (Thauvin-Robinet et al., 2013).PIK3R1 E489K-containing primary cells were previously suggested to show basal hyperactivation (Thauvin-Robinet et al., 2013).
Wild-type and SHORT syndrome mutant holoenzymes were successfully purified for in vitro assay, but despite multiple attempts, ΔEx11 holoenzyme could only be made in small amounts under identical conditions, and moreover was unstable on storage, precluding further study.In keeping with previous reports (Chudasama et al., 2013;Dornan et al., 2017;Dornan et al, 2020), p85α R649W showed severely reduced phosphotyrosine-stimulated activity in complex with p110α, with highly significant but lesser loss of function seen for

Binding of p110α by mutant p85α
Given the potent dominant negativity of APDS2-related p85α ΔEx11 in cells, and the instability of p85α ΔEx11-containing PI3K holoenzyme in vitro, we next used immunoprecipitation to assess binding of p110α by this and other p85α variants in cells.
p85α was easily detected in anti-p110α immunoprecipitates from all cell lines at baseline (Fig 4).No increase was seen on conditional overexpression of wild-type or R649W p85α.
Given the known instability of monomeric p110α, this suggests that all p110α is bound to p85α before overexpression.Although wild-type endogenous p85α may have been replaced by heterologously overexpressed p85α in these cells, this could not be detected without a size difference of the variant from wild-type.For cells overexpressing the SHORT syndromeassociated p85α Y657X, the truncated variant was strongly co-immunoprecipitated, accounting for nearly all of the p85α signal in anti-p110α immunoprecipitate.This This argues against destabilisation of p110α as the mechanism of the observed dominant negative activity.

Effect of PIK3R1 mutations on insulin-induced PI3K recruitment to IRS1/2
As APDS2 p85α ΔEx11 does not appear to displace wild-type p85α from p110α, despite strong overexpression, it is likely that there are high levels of the monomeric truncated p85α in the cell.This may be important as it is p85α that mediates recruitment of PI3K to activated tyrosine kinase receptors and their tyrosine phosphorylated substrates, including the insulin-receptor substrate proteins Irs1 and Irs2.Excess free regulatory subunits compete with heterodimeric PI3K holenzyme (Ueki et al., 2002), raising the possibility that excess free, truncated APDS2 p85α ΔEx11 may exert its inhibitory action by competing with PI3K holenzyme.
To assess this possibility, we again used the 3T3-L1 cellular model to determine whether overexpression of disease-causing p85α variants impairs recruitment of p110α to Irs1 and

Discussion
Murine genetic studies of Pik3r1 have proved complicated to interpret, due in part to functional redundancy among PI3K regulatory subunits, and in part to unanticipated effects of perturbing PI3K subunit stoichiometry.This has left several important questions about in vivo functions of Pik3r1 unresolved.Identification of a series of human genetic disorders caused by constitutional PIK3R1 mutations over the past 10 years has given fresh impetus to the field and has been mechanistically illuminating.
Homozygous truncating PIK3R1 mutations abolishing p85α expression while preserving p55α and p50α produce agammaglobulinaemia (Conley et al, 2012;Tang et al, 2018).This resembles the immunodeficiency reported in Pik3r1 knockout mice (Fruman et al, 1999;Suzuki et al, 1999) and suggests an essential, non-redundant function of p85α in B cell development in humans.Thereafter, human genetics has provided more novel insights.
PIK3R1 mutations were identified in SHORT syndrome in 2013 (Chudasama et al., 2013;Dyment et al., 2013;Thauvin-Robinet et al., 2013), nearly all in the C-terminal SH2 domain, which together with the N-terminal SH2 domain enables PI3K recruitment to activated RTKs (Rordorf-Nikolic et al, 1995).SHORT syndrome features short stature and insulin resistance consistent with impaired ligand-induced p110α action, a phenotype distinct from the enhanced insulin sensitivity produced by genetic ablation of one or more Pik3r1 products in mice (Chen et al., 2004;Fruman et al., 2000;Mauvais-Jarvis et al., 2002).Mice with SHORT syndrome mutations knocked in were generated after human findings, and faithfully reproduce the human phenotype (Kwok et al., 2020;Solheim et al, 2018;Winnay et al, 2016), confirming that expression of a signalling-impaired PIK3R1 has different consequences to Pik3r1 knockout.No immunodeficiency has been described associated with classic SHORT syndrome mutations, however, suggesting a selective effect on PI3Kα, but the basis of this selectivity has not previously been investigated.
In contrast to SHORT syndrome, mutations in the inter SH2 domain of PIK3R1, mostly leading to skipping of exon 11, were shown in 2014 to activate PI3K in vitro and to cause immunodeficiency (APDS2) (Deau et al., 2014;Lucas et al., 2014b) similar to that caused by activating mutations in p110δ (APDS1) (Angulo et al., 2013;Lucas et al., 2014a).Neither overgrowth nor metabolic features of APDS2 have been described to suggest p110α hyperactivation, and indeed short stature is common in APDS2 (Elkaim et al., 2016;Jamee et al., 2020;Maccari et al., 2023;Olbrich et al., 2016;Petrovski et al., 2016), with a growing number of APDS2 patients described with features of SHORT syndrome (Bravo Garcia-Morato et al., 2017;Maccari et al., 2023;Nguyen et al., 2023;Petrovski et al., 2016;Ramirez et al., 2020;Szczawinska-Poplonyk et al., 2022).Moreover mice with the common APDS2 causal Pik3r1 variant knocked in show impaired growth and in utero survival, unclike APDS2 murine models (Nguyen et al., 2023).Thus, study of distinct PIK3R1-related syndromes shows that impaired signalling caused by PIK3R1 mutations produces phenotypes related to impaired PI3Kα function, without affecting PI3Kδ.Conversely, mutations of PIK3R1 that produce phenotypes related to increased PI3Kδ signalling do not increase PI3Kα signalling.Indeed, they surprisingly have features characteristic of impaired PI3Kα function.
Lack of overgrowth in APDS2 has been attributed to greater ability of APDS2 PIK3R1 variants to activate p110δ than p110α (Dornan et al., 2017), but the co-ocurrence of gainand loss-of function phenotypes has not been explained to date.Our findings suggest that the explanation may lie in competing effects of APDS2 PIK3R1 variants to activate PI3Kδ on one hand, as previously shown (Angulo et al., 2013;Dornan et al., 2017;Lucas et al., 2014b), while on the other hand interfering with PI3Kα through the dominant negative effect of non p110α-bound mutant p85α.The low expression of truncated p85α ΔEx11 we described in dermal fibroblasts is similar to observations made in lymphocytes, however, in lymphocytes this is associated with increased basal AKT phosphorylation (Deau et al., 2014;Lucas et al., 2014b) that is abolished by p110δ inhibition (Lucas et al., 2014b).P110δ is also expressed in fibroblasts, but protein levels were reduced in the cells studied, consistent with the previously reported observation that when expressed in insect cells the PI3K holoenzyme containing p85α ΔEx11 has a low yield and is unstable (Dornan et al., 2017).We speculate that in cells with low endogenous p110δ protein expression, the destabilising effect of mutant PIK3R1 predominates over its activating effect.
The balance between expression and signalling may be a fine one, however, as transient overexpression of FLAG-tagged p85α ΔEx11 did increase AKT phosphorylation in 3T3 fibroblasts in a previous study, although expression of p110α, β and δ was not determined (Deau et al., 2014).We find, in contrast, that overexpression of untagged p85α ΔEx11 has a strong inhibitory effect on insulin signalling, and we were unable to identify a window of overexpression where increased AKT phosphorylation could be observed.We further demonstrated that most or all of the mutant p85α expressed is not bound to p110α, while unbound mutant p85α still binds to Irs1/2, effectively competing with PI3K holoenzyme.The observation that p85α ΔEx11 can associate with Irs1/2 is in agreement with reports that p85α ΔEx11-containing PI3Kα and PI3Kδ can be stimulated by pY2-peptides (Dornan et al., 2017;Dornan et al., 2020) and that p85α ΔEx11 is recruited to tyrosine-phosphorylated LAT in T cells (Lucas et al., 2014b).The competition we suggest between unbound mutant p85α and PI3K holoenzyme for binding to the activated RTKs is in keeping with longstanding evidence that free p85 downregulates PI3K signalling through the same competitive mechanism (Thorpe et al, 2017;Ueki et al., 2002), with a single study suggesting in addition that overexpression of tagged p85α leads to Irs1 sequestration with free p85α in cytosolic foci where PI(3,4,5)P3 production does not occur (Luo et al, 2005).
The current study has limitations.We have studied primary cells from only a single APDS2 patient, and in the 3T3-L1 cell model, we did not determine whether p110δ protein could be detected.If not, this could explain the lack of detectable AKT phosphorylation with induction of Pik3r1 ΔEx11.Our study moreover raises further questions.Full length p85α subunits can homodimerize (Cheung et al, 2015;Harpur et al, 1999;LoPiccolo et al, 2015), and it is speculated that homodimers may outcompete p85α/p110 heterodimers for binding to activated Irs1 due to configuration of the four SH2 domains.In the 3T3-L1 preadipocyte APDS2 models, p85α ΔEx11 expression was high despite impaired p110α association, but whether this is composed of monomeric or homodimeric p85α, and whether mutant p85α expression leads to sequestration of Irs1 remote from the insulin receptor, as previously suggested for tagged wild-type p85α, is undetermined.
In summary, it is already established that: A. genetic activation of PIK3CD causes immunodeficiency without disordered growth, while B. inhibition of PIK3R1 recruitment to RTKs and their substrates impairs growth and insulin action, without immunodeficiency, despite all catalytic subunits being affected and C. loss of p85α alone causes immunodeficiency.The current study, coupled with prior reports, suggests that the common APDS2 mutation in PIK3R1 has mixed consequences, producing greater hyperactivation of p110δ than p110α, based on subtle differences in the inhibitory interactions of regulatory and catalytic subunits, while also destabilising PI3K holoenzyme and exerting dominant negative activity on wild-type PIK3R1 function.We suggest that these competing activating and inhibitory consequences are finely balanced, potentially differing among tissues, leading to mixed clinical profiles of gain-and loss-of function features.The patient studied gave written informed consent for NIH IRB-approved research protocols 06-I-0015 and 09-I-0133 and was reported previously (Lucas et al., 2014b).Dermal fibroblasts were isolated from a skin punch biopsy and cultured as previously described (Huang-Doran et al., 2016).

Baculovirus Generation
p85α point mutation expression constructs were created by site-directed mutagenesis of a pACEBac1_Homo sapiens p85α plasmid using a QuikChange II XL Site-Directed Mutagenesis Kit.In-Fusion cloning was used to generate pACEBac1_p85α ΔEx11.Purified plasmid inserts were sequenced and verified by restriction enzyme digest, exploiting loss of a DpnI site within excised exon 11. pACEBac1_p85α and pFastBac™HT B plasmids encoding N terminally tandem His-tagged p110 subunits were used to generate bacmid DNA.Bacmids were expanded using MAX Efficiency DH10Bac™ Competent or EMBacY cells and 2-4μg was transfected into Sf9 cells using Insect-XPRESS (Promega E2311).Pooled virus from low titre supernatant (P1 stock) was used to produce high titre P2 stock by transfection of 1.5x10 6 Sf9 cells/mL in 450mL using.For catalytic subunits, 1-2mL P2 stock was used for a further round of Sf9 transfection and expansion to generate P3 stock.

Purification of PI3K holoenzymes
1.5-2L of 1.5x10 6 Sf9 cells/mL were co-infected with 18mL P3 catalytic subunit and 4mL P2 regulatory subunit baculovirus.Cells were cultured at 27°C, harvested 48 h post-infection, pelleted, washed and stored at -80°C.Pellets were later lysed by sonication before ultracentrifugation.p85α/p110 heterodimers were pulled down via the tandem p110 Nterminal His tags, preventing purification of monomeric p85α, using tandem Ni 2+ HisTrap Fast Flow columns.Further purification utilised a heparin HiTrap Q HP column.Eluted fractions were concentrated to ≥1mg/mL and 1mL concentrated fractions were gel filtered on Superdex 200 16/60 columns.The p110 tandem His tag was retained for proteins used in functional analyses.Purity of eluted complexes was verified by SDS-PAGE, and purified proteins were quantified using a Nanodrop 1000 Spectrophotometer (Thermo Scientific), with concentrations calculated using the molecular extinction coefficient (assuming full cysteine reduction) determined by sequence input to the ProtParam tool (ExPASy).Total injection volume onto gel filtration columns was kept at 1 mL.Yields of PI3K complexes were determined using the area under the curve (280nm mAU per mL eluted protein) from gel filtration chromatograms, and normalised to the volume of Sf9 cells infected.

Concentrations of thawed protein aliquots were assessed as 280 nm absorbance by
Nanodrop 1000 Spectrophotometer (Thermo Scientific), and samples of preparations used in assays were resolved by SDS-PAGE to confirm equal amounts of each complex.Three technical replicates were used per assay on at least three different occasions.Relevant wildtype p85α/p110 catalytic isoforms were included in each assay as controls.Other controls are detailed in the Appendix.Standard curves of diC8-PI(3,4,5)P 3 in the presence of liposomes were included in every experiment and used to infer PIP 3 generated by purified PI3K.Standard curves for each experiment provided the linear range within which PIP 3 could be accurately determined.Quadruplicate interpolated PIP 3 concentrations were averaged and normalised by reaction time (30 min) and enzyme concentration in pM to yield PI3K activity in pmol PIP 3 /min/pM enzyme.Specific activities were then further normalised to activity of p85α wild-type controls in the presence of pY2 for each experiment.

Insulin signalling studies
3T3-L1 cells serum starved in DMEM containing 0.5 % BSA for 16h were stimulated with 100nM Actrapid insulin (Novo Nordisk) for 10min.Monolayers were snap frozen in liquid N2 and stored at -80°C.200-600μL co-IP lysis or RIPA buffer was added to frozen before scraping of lysate into pre-chilled tubes, incubation on ice for 30min and clearing by centrifugation.Protein was quantified using the Bio-Rad DC assay.
Lysates mixed with NuPAGE® SDS Loading bufferwith 5% β-mercaptoethanol were boiled before SDS-PAGE, with 10μg boiled lysate used to represent co-immunoprecipitation (Co-IP) input.For Co-IP, lysates were incubated with immunoprecipitating antibody overnight at 4°C. before incubation for 2h with Protein G Dynabeads®, followed by centrifugation and bead removal using a DynaMag™-2 Magnet.Supernatants mixed 1:1 with Co-IP Elution Buffer were boiled for 10 min.Protein was eluted from washed beads and boiled for 10min.
Input, supernatant and co-IP samples underwent SDS-PAGE using NuPAGE® 4-12% gradient Bis-Tris gels in NuPAGE® MOPS SDS Running Buffer.Transfer to nitrocellulose was performed using the iBlot™ Dry Blotting System (Invitrogen), with preincubation in Equilibration Transfer Buffer for proteins above 150kDa.
For immunoblotting, blocked membranes were incubated overnight at 4°C in primary antibody, washed and incubated with HRP-linked anti-rabbit or anti-mouse IgG secondary antibody diluted 1:5000 in blocking buffer.Proteins were visualized using the ChemiDoc™ MP System (Bio-Rad) and band intensities quantified using Image Lab software (Bio-Rad).

Statistical Analysis
For quantitative data, all biological replicates (i.e.results of independent experiments on different days) are represented in figures, with paired points (e.g.with/without insulin in the same experiment) connected by lines.For fluorescence polarisation assays, each biological replicate shown is the mean of 3 technical replicates.Sample size for individual experiments was not pre-determined.To avoid the pitfalls of dichotomous significance testing on lowthroughput biological datasets, we used estimation statistics (Data Analysis using Bootstrap-Coupled ESTimation) with default settings (5000 resamples, BCa boostrap) (Ho et al, 2019).
This focuses on effect sizes and derives confidence intervals derived from bootstrapping for differences in means; the small (but typical) sample size of the experiments analysed limits reliable bootstrapping, but it offers additional confidence to the consistent patterns seen in .CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.It is made The copyright holder for this preprint this version posted November 2, 2023.; https://doi.org/10.1101/2023.11.02.565250 doi: bioRxiv preprint independent replicates.We have indicated significance in main text figures with an asterisk, and show the 95% confidence intervals for mean differences in extended view figures.
Expanded details of methods, including sources and concentrations of antibodies, and buffer compositions, are listed in the Appendix.inhibiting PI3Kα activity.We suggest that this explains the mixed PI3K gain-and loss-of function features that co-exist in APDS2.Our findings imply that PI3Kδ inhibition alone may not be sufficient to reverse all components of APDS2, and that additional therapeutic strategies targeting the mutant PI3KR1 products may be warranted.Exposure was for 72 hours in all cases.The truncated p85α variant can be seen below the WT p85α for the APDS2 ΔEx11 mutant.

For
length wild-type p85α (Fig 1A, FigEV1B,C).Despite this, no increase in basal or insulinstimulated AKT phosphorylation was seen in APDS2 cells compared to cells from wild-type volunteers or from people with PROS and activating PIK3CA mutations H1047L or H1047R (Fig 1A-C, Fig EV2A,B).Indeed, insulin-induced phosphorylation of AKT was lower in fibroblasts from this APDS2 patient compared to controls.
PROS cells (Fig1A,D, Fig EV2D).p110α was unchanged in APDS2 cells but modestly increased in PROS cells (Fig 1A,E, Fig EV2C).Collectively these findings suggest that any ability of the APDS2 PIK3R1 variant in skin cells to activate PI3K may be overcome by reduced protein levels of p110δ, likely through reduced binding and/or reduced stabilisation of p110δ by the mutant regulatory subunit.
or p110α transgenes (Fig 2A), before assessing basal and insulin-stimulated Akt phosphorylation.As expected, overexpression of oncogenic H1047R p110α strongly increased basal Akt phosphorylation (Fig 2A-C, Fig EV3A,C) with no additional increase on insulin stimulation (Fig 2A-C, Fig EV3B,D).Surprisingly, however, not only did overexpression of the APDS2 ΔEx11 p85α not increase basal PI3Kα signalling, but it potently inhibited insulin-induced Akt phosphorylation, consistent with a strong dominant negative action on pathway activation (Fig 2A-C, Fig EV3).Overexpressing wild-type p85α mildly reduced insulin-stimulated Akt phosphorylation (Fig 2A-C, Fig EV3B,C), although WT p85α was not expressed to as high a level as ΔEx11 p85α.In keeping with impaired PI3Kα activity, overexpression of the ΔEx11 variant also severely impaired adipocyte differentiation, assessed by triglyceride accumulation in response to a standard differentiation protocol (Fig 2D).A similar effect was seen on overexpressing SHORT syndrome variants R649W and Y657X (Fig 2D).
Y657X and E489K (Fig 3A, Fig EV5A).No increase in basal activity was seen for any variant.We also assessed whether any of the SHORT syndrome variants affect function of PI3Kβ and found that all variants impaired phosphotyrosine-stimulated activity of PI3Kβ.Again, this impairment was less for p85α Y657X than for R649W, and in this case only very mild for E489K (Fig 3B, Fig EV5B).For PI3Kδ, p85α R649W conferred severe loss of function, as for other isoforms (Fig 3C, Fig EV5C), suggesting that the absence of immunodeficiency in SHORT syndrome is not accounted for by selective inhibition of PI3Kα function by causal mutations.
demonstrates preserved binding of p110α by mutant p85α (Fig 4).In sharp contrast, although truncated p85α ΔEx11 was easily detected in cell lysates before immunoprecipitation and in supernatant after immunoprecipitation (arrowed in Fig 4), no truncated p85α ΔEx11 was seen in p110α immunoprecipitates, and no change in p110α expression was detected (Fig 4).This suggests that this truncated APDS2 causal variant does not supplant endogenous, full-length p85α binding to p110α, despite overexpression.

Irs2.
Irs1 was immunoprecipitated with or without conditional p85α overexpression and with or without insulin stimulation.Overexpression of wild-type p85α had no effect on basal or insulin-induced p110α recruitment to Irs1 (Fig 5A,B, Fig EV6A,B).In contrast, overexpression of either p85α R649W or Y657X sharply reduced insulin-stimulated p110α recruitment to Irs1 on insulin stimulation (Fig 5A,B, Fig EV6A,B).In keeping with the inability of the R649W cSH2 domain to bind phosphotyrosines, p85α R649W was also not recruited to Irs1, while overexpression of Y657X increased stimulated but not basal p85α recruitment (Fig 5A,C, Fig EV6C,D).This suggests a pure defect in PI3K holoenzyme recruitment for R649W.For Y657X the signalling defect may have mixed mechanisms, with reduced activation by phosphotyrosine seen in in vitro studies coupled to increased abundance of monomeric mutant p85α, leading to recruitment of non p110α-bound mutant p85α to Irs1.In keeping with our finding of severely attenuated insulin signalling upon p85α ΔEx11 overexpression (Fig 2), overexpression of this mutant p85α abolished p110α recruitment to Irs1 (Fig 5A,B, Fig EV6A,B).However, non p110α-bound p85α ΔEx11 was strongly recruited to Irs1 even in the absence of insulin stimulation (Fig 5A,C, Fig EV6C,D).This suggests that although p85α ΔEx11 does not effectively compete with wild-type p85α for binding to p110α, it has preserved or possibly enhanced ability to bind Irs1.This gives the mutant p85α properties that render it a more potent endogenous inhibitor of PI3K signalling

Figure 4 .
Figure 4. Ability of pathogenic p85α variants to bind p110α, assessed by co-

Figure 5 .
Figure 5. Attenuated insulin-induced association of p110α with Irs1 in the presence of

Figure EV3 .
Figure EV3.Full statistical analysis of data presented in main Figure 2. Analysis of the effects of doxycycline-induced expression of wild-type (WT) or ΔEx11 (APDS2) p85α, or of

Figure EV4 .
Figure EV4.The effect of graded expression of wild-type or disease-associated p85α

Figure EV5 .
Figure EV5.Full statistical analysis of data presented in main Figure 3. Analysis of fluorescence polarisation assay of phosphoinositide 3-kinase (PI3K) activity of in vitro synthesised wild-type (WT) or mutant (E489K, R649W or Y657X) p85α.Results for p110α-

Figure EV6 .
Figure EV6.Full statistical analysis of data presented in main Figure 5. Analysis of the effects of doxycycline (dox)-induced expression of wild-type (WT), Y657X, R649W or ΔEx11

Figure EV7 .
Figure EV7.Attenuated insulin-induced association of p110α with Irs2 in the presence

Figure EV8 .
Figure EV8.Full statistical analysis of data presented in Figure EV7.Analysis of the effects of doxycycline (dox)-induced expression of wild-type (WT), Y657X, R649W or ΔEx11 (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.It is made (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.It is made (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.It is made (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.It is made (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.It is made (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.It is made (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.It is made (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.It is made