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Different inhibition of G??-stimulated class IB phosphoinositide 3-kinase (PI3K) variants by a monoclonal antibody. Specific function of p101 as a G??-dependent regulator of PI3K? enzymatic activity.

ABSTRACT: Class IB phosphoinositide 3-kinases ? (PI3K?) are second-messenger-generating enzymes downstream of signalling cascades triggered by G-protein-coupled receptors (GPCRs). PI3K? variants have one catalytic p110? subunit that can form two different heterodimers by binding to one of a pair of non-catalytic subunits, p87 or p101. Growing experimental data argue for a different regulation of p87-p110? and p101-p110? allowing integration into distinct signalling pathways. Pharmacological tools enabling distinct modulation of the two variants are missing. The ability of an anti-p110? monoclonal antibody [mAb(A)p110?] to block PI3K? enzymatic activity attracted us to characterize this tool in detail using purified proteins. In order to get insight into the antibody-p110? interface, hydrogen-deuterium exchange coupled to MS (HDX-MS) measurements were performed demonstrating binding of the monoclonal antibody to the C2 domain in p110?, which was accompanied by conformational changes in the helical domain harbouring the G??-binding site. We then studied the modulation of phospholipid vesicles association of PI3K? by the antibody. p87-p110? showed a significantly reduced G??-mediated phospholipid recruitment as compared with p101-p110?. Concomitantly, in the presence of mAb(A)p110?, G?? did not bind to p87-p110?. These data correlated with the ability of the antibody to block G??-stimulated lipid kinase activity of p87-p110? 30-fold more potently than p101-p110?. Our data argue for differential regulatory functions of the non-catalytic subunits and a specific G??-dependent regulation of p101 in PI3K? activation. In this scenario, we consider the antibody as a valuable tool to dissect the distinct roles of the two PI3K? variants downstream of GPCRs.

SUBMITTER: Shymanets A

PROVIDER: S-EPMC4655608 | biostudies-literature | 2015 Jul

REPOSITORIES: biostudies-literature

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Different inhibition of Gβγ-stimulated class IB phosphoinositide 3-kinase (PI3K) variants by a monoclonal antibody. Specific function of p101 as a Gβγ-dependent regulator of PI3Kγ enzymatic activity.

Shymanets Aliaksei A Prajwal Vadas Oscar O Czupalla Cornelia C LoPiccolo Jaclyn J Brenowitz Michael M Ghigo Alessandra A Hirsch Emilio E Krause Eberhard E Wetzker Reinhard R Williams Roger L RL Harteneck Christian C Nürnberg Bernd B

The Biochemical journal 20150701 1

Class IB phosphoinositide 3-kinases γ (PI3Kγ) are second-messenger-generating enzymes downstream of signalling cascades triggered by G-protein-coupled receptors (GPCRs). PI3Kγ variants have one catalytic p110γ subunit that can form two different heterodimers by binding to one of a pair of non-catalytic subunits, p87 or p101. Growing experimental data argue for a different regulation of p87-p110γ and p101-p110γ allowing integration into distinct signalling pathways. Pharmacological tools enabling ...[more]

PMID: 26173259

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Project description:We have addressed the differential roles of class I Phosphoinositide 3-kinases (PI3K) in human breast-derived MCF10a (and iso-genetic derivatives) and MDA-MB 231 and 468 cells. Class I PI3Ks are heterodimers of p110 catalytic (?, ?, ? and ?) and p50-101 regulatory subunits and make the signaling lipid, phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) that can activate effectors, eg protein kinase B (PKB), and responses, eg migration. The PtdIns(3,4,5)P3-3-phosphatase and tumour-suppressor, PTEN inhibits this pathway. p110?, but not other p110s, has a number of onco-mutant variants that are commonly found in cancers. mRNA-seq data shows that MCF10a cells express p110?>>?>? with undetectable p110?. Despite this, EGF-stimulated phosphorylation of PKB depended upon p110?-, but not ?- or ?- activity. EGF-stimulated chemokinesis, but not chemotaxis, was also dependent upon p110?, but not ?- or ?- activity. In the presence of single, endogenous alleles of onco-mutant p110? (H1047R or E545K), basal, but not EGF-stimulated, phosphorylation of PKB was increased and the effect of EGF was fully reversed by p110? inhibitors. Cells expressing either onco-mutant displayed higher basal motility and EGF-stimulated chemokinesis.This latter effect was, however, only partially-sensitive to PI3K inhibitors. In PTEN(-/-) cells, basal and EGF-stimulated phosphorylation of PKB was substantially increased, but the p110-dependency was variable between cell types. In MDA-MB 468s phosphorylation of PKB was significantly dependent on p110?, but not ?- or ?- activity; in PTEN(-/-) MCF10a it remained, like the parental cells, p110?-dependent. Surprisingly, loss of PTEN suppressed basal motility and EGF-stimulated chemokinesis. These results indicate that; p110? is required for EGF signaling to PKB and chemokinesis, but not chemotaxis; onco-mutant alleles of p110? augment signaling in the absence of EGF and may increase motility, in part, via acutely modulating PI3K-activity-independent mechanisms. Finally, we demonstrate that there is not a universal mechanism that up-regulates p110? function in the absence of PTEN.

Project description:Background Endometrial cancer (EC) is a common gynecologic malignancy and patients with advanced and recurrent EC have a poor prognosis. Although chemotherapy is administered for those patients, the efficacy of current chemotherapy is limited. Therefore, it is necessary to develop novel therapeutic agents for EC. In this study, we focused on lipolysis-stimulated lipoprotein receptor (LSR), a membrane protein highly expressed in EC cells, and developed a chimeric chicken–mouse anti-LSR monoclonal antibody (mAb). This study investigated the antitumor effect of an anti-LSR mAb and the function of LSR in EC. Methods We examined the expression of LSR in 228 patients with EC using immunohistochemistry and divided them into two groups: high-LSR (n = 153) and low-LSR groups (n = 75). We developed a novel anti-LSR mAb and assessed its antitumor activity in an EC cell xenograft mouse model. Pathway enrichment analysis was performed using protein expression data of EC samples. LSR-knockdown EC cell lines (HEC1 and HEC116) were generated by transfected with small interfering RNA and used for assays in vitro. Results High expression of LSR was associated with poor overall survival (hazard ratio: 3.53, 95% confidence interval: 1.35–9.24, p = 0.01), advanced stage disease (p = 0.045), deep myometrial invasion (p = 0.045), and distant metastasis (p < 0.01). In EC with deep myometrial invasion, matrix metalloproteinase (MMP) 2 was highly expressed along with LSR. Anti-LSR mAb significantly inhibited the tumor growth in EC cell xenograft mouse model (tumor volume, 407.1 mm3versus 726.3 mm3, p = 0.019). Pathway enrichment analysis identified the mitogen-activated protein kinase (MAPK) pathway as a signaling pathway associated with LSR expression. Anti-LSR mAb suppressed the activity of MAPK in vivo. In vitro assays using EC cell lines demonstrated that LSR regulated cell proliferation, invasion, and migration through MAPK signaling, particularly MEK/ERK signaling and membrane-type 1 MMP (MT1-MMP) and MMP2. Moreover, ERK1/2-knockdown suppressed cell proliferation, invasion, migration, and the expression of MT1-MMP and MMP2. Conclusions Our results suggest that LSR contributes to tumor growth, invasion, metastasis, and poor prognosis of EC through MAPK signaling. Anti-LSR mAb is a potential therapeutic agent for EC. Supplementary Information The online version contains supplementary material available at 10.1186/s12885-022-09789-6.

Dataset Information

Different inhibition of G??-stimulated class IB phosphoinositide 3-kinase (PI3K) variants by a monoclonal antibody. Specific function of p101 as a G??-dependent regulator of PI3K? enzymatic activity.

Publications

Different inhibition of Gβγ-stimulated class IB phosphoinositide 3-kinase (PI3K) variants by a monoclonal antibody. Specific function of p101 as a Gβγ-dependent regulator of PI3Kγ enzymatic activity.

Similar Datasets

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