p87 and p101 Subunits Are Distinct Regulators Determining Class IB Phosphoinositide 3-Kinase (PI3K) Specificity

Shymanets, Aliaksei; Prajwal,; Bucher, Kirsten; Beer‐Hammer, Sandra; Harteneck, Christian; Nürnberg, Bernd

doi:10.1074/jbc.m113.508234

Cited by 43 publications

(62 citation statements)

References 51 publications

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“…This is in sharp contrast to the 552 DD-p110γ/p101 complex ( Fig. 2A), suggesting that p87 makes much less contribution to Gβγ interaction compared with p101, which is consistent with previous reports (22,45,46). (28,36,45,47).…”

Section: Resultssupporting

confidence: 53%

Molecular determinants of PI3Kγ-mediated activation downstream of G-protein–coupled receptors (GPCRs)

Vadas

Dbouk²,

Shymanets

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Phosphoinositide 3-kinase gamma (PI3Kγ) has profound roles downstream of G-protein-coupled receptors in inflammation, cardiac function, and tumor progression. To gain insight into how the enzyme's activity is shaped by association with its p101 adaptor subunit, lipid membranes, and Gβγ heterodimers, we mapped these regulatory interactions using hydrogen-deuterium exchange mass spectrometry. We identify residues in both the p110γ and p101 subunits that contribute critical interactions with Gβγ heterodimers, leading to PI3Kγ activation. Mutating Gβγ-interaction sites of either p110γ or p101 ablates G-protein-coupled receptor-mediated signaling to p110γ/p101 in cells and severely affects chemotaxis and cell transformation induced by PI3Kγ overexpression. Hydrogen-deuterium exchange mass spectrometry shows that association with the p101 regulatory subunit causes substantial protection of the RBD-C2 linker as well as the helical domain of p110γ. Lipid interaction massively exposes that same helical site, which is then stabilized by Gβγ. Membrane-elicited conformational change of the helical domain could help prepare the enzyme for Gβγ binding. Our studies and others identify the helical domain of the class I PI3Ks as a hub for diverse regulatory interactions that include the p101, p87 (also known as p84), and p85 adaptor subunits; Rab5 and Gβγ heterodimers; and the β-adrenergic receptor kinase.

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Section: Resultssupporting

confidence: 53%

Molecular determinants of PI3Kγ-mediated activation downstream of G-protein–coupled receptors (GPCRs)

Vadas

Dbouk²,

Shymanets

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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119

177

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“…Expression and Purification of Recombinant G␤ 1 His-␥ 2 -Recombinant purified G␤␥ dimers were produced as described elsewhere (48). Essentially, fall armyworm ovary cells (Sf9, from Gibco BRL, Eggenstein, Germany) were cultured in suspension with TNM-FH medium (Sigma, Deisenhofen, Germany) supplemented with 10% (v/v) fetal calf serum (Gibco), Lipid Medium Supplement (1:100; Sigma), penicillin (100 units/ml), and streptomycin (0.1 mg/ml).…”

Section: Methodsmentioning

confidence: 99%

p21-activated Kinases (PAKs) Mediate the Phosphorylation of PREX2 Protein to Initiate Feedback Inhibition of Rac1 GTPase

Barrows

Schoenfeld

Hodakoski

et al. 2015

Journal of Biological Chemistry

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Background:The role of phosphorylation for regulating the Rac1 GEF PREX2 is not understood. Results: PAK phosphorylation of PREX2 downstream of PIP 3 and G␤␥ reduces PREX2 GEF activity. Conclusion: Second messengers can initiate negative feedback to decrease Rac1 activation through PAK phosphorylation of PREX2. Significance: PAK negative regulation of GEFs could represent a broadly utilized mechanism to attenuate Rac1 activation and its outputs.

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“…The former view of p87 and p101 being redundant adapters in Gβγ-mediated recruitment of PI3Kγ variants to the membrane compartment [27–29] has been challenged by recent data showing a different contribution of Gβγ and Ras on the two PI3Kγ variants [38]. In particular, distinct Gβγ-binding affinities of the non-catalytic subunits for p110γ are intriguing [38,40,41]. These findings support data showing that PI3Kγ variants integrate into different and independent signalling cascades [39,42–44].…”

Section: Introductionmentioning

confidence: 99%

“…We have recently reported specific features for p87 and p101, such as diverse spatial and temporal distribution in human tissues and a different regulatory impact on p110γ activity, which may contribute to the differential regulation of the PI3Kγ variants [40,41]. These findings, in combination with the fact that only a single class I B catalytic subunit is present in cells led us to postulate that p87 and p101 serve as signal-discriminating regulatory subunits defining specific functions for both p87-p110γ and p101-p110γ variants [41]. However, the exact molecular mechanisms that maintain the specificity and selectivity of the two PI3Kγ variants are still unknown.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2015

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Class IB phosphoinositide 3-kinases (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 distinct modulation of the two variants are missing. The ability of an anti-p110γ monoclonal antibody (mAb(A)p110γ) to block PI3Kγ enzymatic activity attracted us to characterize this tool in detail using purified proteins. In order to get insight into the antibody-p110γ-interface, hydrogen-deuterium exchange coupled to mass spectrometry measurements were performed demonstrating binding of the monoclonal antibody to the C2 domain in p110γ, which was accompanied by conformational changes in the helical domain harbouring the Gβγ-binding site. We then studied the modulation of phospholipid vesicles association of PI3Kγ by the antibody. p87-p110γ showed a significantly reduced Gβγ-mediated phospholipid recruitment as compared with p101-p110γ. Concomitantly, in the presence of mAb(A)p110γ Gβγ did not bind to p87-p110γ. These data correlated with the ability of the antibody to block Gβγ-stimulated lipid kinase activity of p87-p110γ 30 times more potently than p101-p110γ. Our data argue for differential regulatory functions of the non-catalytic subunits and a specific Gβγ-dependent regulation of p101 in PI3Kγ activation. In this scenario, we consider the antibody as a valuable tool to dissect the distinct roles of the two PI3Kγ variants downstream of GPCRs.

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p87 and p101 Subunits Are Distinct Regulators Determining Class IB Phosphoinositide 3-Kinase (PI3K) Specificity

Cited by 43 publications

References 51 publications

Molecular determinants of PI3Kγ-mediated activation downstream of G-protein–coupled receptors (GPCRs)

Molecular determinants of PI3Kγ-mediated activation downstream of G-protein–coupled receptors (GPCRs)

p21-activated Kinases (PAKs) Mediate the Phosphorylation of PREX2 Protein to Initiate Feedback Inhibition of Rac1 GTPase

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

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