Structure of the phosphoinositide 3-kinase (PI3K) p110γ-p101 complex reveals molecular mechanism of GPCR activation.
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ABSTRACT:
SUBMITTER: Rathinaswamy MK
PROVIDER: S-EPMC8397274 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The class IB phosphoinositide 3-kinase (PI3K), PI3K, is a master regulator of immune cell function, and is a promising drug target for both inflammatory diseases and cancer. Critical to PI3K function is the association of the p110 catalytic subunit to either a p101 or p84 regulatory subunit, which mediates regulation by G-protein coupled receptors (GPCRs). Here, we report the first structure of a heterodimeric PI3K complex, p110-p101. This structure revealed a unique mode of assembly of catalytic and regulatory subunits distinct from that of other class I PI3K complexes. Multiple oncogenic mutations mapped to these novel interfaces led to increased activation by G. p101 mediates activation through its G binding domain, recruiting the complex to the membrane and allowing for engagement of a secondary site in p110. A nanobody that specifically binds to this p101-G interface blocks activation providing a novel tool to study p101-specific signaling events in vivo.
2021-09-22 | PXD025209 | Pride
Project description:Receptor-regulated class I phosphoinositide 3-kinases (PI3K) phosphorylate the membrane lipid phosphatidylinositol (PtdIns)-4,5-P2 to PtdIns-3,4,5-P3. This, in turn, recruits and activates cytosolic effectors with PtdIns-3,4,5-P3-binding pleckstrin homology (PH) domains, thereby controlling important cellular functions such as proliferation, survival, or chemotaxis. The class IB p110 gamma/p101 PI3K gamma is activated by G beta gamma on stimulation of G protein-coupled receptors. It is currently unknown whether in living cells G beta gamma acts as a membrane anchor or an allosteric activator of PI3K gamma, and which role its noncatalytic p101 subunit plays in its activation by G beta gamma. Using GFP-tagged PI3K gamma subunits expressed in HEK cells, we show that G beta gamma recruits the enzyme from the cytosol to the membrane by interaction with its p101 subunit. Accordingly, p101 was found to be required for G protein-mediated activation of PI3K gamma in living cells, as assessed by use of GFP-tagged PtdIns-3,4,5-P3-binding PH domains. Furthermore, membrane-targeted p110 gamma displayed basal enzymatic activity, but was further stimulated by G beta gamma, even in the absence of p101. Therefore, we conclude that in vivo, G beta gamma activates PI3K gamma by a mechanism assigning specific roles for both PI3K gamma subunits, i.e., membrane recruitment is mediated via the noncatalytic p101 subunit, and direct stimulation of G beta gamma with p110 gamma contributes to activation of PI3K gamma.
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Project description:UnlabelledLong-term potentiation (LTP), a long-lasting enhancement in communication between neurons, is considered to be the major cellular mechanism underlying learning and memory. LTP triggers high-frequency calcium pulses that result in the activation of Calcium/Calmodulin (CaM)-dependent kinase II (CaMKII). CaMKII acts as a molecular switch because it remains active for a long time after the return to basal calcium levels, which is a unique property required for CaMKII function. Here we describe the crystal structure of the human CaMKIIdelta/Ca2+/CaM complex, structures of all four human CaMKII catalytic domains in their autoinhibited states, as well as structures of human CaMKII oligomerization domains in their tetradecameric and physiological dodecameric states. All four autoinhibited human CaMKIIs were monomeric in the determined crystal structures but associated weakly in solution. In the CaMKIIdelta/Ca2+/CaM complex, the inhibitory region adopted an extended conformation and interacted with an adjacent catalytic domain positioning T287 into the active site of the interacting protomer. Comparisons with autoinhibited CaMKII structures showed that binding of calmodulin leads to the rearrangement of residues in the active site to a conformation suitable for ATP binding and to the closure of the binding groove for the autoinhibitory helix by helix alphaD. The structural data, together with biophysical interaction studies, reveals the mechanism of CaMKII activation by calmodulin and explains many of the unique regulatory properties of these two essential signaling molecules.Enhanced versionThis article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3-D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the Web plugin are available in Text S1.
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Project description: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.
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