Molecular basis for differential activation of p101 and p84 complexes of PI3Kγ by Ras and GPCRs

Rathinaswamy, Manoj Kumar; Jenkins, Meredith L.; Duewell, Benjamin R; Zhang, Xuxiao; Harris, Noah J; Evans, John T; Stariha, Jordan T B; Dalwadi, Udit; Fleming, Kaelin D; Ranga-Prasad, Harish; Yip, Calvin K.; Williams, Roger L.; Hansen, Scott D.; Burke, John E.

doi:10.1016/j.celrep.2023.112172

Cited by 14 publications

(24 citation statements)

References 73 publications

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“…Therefore, rigidifying the regulatory motif likely explains the molecular basis for how it prevents kinase activity. The nanobody interface is distinct from the predicted Gbg interface (Rathinaswamy et al, 2023) and the experimentally resolved Ras interface (Pacold et al, 2000), explaining why it can still be membrane recruited by these stimuli.…”

Section: Molecular Mechanism Of Nanobody Inhibition Of P110gmentioning

confidence: 83%

“…The p110g-p84 complex forms a more dynamic complex compared to p110g-p101 (Rathinaswamy et al, 2023;Shymanets et al, 2013), however, no clear unique regulatory role of this difference in dynamics has been identified.…”

Section: Introductionmentioning

confidence: 99%

“…This leads to the different complexes driving unique immune responses, with p110g-p101involved in chemotaxis in neutrophils (Bohnacker et al, 2009;Deladeriere et al, 2015), and p110g-p84 involved in reactive oxide production. Differential activation of unique PI3Kg complexes downstream of GPCRs and Ras is caused by the ability of p101 to directly bind to Gbg subunits downstream of activated GPCRs, with this being lost in p84, making p110g-p84 activation by Gbg dependent on Ras mediated membrane recruitment (Rathinaswamy et al, 2023;Kurig et al, 2009;Rynkiewicz et al, 2020).…”

Section: Introductionmentioning

confidence: 99%

“…Extensive biophysical approaches including cryo electron microscopy (cryo-EM), X-ray crystallography, and hydrogen deuterium exchange mass spectrometry (HDX-MS) have provided extensive insight into the molecular underpinnings of how p110g associates with both p101 and p84, how they are differentially activated by Ras and GPCR signals, and how they can be activated on lipid membranes (Pacold et al, 2000;Walker et al, 1999;Rathinaswamy et al, 2021cRathinaswamy et al, , 2021aGangadhara et al, 2019;Vadas et al, 2013;Rathinaswamy et al, 2023). The p110g catalytic subunit is composed of an adaptor binding domain (ABD), a Ras binding domain (RBD), a C2 domain, a helical domain, and a bi-lobal kinase domain (Rathinaswamy et al, 2021a;Walker et al, 1999).…”

Section: Introductionmentioning

confidence: 99%

“…The regulatory motif is a common site of activating mutations in the other class I PI3K isoforms (Jenkins et al, 2023), with p110g having rare activating oncogenic mutants in this region (Rathinaswamy et al, 2021c). The p110g subunit interacts with both p84 and p101 at an interface composed of the C2 domain, and the linkers between the RBD-C2 and C2-helical domains (Rathinaswamy et al, 2023(Rathinaswamy et al, , 2021a.…”

Section: Introductionmentioning

confidence: 99%

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Allosteric activation or inhibition of PI3Kγ mediated through conformational changes in the p110γ helical domain

Harris

Jenkins

Nam

et al. 2023

Preprint

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PI3Kγ is a critical immune signaling enzyme activated downstream of diverse cell surface molecules, including Ras, PKCβ activated by the IgE receptor, and Gβγ subunits released from activated GPCRs. PI3Kγ can form two distinct complexes, with the p110γ catalytic subunit binding to either a p101 or p84 regulatory subunit, with these complexes being differentially activated by upstream stimuli. Here using a combination of Cryo electron microscopy, HDX-MS, and biochemical assays we have identified novel roles of the helical domain of p110γ in regulating lipid kinase activity of distinct PI3Kγ complexes. We defined the molecular basis for how an allosteric inhibitory nanobody potently inhibits kinase activity through rigidifying the helical domain and regulatory motif of the kinase domain. The nanobody did not block either p110γ membrane recruitment or Ras/Gβγ binding, but instead decreased ATP turnover. We also identified that p110γ can be activated by dual PKCβ helical domain phosphorylation leading to partial unfolding of an N-terminal region of the helical domain. PKCβ phosphorylation is selective for p110γ-p84 compared to p110γ-p101, driven by differential dynamics of the helical domain of these different complexes. Nanobody binding prevented PKCβ mediated phosphorylation. Overall, this works shows an unexpected allosteric regulatory role of the helical domain of p110γ that is distinct between p110γ-p84 and p110γ-p101, and reveals how this can be modulated by either phosphorylation or allosteric inhibitory binding partners. This opens possibilities of future allosteric inhibitor development for therapeutic intervention.

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Section: Molecular Mechanism Of Nanobody Inhibition Of P110gmentioning

confidence: 83%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Allosteric activation or inhibition of PI3Kγ mediated through conformational changes in the p110γ helical domain

Harris

Jenkins

Nam

et al. 2023

Preprint

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Defining bone fide effectors of RAS GTPases

Smith

2023

BioEssays

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RAS GTPases play essential roles in normal development and are direct drivers of human cancers. Three decades of study have failed to wholly characterize pathways stimulated by activated RAS, driven by engagement with ‘effector’ proteins that have RAS binding domains (RBDs). Bone fide effectors must bind directly to RAS GTPases in a nucleotide‐dependent manner, and this interaction must impart a clear change in effector activity. Despite this, for most proteins currently deemed effectors there is little mechanistic understanding of how binding to the GTPase alters protein function. There has also been limited effort to comprehensively resolve the specificity of effector binding to the full array of RAS superfamily GTPase proteins. This review will summarize what is known about RAS‐driven activation for an array of potential effector proteins, focusing on structural and mechanistic effects and highlighting how little is still known regarding this key paradigm of cellular signal transduction.

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