Cryo-EM structures of cancer-specific helical and kinase domain mutations of PI3Kα

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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“…S2). This is consistent with the concept that ABD flexibility plays an important role in class I PI3K regulation (Liu et al, 2022).…”

Section: Molecular Mechanism Of Nanobody Inhibition Of P110gsupporting

confidence: 92%

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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“…1D, E) 17 . However, many features of this structure are likely artefactual, and driven by crystal packing, as recent Cryo-EM structures did not observe this difference 32 .…”

Section: Biochemical Analysis Of Oncogenic Mutations In the Inhibitor...mentioning

confidence: 71%

“…The E545K disrupts the nSH2-helical interface 15 while H1047R alters the membrane binding surface of the kinase domain, and how the kinase domain packs against the activation loop (Fig. 1D, E) 17,32 . However, there also are relatively high-frequency mutations at the ABD-kinase interface, the ABD-RBD linker, the C2-iSH2/C2-nSH2 interfaces, the putative membrane interface of the N-lobe with membranes, and in a region of the kinase domain C-terminal to the activation loop, referred to as the regulatory motif (Fig.…”

mentioning

confidence: 99%

Oncogenic mutations of PIK3CA lead to increased membrane recruitment driven by reorientation of the ABD, p85 and C-terminus

et al. 2023

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PIK3CA encoding the phosphoinositide 3-kinase (PI3K) p110α catalytic subunit is frequently mutated in cancer, with mutations occurring widely throughout the primary sequence. The full set of mechanisms underlying how PI3Ks are activated by all oncogenic mutations on membranes are unclear. Using a synergy of biochemical assays and hydrogen deuterium exchange mass spectrometry (HDX-MS), we reveal unique regulatory mechanisms underlying PI3K activation. Engagement of p110α on membranes leads to disengagement of the ABD of p110α from the catalytic core, and the C2 domain from the iSH2 domain of the p85 regulatory subunit. PI3K activation also requires reorientation of the p110α C-terminus, with mutations that alter the inhibited conformation of the C-terminus increasing membrane binding. Mutations at the C-terminus (M1043I/L, H1047R, G1049R, and N1068KLKR) activate p110α through distinct mechanisms, with this having important implications for mutant selective inhibitor development. This work reveals unique mechanisms underlying how PI3K is activated by oncogenic mutations, and explains how double mutants can synergistically increase PI3K activity.

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“…After phosphorylation, IRSs bind to PI3K that is activated, phosphorylating Akt ( Figure 1 ). Mutations in the regulatory (p85) and catalytic (p110) subunits of PI3K represent a driving force in the beginning and expansion of cancer [ 47 ].…”

Section: Regulation Of Leptin Efficiencymentioning

confidence: 99%

Recent Advances in the Knowledge of the Mechanisms of Leptin Physiology and Actions in Neurological and Metabolic Pathologies

Casado

Collado-Pérez

Frago

et al. 2023

IJMS

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Excess body weight is frequently associated with low-grade inflammation. Evidence indicates a relationship between obesity and cancer, as well as with other diseases, such as diabetes and non-alcoholic fatty liver disease, in which inflammation and the actions of various adipokines play a role in the pathological mechanisms involved in these disorders. Leptin is mainly produced by adipose tissue in proportion to fat stores, but it is also synthesized in other organs, where leptin receptors are expressed. This hormone performs numerous actions in the brain, mainly related to the control of energy homeostasis. It is also involved in neurogenesis and neuroprotection, and central leptin resistance is related to some neurological disorders, e.g., Parkinson’s and Alzheimer’s diseases. In peripheral tissues, leptin is implicated in the regulation of metabolism, as well as of bone density and muscle mass. All these actions can be affected by changes in leptin levels and the mechanisms associated with resistance to this hormone. This review will present recent advances in the molecular mechanisms of leptin action and their underlying roles in pathological situations, which may be of interest for revealing new approaches for the treatment of diseases where the actions of this adipokine might be compromised.

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Cryo-EM structures of cancer-specific helical and kinase domain mutations of PI3Kα

Cited by 20 publications

References 60 publications

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

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

Oncogenic mutations of PIK3CA lead to increased membrane recruitment driven by reorientation of the ABD, p85 and C-terminus

Recent Advances in the Knowledge of the Mechanisms of Leptin Physiology and Actions in Neurological and Metabolic Pathologies

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