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1.
Elife ; 122024 May 07.
Article in English | MEDLINE | ID: mdl-38713746

ABSTRACT

Phosphoinositide 3-kinase (PI3K) beta (PI3Kß) is functionally unique in the ability to integrate signals derived from receptor tyrosine kinases (RTKs), G-protein coupled receptors, and Rho-family GTPases. The mechanism by which PI3Kß prioritizes interactions with various membrane-tethered signaling inputs, however, remains unclear. Previous experiments did not determine whether interactions with membrane-tethered proteins primarily control PI3Kß localization versus directly modulate lipid kinase activity. To address this gap in our knowledge, we established an assay to directly visualize how three distinct protein interactions regulate PI3Kß when presented to the kinase in a biologically relevant configuration on supported lipid bilayers. Using single molecule Total Internal Reflection Fluorescence (TIRF) Microscopy, we determined the mechanism controlling PI3Kß membrane localization, prioritization of signaling inputs, and lipid kinase activation. We find that auto-inhibited PI3Kß prioritizes interactions with RTK-derived tyrosine phosphorylated (pY) peptides before engaging either GßGγ or Rac1(GTP). Although pY peptides strongly localize PI3Kß to membranes, stimulation of lipid kinase activity is modest. In the presence of either pY/GßGγ or pY/Rac1(GTP), PI3Kß activity is dramatically enhanced beyond what can be explained by simply increasing membrane localization. Instead, PI3Kß is synergistically activated by pY/GßGγ and pY/Rac1 (GTP) through a mechanism consistent with allosteric regulation.


Subject(s)
Class I Phosphatidylinositol 3-Kinases , rac1 GTP-Binding Protein , rho GTP-Binding Proteins , Humans , GTP-Binding Protein beta Subunits/metabolism , GTP-Binding Protein beta Subunits/chemistry , GTP-Binding Protein gamma Subunits/metabolism , GTP-Binding Protein gamma Subunits/chemistry , GTP-Binding Protein gamma Subunits/genetics , Microscopy, Fluorescence , Phosphatidylinositol 3-Kinases/metabolism , Protein Binding , Receptor Protein-Tyrosine Kinases/metabolism , Receptor Protein-Tyrosine Kinases/chemistry , rho GTP-Binding Proteins/metabolism , rho GTP-Binding Proteins/chemistry , Signal Transduction , Class I Phosphatidylinositol 3-Kinases/chemistry , Class I Phosphatidylinositol 3-Kinases/metabolism , rac1 GTP-Binding Protein/chemistry , rac1 GTP-Binding Protein/metabolism
2.
bioRxiv ; 2023 Dec 21.
Article in English | MEDLINE | ID: mdl-37205345

ABSTRACT

The class 1A phosphoinositide 3-kinase (PI3K) beta (PI3Kß) is functionally unique in the ability to integrate signals derived from receptor tyrosine kinases (RTKs), heterotrimeric guanine nucleotide-binding protein (G-protein)-coupled receptors (GPCRs), and Rho-family GTPases. The mechanism by which PI3Kß prioritizes interactions with various membrane tethered signaling inputs, however, remains unclear. Previous experiments have not been able to elucidate whether interactions with membrane-tethered proteins primarily control PI3Kß localization versus directly modulate lipid kinase activity. To address this gap in our understanding of PI3Kß regulation, we established an assay to directly visualize and decipher how three distinct protein interactions regulate PI3Kß when presented to the kinase in a biologically relevant configuration on supported lipid bilayers. Using single molecule Total Internal Reflection Fluorescence (TIRF) Microscopy, we determined the mechanism controlling membrane localization of PI3Kß, prioritization of signaling inputs, and lipid kinase activation. We find that auto-inhibited PI3Kß prioritizes interactions with RTK-derived tyrosine phosphorylated (pY) peptides before engaging either GßGγ or Rac1(GTP). Although pY peptides strongly localize PI3Kß to membranes, stimulation of lipid kinase activity is modest. In the presence of either pY/GßGγ or pY/Rac1(GTP), PI3Kß activity is dramatically enhanced beyond what can be explained by simply increasing the strength of membrane localization. Instead, PI3Kß is synergistically activated by pY/GßGγ and pY/Rac1(GTP) through a mechanism consistent with allosteric regulation.

3.
Cell Rep ; 42(3): 112172, 2023 03 28.
Article in English | MEDLINE | ID: mdl-36842083

ABSTRACT

Class IB phosphoinositide 3-kinase (PI3Kγ) is activated in immune cells and can form two distinct complexes (p110γ-p84 and p110γ-p101), which are differentially activated by G protein-coupled receptors (GPCRs) and Ras. Using a combination of X-ray crystallography, hydrogen deuterium exchange mass spectrometry (HDX-MS), electron microscopy, molecular modeling, single-molecule imaging, and activity assays, we identify molecular differences between p110γ-p84 and p110γ-p101 that explain their differential membrane recruitment and activation by Ras and GPCRs. The p110γ-p84 complex is dynamic compared with p110γ-p101. While p110γ-p101 is robustly recruited by Gßγ subunits, p110γ-p84 is weakly recruited to membranes by Gßγ subunits alone and requires recruitment by Ras to allow for Gßγ activation. We mapped two distinct Gßγ interfaces on p101 and the p110γ helical domain, with differences in the C-terminal domain of p84 and p101 conferring sensitivity of p110γ-p101 to Gßγ activation. Overall, our work provides key insight into the molecular basis for how PI3Kγ complexes are activated.


Subject(s)
Phosphatidylinositol 3-Kinases , Signal Transduction , Signal Transduction/physiology , Phosphatidylinositol 3-Kinases/metabolism , Receptors, G-Protein-Coupled , Models, Molecular , Phosphatidylinositol 3-Kinase
4.
Elife ; 112022 08 17.
Article in English | MEDLINE | ID: mdl-35976097

ABSTRACT

The phosphatidylinositol 4-phosphate 5-kinase (PIP5K) family of lipid-modifying enzymes generate the majority of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] lipids found at the plasma membrane in eukaryotic cells. PI(4,5)P2 lipids serve a critical role in regulating receptor activation, ion channel gating, endocytosis, and actin nucleation. Here, we describe how PIP5K activity is regulated by cooperative binding to PI(4,5)P2 lipids and membrane-mediated dimerization of the kinase domain. In contrast to constitutively dimeric phosphatidylinositol 5-phosphate 4-kinase (PIP4K, type II PIPK), solution PIP5K exists in a weak monomer-dimer equilibrium. PIP5K monomers can associate with PI(4,5)P2-containing membranes and dimerize in a protein density-dependent manner. Although dispensable for cooperative PI(4,5)P2 binding, dimerization enhances the catalytic efficiency of PIP5K through a mechanism consistent with allosteric regulation. Additionally, dimerization amplifies stochastic variation in the kinase reaction velocity and strengthens effects such as the recently described stochastic geometry sensing. Overall, the mechanism of PIP5K membrane binding creates a broad dynamic range of lipid kinase activities that are coupled to the density of PI(4,5)P2 and membrane-bound kinase.


Subject(s)
Phosphates , Phosphotransferases (Alcohol Group Acceptor) , Cell Membrane/metabolism , Dimerization , Phosphates/metabolism , Phosphatidylinositol 4,5-Diphosphate/metabolism , Phosphatidylinositols/metabolism , Phosphorylation , Phosphotransferases (Alcohol Group Acceptor)/metabolism
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