Membrane-mediated dimerization potentiates PIP5K lipid kinase activity

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.

Virulence adaptation ofPseudomonas aeruginosaphospholipase mutant with altered membrane phospholipid composition

“…This result indicates that SKs accumulated in Δ plaF might have impaired kinase function or enhanced phosphatase function. It remains to be answered whether the function of the SKs identified here could be regulated by PlaF-mediated alteration of PLs, as recently described for lipid kinases [67].…”

Molecular basis of product recognition during PIP5K-mediated production of PI(4,5)P2with positive feedback

“…Kinetics measurements of PI(4,5)P 2 lipid phosphorylation. The kinetics of PI(4)P phosphorylation were measured on SLBs formed in IBIDI chambers and visualized using TIRF microscopy as previously described (Hansen et al 2019(Hansen et al , 2022. Reaction buffer contained 20 mM HEPES [pH 7.0], 150 mM NaCl, 1 mM ATP, 5 mM MgCl 2 , 0.5 mM EGTA, 20 mM glucose, 200 µg/mL beta casein (ThermoScientific, Cat# 37528), 20 mM BME, 320 µg/mL glucose oxidase (Serva, #22780.01 Aspergillus niger), 50 µg/mL catalase (Sigma, #C40-100MG Bovine Liver), and 2 mM Trolox (UV treated, see methods below).…”

“…We discovered that human PIP5KB binds cooperatively to its product, PI(4,5)P 2 (Hansen et al 2019), which enhances PIP5K membrane localization through a PI(4,5)P 2 -mediated positive feedback loop. When PIP5K crosses a threshold membrane surface density it can also dimerize, which potentiates lipid kinase activity (Hansen et al 2022). This suggests that the site of PI(4,5)P 2 binding may be an important step for plasma membrane localization in cells, as PI(4,5)P 2 lipids exist almost exclusively at the plasma membrane.…”

Beyond PI3Ks: targeting phosphoinositide kinases in disease

“…74,104 ). Optimization of this molecule led to the derivative compound 30, which showed enhanced selectivity 111 In contrast to the stable PI5P4K dimers, PI4P5Ks exist in a monomer-dimer equilibrium in solution, with dimerization promoted by binding to PI(4,5)P 2 -containing membrane surfaces, leading to enhanced catalytic efficiency 238 . Like all lipid kinases, the kinase domain contains an activation loop that determines substrate specificity and also has a role in membrane recruitment.…”

“…a, Structure of the zebrafish homologue of phosphoinositide 4-phosphate 5-kinase-α (PI4P5Kα), with domains annotated on the figure. PI4P5Kα (PDB:4TZ7) shows a putative dimeric interface composed of the dimerization domain as well as the N-lobe of the kinase domain, which is unique compared with the phosphoinositide 5-phosphate 4-kinases (PI5P4Ks; see panel b).In contrast to the stable PI5P4K dimers, PI4P5Ks exist in a monomer-dimer equilibrium in solution, with dimerization promoted by binding to PI(4,5)P 2 -containing membrane surfaces, leading to enhanced catalytic efficiency238 . Like all lipid kinases, the kinase domain contains an activation loop that determines substrate specificity and also has a role in membrane recruitment.…”