Molecular basis of product recognition during PIP5K-mediated production of PI(4,5)P 2 with positive feedback.

Autor: Duewell BR; Department of Chemistry and Biochemistry, University of Oregon, Eugene, Oregon, USA; Institute of Molecular Biology, University of Oregon, Eugene, Oregon, USA., Faris KA; Department of Chemistry and Biochemistry, University of Oregon, Eugene, Oregon, USA; Institute of Molecular Biology, University of Oregon, Eugene, Oregon, USA., Hansen SD; Department of Chemistry and Biochemistry, University of Oregon, Eugene, Oregon, USA; Institute of Molecular Biology, University of Oregon, Eugene, Oregon, USA. Electronic address: shansen5@uoregon.edu.
Jazyk: angličtina
Zdroj: The Journal of biological chemistry [J Biol Chem] 2024 Sep; Vol. 300 (9), pp. 107631. Date of Electronic Publication: 2024 Aug 03.
DOI: 10.1016/j.jbc.2024.107631
Abstrakt: The ability for cells to localize and activate peripheral membrane-binding proteins is critical for signal transduction. Ubiquitously important in these signaling processes are phosphatidylinositol phosphate (PIP) lipids, which are dynamically phosphorylated by PIP lipid kinases on intracellular membranes. Functioning primarily at the plasma membrane, phosphatidylinositol-4-phosphate 5-kinases (PIP5K) catalyzes the phosphorylation of PI(4)P to generate most of the PI(4,5)P 2 lipids found in eukaryotic plasma membranes. Recently, we determined that PIP5K displays a positive feedback loop based on membrane-mediated dimerization and cooperative binding to its product, PI(4,5)P 2 . Here, we examine how two motifs contribute to PI(4,5)P 2 recognition to control membrane association and catalysis of PIP5K. Using a combination of single molecule TIRF microscopy and kinetic analysis of PI(4)P lipid phosphorylation, we map the sequence of steps that allow PIP5K to cooperatively engage PI(4,5)P 2 . We find that the specificity loop regulates the rate of PIP5K membrane association and helps orient the kinase to more effectively bind PI(4,5)P 2 lipids. After correctly orienting on the membrane, PIP5K transitions to binding PI(4,5)P 2 lipids near the active site through a motif previously referred to as the substrate or PIP-binding motif (PIPBM). The PIPBM has broad specificity for anionic lipids and serves a role in regulating membrane association in vitro and in vivo. Overall, our data supports a two-step membrane-binding model where the specificity loop and PIPBM act in concert to help PIP5K orient and productively engage anionic lipids to drive the positive feedback during PI(4,5)P 2 production.
Competing Interests: Conflicts of interest The authors declare that they have no conflicts of interest with the contents of this article.
(Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)
Databáze: MEDLINE
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