Septins tune lipid kinase activity and PI(4,5)P2 turnover during G-protein-coupled PLC signalling in vivo.

Aastha Kumari,Padinjat Raghu,Sourav Kolay,Avishek Ghosh

doi:10.26508/lsa.202101293

Abstract

Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] hydrolysis by phospholipase C (PLC) is a conserved mechanism of signalling. Given the low abundance of PI(4,5)P2, its hydrolysis needs to be coupled to resynthesis to ensure continued PLC activity; however, the mechanism by which depletion is coupled to resynthesis remains unknown. PI(4,5)P2 synthesis is catalyzed by the phosphorylation of phosphatidylinositol 4 phosphate (PI4P) by phosphatidylinositol 4 phosphate 5 kinase (PIP5K). In Drosophila photoreceptors, photon absorption is transduced into PLC activity and during this process, PI(4,5)P2 is resynthesized by a PIP5K. However, the mechanism by which PIP5K activity is coupled to PI(4,5)P2 hydrolysis is unknown. In this study, we identify a unique isoform dPIP5KL, that is both necessary and sufficient to mediate PI(4,5)P2 synthesis during phototransduction. Depletion of PNUT, a non-redundant subunit of the septin family, enhances dPIP5KL activity in vitro and PI(4,5)P2 resynthesis in vivo; co-depletion of dPIP5KL reverses the enhanced rate of PI(4,5)P2 resynthesis in vivo. Thus, our work defines a septin-mediated mechanism through which PIP5K activity is coupled to PLC-mediated PI(4,5)P2 hydrolysis.

Highlights

In many eukaryotic cells the binding of extracellular ligands to plasma membrane receptors is transduced by the activation of phospholipase C (PLC)
When reconstituted individually into dPIP5K18 photoreceptors, we found that dPIP5KL localizes to the plasma membrane at the base of the microvilli (Fig 1G), whereas dPIP5KS was distributed throughout the cell body of photoreceptors, away from the microvillar membrane (Fig 1H)
This situation may arise in settings such as neurons where GPCRs activate PLC at high rates or T-cell receptor activation where sustained activation of PLC occurs over a long period of time

Summary

Introduction

In many eukaryotic cells the binding of extracellular ligands to plasma membrane receptors is transduced by the activation of phospholipase C (PLC). The utilization of PI(4,5)P2 by PLC is coupled to the synthesis of this lipid; in eukaryotes, the principal route of PI(4,5)P2 synthesis is through the sequential phosphorylation of phosphatidylinositol (PI) at positions 4 and 5 by phosphatidylinositol 4 kinase (Nakatsu et al, 2012; Balakrishnan et al, 2018) and phosphatidylinositol 4 phosphate 5-kinase (Stephens et al, 1991; Chakrabarti et al, 2015) (reviewed in Kolay et al [2016]) For these enzymes to resynthesize PI(4,5)P2 in response to its utilization by PLC activity, it is necessary that plasma membrane PI(4,5)P2 levels are sensed and any drop in the levels of this lipid be communicated to the enzymatic machinery that regulates its synthesis

Methods

Results

Conclusion