Abstract
Type I phosphatidylinositol-4-phosphate 5-kinase (PIPKI) is the main enzyme generating the lipid second messenger phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2], which has critical functions in many cellular processes, such as cytoskeletal reorganization, membrane trafficking, and signal transduction. All three members of the PIPKI family are activated by phosphatidic acid (PA). However, how PA regulates the activity and functions of PIPKI have not been fully elucidated. In this study, we identify a PA-binding site on PIPKIγ. Mutation of this site inhibited the PA-stimulated activity and membrane localization of PIPKIγ as well as the formation of actin comets and foci induced by PIPKIγ. We also demonstrate that phospholipase D (PLD) generates a pool of PA involved in PIPKIγ regulation by showing that PLD inhibitors blocked the membrane localization of PIPKIγ and its ability to induce actin cytoskeletal reorganization. Targeting the PIPKIγ PA-binding-deficient mutant to membranes by a membrane localization sequence failed to restore the actin reorganization activity of PIPKIγ, suggesting that PA binding is not only involved in recruiting PIPKIγ to membranes but also may induce a conformational change. Taken together, these results reveal a new molecular mechanism through which PA regulates PIPKI and provides direct evidence that PA is important for the localization and functions of PIPKI in intact cells.
Highlights
Type I phosphatidylinositol-4-phosphate 5-kinase (PIPKI) is the main enzyme generating the lipid second messenger phosphatidylinositol-4,5-bisphosphate [PI[4,5] P2], which has critical functions in many cellular processes, such as cytoskeletal reorganization, membrane trafficking, and signal transduction
Using the phosphatidic acid (PA)-binding-deficient mutant created in this study and pharmacological inhibitors, we further show that phospholipase D (PLD)-generated PA is required for the formation of actin comets and foci induced by PIPKI␥
Because PA stimulates the activity of all PIPKI family members but not that of PIPKII or PIPKIII members [17, 18], we hypothesized that the potential PA-interacting residues should fit the following three criteria: i) located close to or within the putative flat membrane interacting surface, ii) conserved in the three PIPKI members but not in PIPKII, and iii) positively charged
Summary
Type I phosphatidylinositol-4-phosphate 5-kinase (PIPKI) is the main enzyme generating the lipid second messenger phosphatidylinositol-4,5-bisphosphate [PI[4,5] P2], which has critical functions in many cellular processes, such as cytoskeletal reorganization, membrane trafficking, and signal transduction. To assess the contribution of PA binding to PIPKI membrane localization and actin cytoskeletal reorganization activity, we utilized a GFP-tagged PIPKI␥ construct [31] and subcloned all mutants described above into this plasmid.
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