Oxidative Stress Decreases Phosphatidylinositol 4,5-Bisphosphate Levels by Deactivating Phosphatidylinositol- 4-phosphate 5-Kinase β in a Syk-dependent Manner

Abstract

Phosphatidylinositol 4,5-bisphosphate (PIP 2 ) has many essential functions and its homeostasis is highly regulated. We previously found that hypertonic stress increases PIP 2 by selectively activating the ␤ isoform of the type I phosphatidylinositol phosphate 5-kinase (PIP5K␤) through Ser/Thr dephosphorylation and promoting its translocation to the plasma membrane. Here we report that hydrogen peroxide (H 2 O 2 ) also induces PIP5K␤ Ser/Thr dephosphorylation, but it has the opposite effect on PIP 2 homeostasis, PIP5K␤ function, and the actin cytoskeleton. Brief H 2 O 2 treatments decrease cellular PIP 2 in a PIP5K␤-dependent manner. PIP5K␤ is tyrosine phosphorylated, dissociates from the plasma membrane, and has decreased lipid kinase activity. In contrast, the other two PIP5K isoforms are not inhibited by H 2 O 2 . We identified spleen tyrosine kinase (Syk), which is activated by oxidants, as a candidate PIP5K␤ kinase in this pathway, and mapped the oxidant-sensitive tyrosine phosphorylation site to residue 105. The PIP5K␤Y105E phosphomimetic is catalytically inactive and cytosolic, whereas the Y105F non-phosphorylatable mutant has higher intrinsic lipid kinase activity and is much more membrane associated than wild type PIP5K␤. These results suggest that during oxidative stress, as modeled by H 2 O 2 treatment, Syk-dependent tyrosine phosphorylation of PIP5K␤ is the dominant post-translational modification that is responsible for the decrease in cellular PIP 2 .

Reactive Oxygen Species in Inflammation and Tissue Injury

“…A direct effect of exogenous application of H 2 O 2 on remodeling of actin cytoskeleton of endothelial cells has been documented in several reports. Endothelial monolayer treated with inflammatory agonists such as thrombin, histamine, and VEGF or directly with H 2 O 2 correlated with decreased cortical actin and formation of polymerized F-actin (61,182). Reorganization of actin ctyoskelton into F-actin increased centripetal tension and induced gap formation in endothelial cells (103,349,397).…”

GABARAPs regulate PI4P-dependent autophagosome:lysosome fusion

“…HeLa cells cultured in DMEM containing 10% (vol/vol) FBS were transfected with epitope-tagged cDNAs using Lipofectamine 2000. Previously validated siRNAs that target human PI4KIIα (6), PIP5Kβ (19), and PI4KIIIβ (20) or Ctrl siRNA were from Sigma. The siGABARAP/GATE-16 pool was directed against all members of the family (3).…”

Phosphatidylinositol-4-phosphate 5-Kinase Isoforms Exhibit Acyl Chain Selectivity for Both Substrate and Lipid Activator

“…PIP5K␣ also interacts directly with diacylglycerol kinase (DGK), resulting in the promotion of the formation of PtdIns(4,5)P 2 , probably through the activation of PIP5K by phosphatidic acid (PA), the * This work was supported, in whole or in part, by National Institutes of product of the reaction catalyzed by DGK (13,14). The ␤ isoform of PIP5K is activated by both Ser/Thr and by Tyr phosphorylation that is promoted by oxidative stress (15). This isoform controls neutrophil polarity and directional movement (16,17).…”