Phosphatidic acid regulates the affinity of the murine phosphatidylinositol 4‐phosphate 5‐kinase‐Iβ for phosphatidylinositol‐4‐phosphate

Abstract

Type I phosphatidylinositol 4-phosphate 5-kinase (PI4P5K) catalyzes the phosphorylation of phosphatidylinositol 4 phosphate [PI(4)P] at carbon 5, producing phosphatidylinositol 4,5 bisphosphate [PI(4,5)P2]. Phosphatidic acid (PA) activates PI4P5K in vitro and plays a central role in the activation of PIP5K pathways in vivo. This report demonstrates that actin fiber formation in murine fibroblasts involves PA activation of PIP5Ks and defines biochemical interactions between PA and the PIP5Ks. Inhibition of phospholipase D production of PA results in the loss of actin fibers. Overexpression of the beta isoform of the type I murine phosphatidylinositol 4-phosphate 5-kinase (mPIP5K-Ib) maintains actin fiber structure in the face of phospholipase D inhibition. PA activates mPIP5K-Ib by direct binding to mPIP5K-Ib through both electrostatic and hydrophobic interactions, with the fatty acid acyl chain length and degree of saturation acting as critical determinants of binding and activation. Furthermore, kinetic analysis suggests that phosphorylation of the PI(4)P substrate does not follow classical Michaelis-Menten kinetics. Instead, the kinetic data are consistent with a model in which mPIP5K-Ib initially binds to the lipid micelle and subsequently binds the PI(4)P substrate. In addition, the kinetics indicate substrate inhibition, suggesting that mPIP5K-Ib contains an inhibitory PI(4)P-binding site. These results suggest a model in which mPIP5K-Ib is surrounded by PI(4)P, but is unable to catalyze its conversion to PI(4,5)P2 unless PA is bound.

Irreversible Platelet Activation Requires Protease-Activated Receptor 1-Mediated Signaling to Phosphatidylinositol Phosphates

“…Hence, PAR1 regulation of PIP 2 levels (which would probably affect PLD, because PLD requires PIP 2 for activation) (Brown et al, 1993;Liscovitch et al, 1994) may play a critical role in feed-forward signal amplification. This is consistent with findings that both PIP 2 and PA (shown here to be crucial for stable platelet aggregation) can bind to and activate PI5K in vitro (Jarquin-Pardo et al, 2007). PAderived DAG formation may also play a role in feed-forward signal amplification through PKC (Henage et al, 2006), a cellular modulator of PLD activation, as well as CalDAG-GEF, an activator of Rap1 known to be highly expressed in platelets.…”

Phospholipase D Signaling Pathways and Phosphatidic Acid as Therapeutic Targets in Cancer

“…Early investigations revealed that PtdOH stimulated PIP5K activity from bovine brain membranes (Moritz et al, 1992) and later investigations revealed that only the class I PIP5Ks are stimulated by PtdOH (Jenkins et al, 1994). In vivo regulation of PIP5K by PtdOH has been documented as a significant decrease in PIP 2 levels in lysosomal membranes following butanol treatment (Arneson et al, 1999) and a decrease in PIP5K-dependent actin stress fiber formation after PLD inhibition in murine fibroblasts (Jarquin-Pardo et al, 2007). Recent studies demonstrated reduced PIP5K activity after treatment with small-molecule PLD inhibitors (Roach et al, 2012), further underscoring the importance of PtdOH in PIP5K regulation.…”

PIP5K-driven PtdIns(4,5)P2 synthesis: regulation and cellular functions

“…Furthermore, it has been proposed that PA can regulate the affinity of PIP5K for PtdIns(4)P (Jarquin-Pardo et al, 2007). PA is generated through the hydrolysis of phosphatidylcholine (PC) by phospholipase D (PLD) or through the phosphorylation of DAG by diacylglycerol kinase (DGK) .…”