Phosphatidylinositol (3,4,5)-trisphosphate specifically interacts with the phox homology domain of phospholipase D1 and stimulates its activity

Abstract

Phospholipase D (PLD), which catalyzes the hydrolysis of phosphatidylcholine to phosphatidic acid and choline, plays key roles in cellular signal transduction by mediating extracellular stimuli including hormones, growth factors, neurotransmitters, cytokines and extracellular matrix molecules. The molecular mechanisms by which domains regulate the activity of PLD - especially the phox homology (PX) domain - have not been fully elucidated. In this study, we have examined the properties of the PX domains of PLD1 and PLD2 in terms of phosphoinositide binding and PLD activity regulation. Interestingly, the PX domain of PLD1, but not that of PLD2, was found to specifically interact with phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3). We found that mutation of the conserved arginine at position 179 of the PLD1 PX domain to lysine or to alanine (R179A or R179K, respectively) disrupts PtdIns(3,4,5)P3 binding. In NIH-3T3 cells, the EGFP-PLD1 PX wild-type domain, but not the two mutants, localized to the plasma membrane after 5-minute treatment with platelet-derived growth factor (PDGF). The enzymatic activity of PLD1 was stimulated by adding PtdIns(3,4,5)P3 in vitro. Treatment with PDGF resulted in the significant increase of PLD1 activity and phosphorylation of the downstream extracellular signal-regulated kinases (ERKs), which was blocked by pre-treatment of HEK 293 cells with phosphoinositide 3-kinase (PI3K) inhibitor after the endogenous PLD2 had been depleted by siRNA specific for PLD2. Nevertheless, both PLD1 mutants (which cannot interact with PtdIns(3,4,5)P3) did not respond to treatment with PDGF. Moreover, PLD1 was activated in HepG2 cells stably expressing the Y40/51 mutant of PDGF receptor that is required for the binding with PI3K. Our results suggest that the PLD1 PX domain enables PLD1 to mediate signal transduction via ERK1/2 by providing a direct binding site for PtdIns(3,4,5)P3 and by activating PLD1.

Lipid signalling in disease

“…When activated by PtdIns(3,4,5)P 3 , Btk phosphorylates PLC , which cleaves PtdIns(4,5)P 2 to form diacylglycerol (DAG) and Ins(1,4,5)P 3 , completing the first wave of lipid signalling. Subsequently, Ins(1,4,5)P 3 releases Ca 2+ from internal stores, while phospholipase D (PLD) 145,146 and sphingosine kinase (SphK) are activated downstream of PI3K. SphK reinforces Ca 2+ release, eventually leading to the opening of store-operated Ca 2+ channels (SOCCs) as soon as the intracellular Ca 2+ pools are depleted.…”

Molecular structure of phospholipase D and regulatory mechanisms of its activity in plant and animal cells

“…This domain contains two binding sites of membrane phospholipids: one of them is highly specific to PtdIns(3,4,5)P 3 , PtdIns(3)P, PtdIns(5)P, and other phosphoinositides, whereas the other also binds phosphatidic acid, phosphatidylserine, and other negatively charged lipids. The concurrent binding of phospholipids with the abovementioned sites synergistically increases the affinity of domain PX for the membranes [48,49]. The PX domain of PLD1 plays an important role in regulation of the enzyme translocation into endocytosis sites on the membranes under the influence of extracellular signals [40,49].…”

“…The concurrent binding of phospholipids with the abovementioned sites synergistically increases the affinity of domain PX for the membranes [48,49]. The PX domain of PLD1 plays an important role in regulation of the enzyme translocation into endocytosis sites on the membranes under the influence of extracellular signals [40,49]. In PLD1 and PLD2 this domain can stimulate the GTPase activity of the protein dynamin [50] and ensure the binding with PLD2 and activation of PLD2 of phospholipase Cγ1 [51], tyrosine kinase Syk [52], and protein kinase Cζ [53].…”

“…The PLD2 activity increases under conditions of a simultaneous expression of the genes encoding PLD and the abovementioned phosphoinositide kinase [79]. As discriminated from other phosphoinositides, PtdIns(3,4,5)P 3 binds with residue Arg179 in the PX domain of PLD1 [49], stimulates the enzyme activity [49,81], and also promotes the translocation of PKCζ into the cell nucleus [82], which results in activation of the nuclear PLD.…”

Molecular tools for cell and systems biology

“…The transient appearance of a lipid, for instance in the plasma membrane, will lead to a transient translocation of a lipid binding domain-bearing protein from the cytosol to the plasma membrane. This technique has been successfully used for measuring lipids such as diacylglycerol and phosphatidylinositol polyphosphates (Gray et al, 2003;Halet, 2005;Lee et al, 2005;Varnai et al, 1999). Alternatively, the breakdown of a lipid with a constantly high residual concentration in the plasma membrane, such as phosphatidylinositol 4,5-bisphosphate ͑PIP 2 ͒, can be monitored after stimulating the cell with an agonist that leads to phospholipase C activation (van der Wal et al, 2001).…”