Regulation of phospholipase D Exton, John H
FEBS Letters,
October 30, 2002, Volume:
531, Issue:
1
Book Review, Journal Article
Peer reviewed
Open access
Structural studies of plant and bacterial members of the phospholipase D (PLD) superfamily are providing information about the role of the conserved HKD domains in the structure of the catalytic ...center and the catalytic mechanism of mammalian PLD isozymes (PLD1 and PLD2). Mutagenesis and sequence comparison studies have also defined the presence of pleckstrin homology and phox homology domains in the N-terminus and have demonstrated that a conserved sequence at the C-terminus is required for catalysis. The N- and C-terminal regions of PLD1 also contain interaction sites for protein kinase C, which can directly activate the enzyme through a non-phosphorylating mechanism. Small G proteins of the Rho and ADP-ribosylation factor families also directly regulate the enzyme, with RhoA binding to a sequence in the C-terminus. Certain tyrosine kinases and members of the Ras subfamily of small G proteins can activate the enzyme, but the mechanisms appear to be indirect. The mechanisms by which agonists activate PLD in vivo probably involve multiple pathways.
Endoplasmic reticulum (ER) stress has been implicated in the pathogenesis of many diseases and in cancer therapy. Although
the unfolded protein response is known to alleviate ER stress by reducing ...the accumulation of misfolded proteins, the exact
survival elements and their downstream signaling pathways that directly counteract ER stress-stimulated apoptotic signaling
remain elusive. Here, we have shown that endogenous Akt and ERK are rapidly activated and act as downstream effectors of phosphatidylinositol
3-kinase in thapsigargin- or tunicamycin-induced ER stress. Introduction of either dominant-negative Akt or MEK1 or the inhibitors
LY294002 and U0126 sensitized cells to ER stress-induced cell death in different cell types. Reverse transcription-PCR analysis
of gene expression during ER stress revealed that cIAP-2 and XIAP, members of the IAP family of potent caspase suppressors,
were strongly induced. Transcription of cIAP-2 and XIAP was up-regulated by the phosphatidylinositol 3-kinase/Akt pathway
as shown by its reversal by dominant-negative Akt or LY294002. Ablation of these IAPs by RNA interference sensitized cells
to ER stress-induced death, which was reversed by the caspase inhibitor benzyloxycarbonyl-VAD-fluoromethyl ketone. The protective
role of IAPs in ER stress coincided with Smac release from mitochondria to the cytosol. Furthermore, it was shown that mTOR
was not required for Akt-mediated survival. These results represent the first demonstration that activation of endogenous
Akt/IAPs and MEK/ERK plays a critical role in controlling cell survival by resisting ER stress-induced cell death signaling.
Phospholipase D exists in various forms that differ in their regulation but predominantly hydrolyze phosphatidylcholine. The Ca(2+)-dependent isozymes of protein kinase C regulate phospholipase D in ...vitro and play a major role in its control by growth factors and G protein-linked agonists in vivo. Recent studies have demonstrated that small G proteins of the ADP-ribosylation factor (ARF) and Rho families activate the enzyme in vitro, and evidence is accumulating that they also are involved in its control in vivo. Both types of G protein play important roles in cellular function, and the possible mechanisms by which they are activated by agonists are discussed. There is also emerging evidence of the control of phospholipase D and Rho proteins by soluble tyrosine kinases and novel serine/threonine kinases. The possible role of these kinases in agonist regulation of phospholipase D is discussed. The function of phospholipase D in cells is still poorly defined. Postulated roles of phosphatidic acid produced by phospholipase D action include the activation of Ca(2+)-independent isoforms of protein kinase C, the regulation of growth and the cytoskeleton in fibroblasts, and control of the respiratory burst in neutrophils. Another important function of phosphatidic acid is to act as a substrate for a specific phospholipase A2 to generate lysophosphatidic acid, which is becoming increasingly recognized as a major intercellular messenger. Finally, it is possible that the phospholipid changes induced in various cellular membranes by phospholipase D may per se play an important role in vesicle trafficking and other membrane-associated events.
Mitogens activate protein translation through phosphorylation of p7S6 kinase (p70S6K) and eIF4E binding protein 1 (4E‐BP1) mediated by the mammalian target of rapamycin (mTOR) or phosphoinositide ...3‐kinase (PI3K). A recent report (Science 294, 1942, 2001) has implicated phospholipase D (PLD) in mTOR signaling. We studied the role of PLD in the phosphorylation of p70S6K and 4E‐BP1 induced by lysophosphatidic acid (LPA) and platelet‐derived growth factor (PDGF) using fibroblasts deficient in PLD activity and also 1‐butanol, which inhibits phosphatidic acid production by PLD. The reduction in PLD activity in both situations impaired the effect of LPA on mTOR signaling but did not inhibit the effect of PDGF. PDGF induced marked phosphorylation of Akt (a PI3K target) but this was not affected by PLD deficiency. LPA caused much less phosphorylation of Akt and this was dependent on PLD activity. Toxin B, which inacti¬vates Rho GTPases, markedly impaired PLD1 activa¬tion and phosphorylation of Akt, p70S6K, and 4E‐BP1 induced by LPA but had a minimal or no effect on the actions of PDGF. These results support the hypothesis that LPA activates protein translation through the ac¬tion of PLD1‐generated PA on mTOR and the PI3K/ Akt pathway whereas PDGF acts through P13K/Akt independent of PLD1.—Kam, Y., Exton, J. H. Role of phospholipase D1 in the regulation of mTOR activity by lysophosphatidic acid. FASEB J. 18, 311–319 (2004)
It has been well documented that protein kinase C (PKC) plays an important role in regulation of phospholipase D (PLD) activity. Although PKC regulation of PLD1 activity has been studied extensively, ...the role of PKC in PLD2 regulation remains to be established. In the present study it was demonstrated that phorbol 12-myristate 13-acetate (PMA) induced PLD2 activation in COS-7 cells. PLD2 was also phosphorylated on both serine and threonine residues after PMA treatment. PKC inhibitors Ro-31-8220 and bisindolylmaleimide I inhibited both PMA-induced PLD2 phosphorylation and activation. However, Gö 6976, a PKC inhibitor relatively specific for conventional PKC isoforms, almost completely abolished PLD2 phosphorylation by PMA but only slightly inhibited PLD2 activation. Furthermore, time course studies showed that phosphorylation of PLD2 lagged behind its activation by PMA. Concentration curves for PMA action on PLD2 phosphorylation and activation also showed that PLD2 was activated by PMA at concentrations at which PMA didn't induce phosphorylation. A kinase-deficient mutant of PKCalpha stimulated PLD2 activity to an even higher level than wild type PKCalpha. Co-expression of wild type PKCalpha, but not PKCdelta, greatly enhanced both basal and PMA-induced PLD2 phosphorylation. A PKCdelta-specific inhibitor, rottlerin, failed to inhibit PMA-induced PLD2 phosphorylation and activation. Co-immunoprecipitation studies indicated an association between PLD2 and PKCalpha under basal conditions that was further enhanced by PMA. Time course studies of the effects of PKCalpha on PLD2 showed that as the phosphorylation of PLD2 increased, its activity declined. In summary, the data demonstrated that PLD2 is activated and phosphorylated by PMA and PKCalpha in COS-7 cells. However, the phosphorylation is not required for PKCalpha to activate PLD2. It is suggested that interaction rather than phosphorylation underscores the activation of PLD2 by PKC in vivo and that phosphorylation may contribute to the inactivation of the enzyme.
In mammalian cells, phospholipase D activity is tightly regulated by diverse cellular signals, including hormones, neurotransmitters, and growth factors. Multiple signaling pathways converge upon ...phospholipase D to modulate cellular actions, such as cell growth, shape, and secretion. We examined the kinetics of protein kinase C and G-protein regulation of mammalian phospholipase D1 (PLD1) in order to better understand interactions between PLD1 and its regulators. Activation by Arf-1, RhoA, Rac1, Cdc42, protein kinase Calpha, and phosphatidylinositol 4,5-bisphosphate displayed surface dilution kinetics, but these effectors modulated different kinetic parameters. PKCalpha activation of PLD1 involves N- and C-terminal PLD domains. Rho GTPases were binding activators, enhancing the catalytic efficiency of a purified PLD1 catalytic domain via effects on Km. Arf-1, a catalytic activator, stimulated PLD1 by enhancing the catalytic constant, kcat. A kinetic description of PLD1 activation by multiple modulators reveals a mechanism for apparent synergy between activators. Synergy was observed only when PLD1 was simultaneously stimulated by a binding activator and a catalytic activator. Surprisingly, synergistic activation was steeply dependent on phosphatidylinositol 4,5-bisphosphate and phosphatidylcholine. Together, these findings suggest a role for PLD1 as a signaling node, in which integration of convergent signals occurs within discrete locales of the cellular membrane.
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