Review
Regulation of phospholipase D

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Abstract

Phospholipase D (PLD) is a widely distributed enzyme that is under elaborate control by hormones, neurotransmitters, growth factors and cytokines in mammalian cells. Protein kinase C (PKC) plays a major role in the regulation of the PLD1 isozyme through interaction with its N-terminus. PKC activates this isozyme by a non-phosphorylation mechanism in vitro, but phosphorylation plays a role in the action of PKC on the enzyme in vivo. Although PLD1 can be phosphorylated by PKC in vitro, it is unclear that this occurs in vivo. Small GTPases of the ADP-ribosylation factor (ARF) and Rho families directly activate PLD1 in vitro and there is evidence that Rho proteins are involved in agonist regulation of PLD1 in vivo. ARF proteins stimulate PLD activity in the Golgi apparatus, but the role of these proteins in agonist regulation of the enzyme is less clear. PLD1 undergoes tyrosine phosphorylation in response to H2O2 treatment of cells. The functional consequence of this phosphorylation and soluble tyrosine kinase(s) involved are presently unknown.

Introduction

Although phospholipase D (PLD) activity is distributed widely in animals, plants, fungi and bacteria, studies of its regulation have been mainly confined to mammalian tissues and cells. As shown in Table 1, the enzyme is present in a wide variety of such tissues and cells, and its activity is stimulated by various hormones, neurotransmitters, growth factors, cytokines and other agonists that activate cell surface receptors. The diverse nature of the stimulatory agonists suggests that the enzyme is regulated by several mechanisms and in vitro studies support this.

Table 1 shows that, in many cells and tissues, the enzyme is activated by phorbol esters and/or Ca2+ ionophores. This indication that conventional (Ca2+-sensitive) protein kinase C (PKC) isozymes regulate PLD is supported by much work with intact cells and purified enzymes. However, regulation of the enzyme by physiological changes in cytosolic Ca2+ has not been demonstrated. Many agonists whose receptors are linked to heterotrimeric G proteins activate PLD. The G proteins are either sensitive or insensitive to pertussis toxin and are of the Gq, Gi and G12 families. However, there is no evidence that these G proteins directly regulate PLD. On the contrary, the regulation appears to occur via activation of PKC and/or small G proteins of the Rho family of GTPases.

Growth factors which activate the tyrosine kinase activities of their receptors also stimulate PLD, but there is no evidence that the receptors directly phosphorylate the enzyme, and it seems likely that PKC and/or Rho family proteins are involved. On the other hand, cytokines and antigens that activate soluble tyrosine kinases may induce phosphorylation of the enzyme.

Knowledge of the regulation of PLD has been greatly enhanced recently due to the cloning of several isoforms of the mammalian enzyme [1], [2], [3], [4], [5], [6], [7], [8], [11]. These have been expressed in mammalian (COS) and insect (Sf9) cells and their regulation studied. Two alternatively sliced forms of human PLD (hPLDa, hPLD1b) show identical regulatory properties, e.g. they are both stimulated markedly by phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3), but not other phospholipids, and are equally stimulated by Mg2+ ions [2]. They show very similar responses to two different types of small G proteins, namely ADP-ribosylation factors (ARFs) and members of the Rho family of small GTPases (RhoA, Rac1, Cdc42Hs), and are directly stimulated by the α-isozyme of PKC in the absence of ATP [2]. Similar results have been obtained with an isoform cloned from rat brain (rPLD1) which has 91% amino acid identity with hPLD1b [5], [6].

In contrast to the findings with hPLD1 and rPLD1, another PLD isoform (PLD2) exhibits high basal activity in the presence of PIP2, but does not respond to Rho or PKC and is stimulated weakly [9] or not at all [3] by ARF. It shows about 50% amino acid identity with PLD1 [3], [4], [9], [10] and has a different chromosomal location [7]. Based on the biochemical and regulatory properties of PLD in different tissues or tissue fractions, it seems likely that other PLD isoforms will be identified that are more selective in their regulation by ARF, Rho and PKC.

Section snippets

Regulation by protein kinase C

As described above, the widespread regulation of PLD by phorbol esters in tissues and cells (Table 1) indicates that PKC plays a major role in control of the enzyme. This is supported by numerous studies showing that specific and non-specific inhibitors of PKC partly or totally inhibit the stimulation of PLD by agonists in many cell types. Likewise down-regulation of conventional and novel PKC isoforms induced by prolonged treatment of cells with phorbol esters blocks the ability of many

Regulation by ADP-ribosylation factors

Studies of the regulation of PLD in cell membranes and permeabilized HL60 cells by GTPγS, an hydrolysis-resistant analog of GTP, indicated the requirement for a cytosolic factor [43], [44], [45], [46]. This factor was subsequently purified to homogeneity and shown to have sequences in common with ARF1 and ARF3 [47], [48], and recombinant rARF1 was shown to be effective in activating PLD in the presence of GTPγS [47], [48]. ARF5 and ARF6 have also been shown to be effective [49], [50], and

Regulation by Rho family small G proteins

The involvement of Rho-type GTPases in PLD regulation was first indicated by studies with neutrophils [77], [78]. These showed that the ability of GTPγS to activate PLD depended on factors in both the cytosolic and plasma membrane fractions [77]. The GTPγS-dependent factor was shown to be membrane-associated and to have the properties of a small G protein [77]. Furthermore, a protein that stimulates GDP dissociation from small G proteins and thus enhances GTP-GDP exchange was found to enhance

Regulation by calcium ions

Although Ca2+ ions can stimulate the activity of certain PLD isoforms, the concentrations required are often well above the physiological cytosolic range [12] or the stimulation is not observed in the presence of physiological Mg2+ concentrations [2]. Thus, direct control of the enzyme by physiological changes in cytosolic Ca2+ seems unlikely. However, many studies have shown that depletion of cellular Ca2+ by treatment of cells with Ca2+ chelators (EGTA or BAPTA) results in inhibition of the

Regulation by protein tyrosine kinases

Many agonists whose receptors have tyrosine kinase activity or which activate soluble protein tyrosine kinases stimulate PLD (Table 1). In addition, there is evidence for the involvement of tyrosine kinase(s) in the activation of PLD by agonists whose receptors are coupled to heterotrimeric G proteins. This is indicated by the observation that PLD activation by these agonists is inhibited in several cell types by the tyrosine kinase inhibitors genistein, herbimycin or tyrophostin [12], [118],

Role of inhibitory proteins and ceramide

Several proteins have been shown to inhibit PLD activity in vitro, but their role in the physiological regulation of the enzyme is unclear. Although some of the protein inhibitors have not been characterized [136], others have been identified as fodrin [137], the clathrin assembly protein AP3 [138] and synaptojanin [139]. The latter acts by decreasing the level of PIP2 through its PIP2 5-phosphatase activity [139]. Ceramide, a product of sphingomyclinase activity, also inhibits PLD in vitro

Role of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate

There is much evidence for an important role of PIP2 in the regulation of PLD in vitro [12] It has also been shown that manipulation of PIP2 levels in permeabilized cells [144] alters PLD activity, and that treatment of intact cells with clostridial toxins, which decrease PIP2 by inhibiting PI 4-P 5-kinase, causes inhibition of agonist stimulation of PLD [87]. However, it has not been demonstrated that agonists control PLD activity through variations in PIP2 in intact cells. In fact, most

Conclusions

There is extensive evidence that the Ca2+-dependent isozymes of PKC play an important role in the regulation of PLD by a variety of agonists. In vitro studies demonstrate that the regulation can occur in the absence of phosphorylation, implying that it involves a protein–protein interaction. However, the mechanisms by which PKC controls PLD in intact cells are far from clear. Many studies indicate the involvement of phosphorylation, but the substrate(s) is undefined. Agonist-induced

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