(Received for publication, October 5, 1995)
From the
Activation of phosphatidylcholine-specific phospholipase D (PLD) has been implicated as a critical step in numerous cellular pathways, including signal transduction, membrane trafficking, and the regulation of mitosis. We report here the identification of the first human PLD cDNA, which defines a new and highly conserved gene family. Characterization of recombinant human PLD1 reveals that it is membrane-associated, selective for phosphatidylcholine, stimulated by phosphatidylinositol 4,5-bisphosphate, activated by the monomeric G-protein ADP-ribosylation factor-1, and inhibited by oleate. PLD1 likely encodes the gene product responsible for the most widely studied endogenous PLD activity.
Many cellular signaling events are initiated with the generation
of biologically active molecules by enzymatic hydrolysis of
phospholipids(1) . Although the best understood system involves
inositol lipids, the production of phosphatidic acid (PA) ()from PLD-catalyzed hydrolysis of phosphatidylcholine (PC)
is also a rapid and widespread response of cells challenged with
diverse agonists (Fig. 1A). The range of cell types and
signals reported implicate PLD activity in a broad spectrum of
physiological processes and diseases, including metabolic regulation,
inflammation, secretion, mitogenesis, oncogenesis, neural and cardiac
stimulation, diabetes, and senescence(2) . In vitro,
PA modulates the activity of varied regulatory proteins, including
certain protein serine/threonine and tyrosine kinases, Ras
GTPase-activating protein, several proteins involved in cytoskeletal
organization, and neutrophil respiratory burst oxidase. In
vivo, however, PLD's role is untested.
Figure 1: Overview and sequence analysis of hPLD1. A, regulation and cellular functions of PLD. PKC, protein kinase C; DG, diacylglycerol; AA, arachidonic acid; LPA, lysophosphatidic acid. See text for details. B, hPLD1 protein sequence. Underlined amino acids indicate residues invariant among all known homologs. Boxed amino acids indicate identical peptides of significant length between yeast and human. Asterisk indicates stop codon. C, PLD1-related genes. The human and mouse PLD1 cDNAs are depicted at the top. Aligned underneath are open reading frames or translated expressed sequence tags that have regions of significant similarity. Regions of significant similarity are indicated by shaded areas. The percent identities for the shaded regions of yPLD1 are displayed. The dashed line indicates that the cognate gene does not have an encoded region corresponding to the existing human sequence. Accession numbers: yPLD1, Z28256; castor bean, L33686; Arabidopsis, T76232, T88610, Z33674, Z18424; fruit fly, G00778; Caenorhabditis elegans, D27058, D33536; Streptococcus ORF, M37842; Pseudomonas putida ORF, X55704; Streptococcus PLD, D16444; Bacillus subtilis ORF, Z49782.
PLD and its modes
of regulation have become the subject of intense investigation. PLD
activities from many mammalian tissues and cell lines have been
reported that differ in their subcellular localization, pH optima,
dependence on divalent cations and phosphatidylinositol
4,5-bisphosphate (PIP), susceptibility to inhibition by
fatty acids (e.g. oleate) and
detergents(2, 3) , and extent of activation by small
G-proteins such as ARFs and RhoA and -B, Rac, and
Cdc42(4, 5, 6, 7, 8, 9, 10) .
The essential role of ARF in controlling the binding of the heptameric
coatomer complex to the surface of the Golgi cisternae (11) and
the essential role of PIP
in secretion have focused
attention on PIP
-dependent ARF-activated PLD activity as a
mediator of vesicular trafficking, although the hypothesis has not been
tested directly(12) . RhoA and -B, Rac, and Cdc42 are
implicated in signal transduction pathways that regulate both the actin
cytoskeleton and mitogen-activated protein kinase cascades that lead to
changes in transcription, suggesting that these small G-proteins act as
intermediates in the process by which cell surface receptors activate
PLD, but the molecular mechanisms underlying receptor regulation of PLD
remain poorly defined. A major impediment toward resolution of these
issues has been the absence of molecular definition of PLD genes in
animals, particularly since the partially purified PLD preparations
described to date may well have contained mixtures in different
proportions of PLDs representing distinct gene products. We report here
the cloning and characterization of the first animal PC-specific PLD,
human PLD1 (hPLD1).
Figure 2:
Baculovirus-mediated expression of hPLD1
in sf9 cells. A, proteins from uninfected sf9 cells (U,
lane 1) or cells infected for 48 h with the hPLD1-expressing
baculovirus vector (lane 2) were analyzed by
SDS-polyacrylamide gel electrophoresis on a 7.5% gel and stained with
Coomassie Blue. The positions of molecular weight markers are shown.
The 120-kDa band observed in the hPLD1 lane was not observed
in Sf9 cells infected with native baculovirus vector or PLC-expressing
baculovirus vector (data not shown). The identity of the 120-kDa band
was confirmed using a rabbit anti-hPLD1 antisera (data not shown). B, cytosolic and membrane fractions were prepared using
standard separation techniques from uninfected Sf9 cells or Sf9 cells
infected for 48 h with the hPLD1-expressing baculovirus
vector(13) . PLD activities are normalized for cell number and
dilution and therefore directly comparable. The data shown are means
± S.E. of triplicate determinations. Sf9 cells infected with
native baculovirus vector or PLC-expressing baculovirus vector yielded
PLD activity levels similar to untransfected cells (data not shown). C, product analysis and substrate specificity of hPLD1.
Membranes from Sf9 cells infected with the hPLD1 baculovirus
(containing 0.1 µg of membrane protein) were incubated with
phospholipid vesicles containing [
H]PC under
standard assay conditions, and the water-soluble products formed were
isolated and analyzed by cation-exchange HPLC (closed
circles). Analysis of material from an unincubated sample is also
shown (open circles). The elution positions of standards are
indicated. PCho, phosphorylcholine; GPCho, glycerophosphorylcholine; Cho, choline. D, membranes from Sf9 cells infected
with the hPLD1 baculovirus were incubated with phospholipid vesicles
containing [
P]PC, [
P]PE,
and [
P]PI under standard assay conditions. Some
of the assays contained 2% ethanol. Phospholipids were analyzed by TLC
as described in the text and detected by autoradiography. Markers
indicate the position of PA and phosphatidylethanol (PEtOH).
PC and PI are observed at the bottom of the TLC plate; most of the
uncleaved PE migrates a short distance away from the
origin.
Figure 3:
Effects of PIP, oleate, and
ARF1 on hPLD1 activity. PLD activity associated with membranes from Sf9
cells infected with the hPLD1 baculovirus was determined under standard
assay conditions except that (A) the PIP
content
of the vesicles was varied or (B) varying amounts of sodium
oleate or (C) varying amounts of ARF1 were added to the
incubations. 50 µM GTP
S was included in all assays
containing exogenously added ARF1. The data shown are means ±
S.E. of triplicate determinations. Standard errors fell within the
symbols for some data points.
0.1 µg of membrane protein was
present in each sample. hARF-1, human ARF1. D, ARF1
activation of hPLD1 expressed in mammalian cells. COS-7 cells were
transfected using Lipofectamine (Life Technologies, Inc.) with 3 µg
of LacZ or hPLD1 driven by a cytomegalovirus promoter and assayed after
48 h for PLD activity in the presence or absence or exogenously
supplied ARF1 and GTP
S.
Numerous reports have implicated monomeric G-proteins as regulators of PLD activities in a variety of mammalian cells and tissues(2, 4, 5, 6) . We found that recombinant human ARF1 strongly activates hPLD1 expressed in either baculovirus or mammalian cells (Fig. 3, C and D) and does so with a magnitude comparable with that previously reported.
PLD activities have been detected in essentially all
organisms (2) . Mammalian PLD activities in many tissues and
cell lines have been studied in detail, although no clear biochemical
classification of their properties has emerged. Moreover, attempts to
devise such a classification have been complicated by reports of
activities that differ in their subcellular localization, metal ion
dependence, phospholipid and detergent requirements in exogenous
substrate assays, and activation by various G-proteins and unidentified
accessory proteins(2) . Failure to isolate any of the enzymes
in sufficient purity has left this matter unresolved. The PLD enzyme we
have identified has properties that clearly implicate it as being
responsible for at least part of the PLD activity previously observed
in cell extracts and partially purified preparations. It is possible
that some of the other PLD activities that have been described are
mediated by additional members of the hPLD1 gene family. We have
identified a second mammalian PLD gene (60% identical; data not
shown), presenting an immediate possibility for another part of the
reported human activity. Moreover, the existence of at least two human
genes suggests that at least one reason that related but different
properties have been reported for PLD has been that the activity
obtained from different cell lines or tissues or using different
purification techniques is actually composed of different mixtures of
at least two distinct gene products that may have different biochemical
properties and/or requirements for activation.
ARF-activated PLD is present in Golgi vesicles(22) . The demonstration that hPLD1 is activated by ARF1 lends support to the hypothesis that PLD and specifically hPLD1 are involved in intravesicular membrane trafficking. Previous reports using partially purified PLD had raised the possibility that PLD and ARF interact directly(6, 9) . Our results extend previous efforts by using a single recombinant, purified protein as a PLD source, as well as recombinant, purified ARF as the activator. The data strengthen the hypothesis that interaction between ARF and hPLD1 is direct, although rigorous proof will require further experiments. Preliminary results suggest that at least some members of the Rho/Rac family of small G-proteins activate hPLD1 as well (data not shown). It will be important to determine which of the numerous small G-proteins reported to activate endogenous PLD actually activate hPLD1 to a significant extent.
Preliminary in vivo expression studies of PLD1 (and the second mammalian gene) in mouse embryos indicate that expression is detected at high levels in different selected regions of the brain and spinal cord (data not shown). These results raise the possibility that the PLD genes may also play a role in selected signal transduction events. In addition, there may be other members of the mammalian PLD gene family that are as of yet unidentified. The work presented in this paper paves the way for a molecular definition of the PLD enzymes. Ultimately, this advance should provide essential information for future studies designed to reveal the cellular and physiological function of these enzymes and their products.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U38545[GenBank].