Andrew J. Morris

Phosphatidylinositol 4-Phosphate 5-Kinase α Is a Downstream Effector of the Small G Protein ARF6 in Membrane Ruffle Formation

Synthesis of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], a signaling phospholipid, is primarily carried out by phosphatidylinositol 4-phosphate 5-kinase [PI(4)P5K], which has been reported to be regulated by RhoA and Rac1. Unexpectedly, we find that the GTPgammaS-dependent activator of PI(4)P5Kalpha is the small G protein ADP-ribosylation factor (ARF) and that the activation strictly requires phosphatidic acid, the product of phospholipase D (PLD). In vivo, ARF6, but not ARF1 or ARF5, spatially coincides with PI(4)P5Kalpha. This colocalization occurs in ruffling membranes formed upon AIF4 and EGF stimulation and is blocked by dominant-negative ARF6. PLD2 similarly translocates to the ruffles, as does the PH domain of phospholipase Cdelta1, indicating locally elevated PI(4,5)P2. Thus, PI(4)P5Kalpha is a downstream effector of ARF6 and when ARF6 is activated by agonist stimulation, it triggers recruitment of a diverse but interactive set of signaling molecules into sites of active cytoskeletal and membrane rearrangement.

Phospholipase D2, a distinct phospholipase D isoform with novel regulatory properties that provokes cytoskeletal reorganization.

BACKGROUND Activation of phospholipase D (PLD) is an important but poorly understood component of receptor-mediated signal transduction responses and regulated secretion. We recently reported the cloning of the human gene encoding PLD1; this enzyme has low basal activity and is activated by protein kinase C and the small GTP-binding proteins, ADP-ribosylation factor (ARF), Rho, Rac and Cdc42. Biochemical and cell biological studies suggest, however, that additional and distinct PLD activities exist in cells, so a search was carried out for novel mammalian genes related to PLD1. RESULTS We have cloned the gene for a second PLD family member and characterized the protein product, which appears to be regulated differently from PLD1: PLD2 is constitutively active and may be modulated in vivo by inhibition. Unexpectedly, PLD2 localizes primarily to the plasma membrane, in contrast to PLD1 which localizes solely to peri-nuclear regions (the endoplasmic reticulum, Golgi apparatus and late endosomes), where PLD activity has been shown to promote ARF-mediated coated-vesicle formation. PLD2 provokes cortical reorganization and undergoes redistribution in serum-stimulated cells, suggesting that it may have a role in signal-induced cytoskeletal regulation and/or endocytosis. CONCLUSIONS PLD2 is a newly identified mammalian PLD isoform with novel regulatory properties. Our findings suggest that regulated secretion and morphological reorganization, the two most frequently proposed biological roles for PLD, are likely to be effected separately by PLD1 and PLD2.

Characterization of Two Alternately Spliced Forms of Phospholipase D1 ACTIVATION OF THE PURIFIED ENZYMES BY PHOSPHATIDYLINOSITOL 4,5-BISPHOSPHATE, ADP-RIBOSYLATION FACTOR, AND RHO FAMILY MONOMERIC GTP-BINDING PROTEINS AND PROTEIN KINASE C-α

We previously reported the cloning of a cDNA encoding human phosphatidylcholine-specific phospholipase D1 (PLD1), an ADP-ribosylation factor (ARF)-activated phosphatidylcholine-specific phospholipase D (Hammond, S. M., Tsung, S., Autschuller, Y., Rudge, S. A., Rose, K., Engebrecht, J., Morris, A. J., and Frohman, M. A. (1995) J. Biol. Chem. 270, 29640-29643). We have now identified an evolutionarily conserved shorter splice variant of PLD1 lacking 38 amino acids (residues 585-624) that arises from regulated splicing of an alternate exon. Both forms of PLD1 (PLD1a and 1b) have been expressed in Sf9 cells using baculovirus vectors and purified to homogeneity by detergent extraction and immunoaffinity chromatography. PLD1a and 1b have very similar properties. PLD1a and 1b activity is Mg2+dependent but insensitive to changes in free Ca2+ concentration. Phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate activate PLD1a and 1b but a range of other acidic phospholipids are ineffective. PLD1a and 1b are highly responsive to activation by GTP-γS-liganded ADP-ribosylation factor-1 (ARF-1) and can also be activated to a lesser extent by three purified RHO family monomeric GTP-binding proteins, RHO A, RAC-1, and CDC42. Activation of PLD1a and 1b by the RHO family monomeric GTP-binding proteins is GTP-dependent and synergistic with ARF-1. Purified protein kinase C-α activates PLD1a and 1b in a manner that is stimulated by phorbol esters and does not require ATP. Activation of PLD1a and 1b by protein kinase C-α is synergistic with ARF and with the RHO family monomeric GTP-binding proteins, suggesting that these three classes of regulators interact with different sites on the enzyme.

Mutagenesis of phospholipase D defines a superfamily including a trans-Golgi viral protein required for poxvirus pathogenicity

Phospholipase D (PLD) genes are members of a superfamily that is defined by several highly conserved motifs. PLD in mammals has been proposed to play a role in membrane vesicular trafficking and signal transduction. Using site‐directed mutagenesis, 25 point mutants have been made in human PLD1 (hPLD1) and characterized. We find that a motif (HxKxxxxD) and a serine/threonine conserved in all members of the PLD superfamily are critical for PLD biochemical activity, suggesting a possible catalytic mechanism. Functional analysis of catalytically inactive point mutants for yeast PLD demonstrates that the meiotic phenotype ensuing from PLD deficiency in yeast derives from a loss of enzymatic activity. Finally, mutation of an HxKxxxxD motif found in a vaccinia viral protein expressed in the Golgi complex results in loss of efficient vaccinia virus cell‐to‐cell spreading, implicating the viral protein as a member of the superfamily and suggesting that it encodes a lipid modifying or binding activity. The results suggest that vaccinia virus and hPLD1 may act through analogous mechanisms to effect viral cellular egress and vesicular trafficking, respectively.

Mammalian phospholipase D structure and regulation.

The recent identification of cDNA clones for phospholipase D1 and 2 has opened the door to new studies on its structure and regulation. PLD activity is encoded by at least two different genes that contain catalytic domains that relate their mechanism of action to phosphodiesterases. In vivo roles for PLD suggest that it may be important for multiple specialized steps in receptor dependent and constitutive processes of secretion, endocytosis, and membrane biogenesis.

Phospholipase D1: a key factor for the exocytotic machinery in neuroendocrine cells

Phospholipase D (PLD) has been proposed to mediate cytoskeletal remodeling and vesicular trafficking along the secretory pathway. We recently described the activation of an ADP ribosylation factor‐regulated PLD at the plasma membrane of chromaffin cells undergoing secretagogue‐stimulated exocytosis. We show here that the isoform involved is PLD1b, and, using a real‐time assay for individual cells, that PLD activation and exocytosis are closely correlated. Moreover, overexpressed PLD1, but not PLD2, increases stimulated exocytosis in a phosphatidylinositol 4,5‐bisphosphate‐dependent manner, whereas catalytically inactive PLD1 inhibits it. These results provide the first direct evidence that PLD1 is an important component of the exocytotic machinery in neuroendocrine cells.

Human Type 2 Phosphatidic Acid Phosphohydrolases SUBSTRATE SPECIFICITY OF THE TYPE 2a, 2b, AND 2c ENZYMES AND CELL SURFACE ACTIVITY OF THE 2a ISOFORM

Phosphatidic acid (PA), lysophosphatidic acid, ceramide 1-phosphate (C1P), and sphingosine 1-phosphate (S1P) are lipid mediators generated by phospholipases, sphingomyelinases, and lipid kinases. The major pathway for degradation of these lipids is dephosphorylation catalyzed by members of two classes (types 1 and 2) of phosphohydrolase activities (PAPs). cDNAs encoding two type 2 PAPs, PAP-2a and -2b, have been expressed by transient transfection and shown to catalyze hydrolysis of PA, C1P, and S1P (Kai, M., Wada, I., Imai, S., Sakane, F. and Kanoh, H. (1997) J. Biol. Chem. 272, 24572–24578). We report the cloning and expression of a third type 2 PAP enzyme (288 amino acids, predicted molecular mass of 32.6 kDa), PAP-2c, which exhibits 54 and 43% sequence homology to PAPs 2a and 2b. Expression of HA epitope-tagged PAP-2a, -2b, and 2c in HEK293 cells produced immunoreactive proteins and increased membrane-associated PAP activity. Sf9 insect cells contain very low endogenous PAP activity. Recombinant expression of the three PAP enzymes using baculovirus vectors produces dramatic increases in membrane-associated Mg2+-independent,N-ethylmaleimide-insensitive PAP activity. Expression of PAP-2a but not PAP-2b or -2c resulted in high levels of cell surface PAP activity in intact insect cells. Kinetic analysis of PAP-2a, -2b, and -2c activity against PA, lysophosphatidic acid, C1P, and S1P presented in mixed micelles of Triton X-100 revealed differences in substrate specificity and susceptibility to inhibition by sphingosine, Zn2+, and propranol.

Structure and regulation of phospholipase D

Phosphar:S~:~oline-specific phos>holipase D (PLDs) are phosphodi‘stemses that catalyse the hydrolysis If phosphatidylcholine (PC) to pmjute phosphatidic acid (PA) and :holine. Diverse stimuli mcreasePLD activity in many cells and PA, or a netabolite of this lipid, is probably he orimarv mediator of PLD-initia , ited cell signalltng. Investigations of PLD regulation irt oitro suggest that .hese enzymes may play roles in nhacellular protein trafficking and nitogenic signaIling processes conrolled by members of the ADPribosylatibn factor (ARFj anJ Rho :amilies of monomeric G uroteins. l’he identification of genes encoding veast and human PLD enzymes xomises to provide the molecular tools necessy this reaction concurrmtly generates Ivso-Dhomhatidic acid. which can e of receptor-mechated PLD activation has been presumed to be the plasma membrane there are convincing reports of PLD activities associated with the nucleus and Co& apparat&?

Integral membrane lipid phosphatases/phosphotransferases: common structure and diverse functions

Phospholipids and sphingolipids play critical roles in signal transduction, intracellular membrane trafficking, and control of cell growth and survival. We discuss recent progress in the identification and characterization of a family of integral membrane proteins with central roles in bioactive lipid metabolism and signalling. These five groups of homologous proteins, which we collectively term LPTs (lipid phosphatases/phosphotransferases), are characterized by a core domain containing six transmembrane-spanning alpha-helices connected by extramembrane loops, two of which interact to form the catalytic site. LPT family members are localized to all major membrane compartments of the cell. The transmembrane topology of these proteins places their active site facing the lumen of endomembrane compartments or the extracellular face of the plasma membrane. Sequence conservation between the active site of the LPPs (lipid phosphate phosphatases), SPPs (sphingosine phosphate phosphatases) and the recently identified SMSs (sphingomyelin synthases) with vanadium-dependent fungal oxidases provides a framework for understanding their common catalytic mechanism. LPPs hydrolyse LPA (lysophosphatidic acid), S1P (sphingosine 1-phosphate) and structurally-related substrates. Although LPPs can dephosphorylate intracellularly generated substrates to control intracellular lipid metabolism and signalling, their best understood function is to regulate cell surface receptor-mediated signalling by LPA and S1P by inactivating these lipids at the plasma membrane or in the extracellular space. SPPs are intracellularly localized S1P-selective phosphatases, with key roles in the pathways of sphingolipid metabolism linked to control of cell growth and survival. The SMS enzymes catalyse the interconversion of phosphatidylcholine and ceramide with sphingomyelin and diacylglycerol, suggesting a pivotal role in both housekeeping lipid synthesis and regulation of bioactive lipid mediators. The remaining members of the LPT family, the LPR/PRGs (lipid phosphatase-related proteins/plasticity-related genes) and CSS2s (type 2 candidate sphingomyelin synthases), are presently much less well studied. These two groups include proteins that lack critical amino acids within the catalytic site, and could therefore not use the conserved LPT reaction mechanism to catalyse lipid phosphatase or phosphotransferase reactions. In this review, we discuss recent ideas about their possible biological activities and functions, which appear to involve regulation of cellular morphology and, possibly, lipid metabolism and signalling in the nuclear envelope.

Roles for lipid phosphate phosphatases in regulation of cellular signaling.

Lipid phosphate phosphatases (LPPs) are a family of integral membrane glycoproteins that catalyze the dephosphorylation of a number of bioactive lipid mediators including lysophosphatidic acid (LPA), sphingosine 1-phosphate (S1P) and phosphatidic acid (PA). These mediators exert complex effects on cell function through both actions at cell surface receptors and on intracellular targets. The LPP-catalyzed dephosphorylation of these substrates can both terminate their signaling actions and itself generate further molecules with biological activity. Recent advances have revealed that a family of structurally related genes is responsible for LPP activities in species from yeast to mammals. These genes exhibit distinct but overlapping expression patterns and their products appear to be heterogeneous with respect to their posttranslational modification and subcellular localizations. Here we review the structure and catalytic properties of the LPPs and consider recent developments in understanding their cellular biology and functions.