Vicki A. Sciorra

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.

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.

Identification of a phosphoinositide binding motif that mediates activation of mammalian and yeast phospholipase D isoenzymes

Phosphoinositides are both substrates for second messenger‐generating enzymes and spatially localized membrane signals that mediate vital steps in signal transduction, cytoskeletal regulation and membrane trafficking. Phosphatidylcholine‐specific phospholipase D (PLD) activity is stimulated by phosphoinositides, but the mechanism and physiological requirement for such stimulation to promote PLD‐dependent cellular processes is not known. To address these issues, we have identified a site at which phosphoinositides interact with PLD and have assessed the role of this region in PLD function. This interacting motif contains critical basic amino acid residues that are required for stimulation of PLD activity by phosphoinositides. Although PLD alleles mutated at this site fail to bind to phosphoinositides in vitro, they are membrane‐associated and properly localized within the cell but are inactive against cellular lipid substrates. Analogous mutations of this site in yeast PLD, Spo14p, result in enzymes that localize normally, but with catalytic activity that has dramatically reduced responsiveness to phosphoinositides. The level of responsiveness to phosphoinositides in vitro correlated with the ability of PLD to function in vivo. Taken together, these results provide the first evidence that phosphoinositide regulation of PLD activity observed in vitro is physiologically important in cellular processes in vivo including membrane trafficking and secretion.

Dual role for phosphoinositides in regulation of yeast and mammalian phospholipase D enzymes.

Phospholipase D (PLD) generates lipid signals that coordinate membrane trafficking with cellular signaling. PLD activity in vitro and in vivo is dependent on phosphoinositides with a vicinal 4,5-phosphate pair. Yeast and mammalian PLDs contain an NH2-terminal pleckstrin homology (PH) domain that has been speculated to specify both subcellular localization and regulation of PLD activity through interaction with phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2). We report that mutation of the PH domains of yeast and mammalian PLD enzymes generates catalytically active PI(4,5)P2-regulated enzymes with impaired biological functions. Disruption of the PH domain of mammalian PLD2 results in relocalization of the protein from the PI(4,5)P2-containing plasma membrane to endosomes. As a result of this mislocalization, mutations within the PH domain render the protein unresponsive to activation in vivo. Furthermore, the integrity of the PH domain is vital for yeast PLD function in both meiosis and secretion. Binding of PLD2 to model membranes is enhanced by acidic phospholipids. Studies with PLD2-derived peptides suggest that this binding involves a previously identified polybasic motif that mediates activation of the enzyme by PI(4,5)P2. By comparison, the PLD2 PH domain binds PI(4,5)P2 with lower affinity but sufficient selectivity to function in concert with the polybasic motif to target the protein to PI(4,5)P2-rich membranes. Phosphoinositides therefore have a dual role in PLD regulation: membrane targeting mediated by the PH domain and stimulation of catalysis mediated by the polybasic motif.

Lysophosphatidic acid signaling: how a small lipid does big things

Lysophosphatidic acid (LPA) promotes growth, differentiation, survival and motility in many different cell types. LPA has therefore been suggested to play a central role in a broad range of physiological and pathophysiological processes, including vascular and neuronal function and cancer. Three closely related G-protein-coupled cell-surface receptors mediate some of these effects, but assigning specific functions to particular receptor subtypes has been challenging and several lines of evidence indicate that other LPA signaling mechanisms might exist. Although the signaling actions of LPA have been studied widely, much less is known about how LPA is generated and released into the extracellular space, and how its signaling actions are terminated. Newly identified enzymes that generate and inactivate LPA have novel roles in cancer progression and early development, and a recent study indicates that LPA might regulate nuclear gene transcription directly. These findings provide novel insights into mechanisms involved in the synthesis, actions and inactivation of LPA, and the proteins involved provide new targets that can be exploited to manipulate LPA signaling at both cellular and organismal levels.

G-protein-coupled receptor regulation of phospholipase D.

Phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine (PC) to generate phosphatidic acid (PA) and choline. PA has been implicated in signal transduction, membrane vesicular trafficking, cytoskeleton reorganization, and cell proliferation. PLD activity is present in ninny mammalian cells and tissues and is upregulated in response to a wide variety of agonists that signal through heterotrimeric G-protein-coupled or tyrosine kinase receptors. Receptor stimulation initiates multiple signal transduction cascades, ultimately including activation of protein kinase C (PKC), ADP-ribosylation factor (ARF), and Rho family members, which have been well characterized as activators of PLD in in vitro and in in vivo assay systems. Because ARK, Rho, and PKC stimulate multiple downstream effector pathways that ultimately regulate cellular morphology, proliferation, and secretion, there has been intense interest in determining the relationship of PLD stimulation through each activator to these cell biological events. The regulation of mammalian PLD by G-protein-coupled receptors is complex and not fully understood at present.