Clive P. Morgan

PHOSPHOLIPASE D AND MEMBRANE TRAFFIC. POTENTIAL ROLES IN REGULATED EXOCYTOSIS, MEMBRANE DELIVERY AND VESICLE BUDDING

It is now well-established that phospholipase D is transiently stimulated upon activation by G-protein-coupled and receptor tyrosine kinase cell surface receptors in mammalian cells. Over the last 5 years, a tremendous effort has gone to identify the major intracellular regulators of mammalian phospholipase D and to the cloning of two mammalian phospholipase D enzymes (phospholipase D1 and D2). In this chapter, we review the physiological function of mammalian phospholipase D1 that is synergistically stimulated by ADP ribosylation factor, Rho and protein kinase Calpha. We discuss the function of this enzyme in membrane traffic, emphasising the possible integrated relationships between consumption of vesicles in regulated exocytosis, membrane delivery and constitutive membrane traffic.

ADP-ribosylation Factor and Rho Proteins Mediate fMLP-dependent Activation of Phospholipase D in Human Neutrophils

Activation of intact human neutrophils by fMLP stimulates phospholipase D (PLD) by an unknown signaling pathway. The small GTPase, ADP-ribosylation factor (ARF), and Rho proteins regulate the activity of PLD1 directly. Cell permeabilization with streptolysin O leads to loss of cytosolic proteins including ARF but not Rho proteins from the human neutrophils. PLD activation by fMLP is refractory in these cytosol-depleted cells. Readdition of myr-ARF1 but not non-myr-ARF1 restores fMLP-stimulated PLD activity. C3 toxin, which inactivates Rho proteins, reduces the ARF-reconstituted PLD activity, illustrating that although Rho alone does not stimulate PLD activity, it synergizes with ARF. To identify the signaling pathway to ARF and Rho activation by fMLP, we used pertussis toxin and wortmannin to examine the requirement for heterotrimeric G proteins of the Gi family and for phosphoinositide 3-kinase, respectively. PLD activity in both intact cells and the ARF-restored response in cytosol-depleted cells is inhibited by pertussis toxin, indicating a requirement for Gi2/Gi3 protein. In contrast, wortmannin inhibited only fMLP-stimulated PLD activity in intact neutrophils, but it has no effect on myr-ARF1-reconstituted activity. fMLP-stimulated translocation of ARF and Rho proteins to membranes is not inhibited by wortmannin. It is concluded that activation of Gi proteins is obligatory for ARF/Rho activation by fMLP, but activation of phosphoinositide 3-kinase is not required.

Regulation of PI3K signalling by the phosphatidylinositol transfer protein PITPα during axonal extension in hippocampal neurons

Phosphatidylinositol transfer proteins (PITPs) mediate the transfer of phosphatidylinositol (PtdIns) or phosphatidylcholine (PtdCho) between two membrane compartments, thereby regulating the interface between signalling, phosphoinositide (PI) metabolism and membrane traffic. Here, we show that PITPα is enriched in specific areas of the postnatal and adult brain, including the hippocampus and cerebellum. Overexpression of PITPα, but not PITPβ or a PITPα mutant deficient in binding PtdIns, enhances laminin-dependent extension of axonal processes in hippocampal neurons, whereas knockdown of PITPα protein by siRNA suppresses laminin and BDNF-induced axonal growth. PITPα-mediated axonal outgrowth is sensitive to phosphoinositide 3-kinase (PI3K) inhibition and shows dependency on the Akt/GSK-3/CRMP-2 pathway. We conclude that PITPα controls the polarized extension of axonal processes through the provision of PtdIns for localized PI3K-dependent signalling.

EGF Regulation of PITP Dynamics Is Blocked by Inhibitors of Phospholipase C and of the Ras-MAP Kinase Pathway

Phosphatidylinositol transfer proteins (PITP) function in signal transduction and in membrane traffic. Studies aimed at elucidating the mechanism of action of PITP have yielded a singular theme; the activity of PITP stems from its ability to transfer phosphatidylinositol (PI) from its site of synthesis to sites of cellular activity and to stimulate the local synthesis of phosphorylated forms of PI. The participation of various phosphoinositides in EGF signal transduction and in the trafficking of the EGF receptors is well documented. Using fluorescence lifetime imaging microscopy (FLIM) to measure fluorescence resonance energy transfer (FRET) between EGFP-PITP proteins and fluorescently labeled phospholipids, we report that PITPalpha and PITPbeta can dynamically interact with PI or PC at the plasma membrane when stimulated with EGF. Additionally, PITPbeta is localized at the Golgi, and EGF stimulation resulted in enhanced FRET. Inhibitors of the PLC and the Ras/MAP kinase pathway were both able to inhibit the EGF-stimulated interaction of PITPalpha with PI at the plasma membrane. The mobility of PITP proteins was determined by using fluorescence recovery after photobleaching (FRAP), and EGF stimulation reduced the mobility at the plasma membrane. We conclude that the dynamic behavior of PITPalpha and PITPbeta in vivo is a regulated process involving multiple mechanisms.

Identification of phospholipase B from Dictyostelium discoideum reveals a new lipase family present in mammals, flies and nematodes, but not yeast.

The social amoeba Dictyostelium discoideum exhibits high activities of phospholipase and lysophospholipase [Ferber, Munder, Fischer and Gerisch (1970) Eur. J. Biochem. 14, 253-257]. We assayed Dictyostelium lysates to demonstrate the presence of a highly active phospholipase B (PLB) enzyme that removed both fatty-acid chains from phosphatidylcholine and produced the water-soluble glycerophosphorylcholine. We purified the PLB activity from Dictyostelium cytosol using standard agarose media (size exclusion and ion exchange), and combined this with an affinity purification step using myristoylated ARF1 (ADP-ribosylation factor 1), a protein which has a single fatty acid at its N-terminus. Two proteins co-purified (48 kDa and 65 kDa), and the 48 kDa protein was digested with trypsin, peptide fragments were separated by reverse-phase chromatography, and the resultant peptides were sequenced by Edman degradation. From the peptide sequences obtained, database searches revealed a gene which encodes a protein of 65 kDa with unknown function. The 48 kDa protein therefore appears to be a fragment of the full-length 65 kDa product. Expression of the gene in Escherichia coli confirmed that it encodes a PLB. Characterization of its substrate specificity indicated that, in addition to phosphatidylcholine deacylation, the enzyme also hydrolysed phosphatidylinositol and phosphatidylethanolamine. The PLB identified in the present study is not related to existing PLBs found in bacteria, fungi or mammals. There are, however, genes similar to Dictyostelium PLB in mammals, flies, worms and Giardia, but not in yeast. We therefore have identified a novel family of intracellular PLBs.

Subcellular localisation of ARF1-regulated phospholipase D in HL60 cells

Phospholipase D (PLD) is a ubiquitous enzyme found in cells and tissues from a wide variety of species. PLD is activated in response to the occupation of many cell-surface receptors including those of the heterotrimeric G-protein, and tyrosine kinase families (Cockcroft, 1992; Billah, 1993; Exton, 1994). It is one of a family of phospholipases which include isozymes of the phospholipase A2 (PLA2), and phospholipase C (PLC) families. PLD catalyses the hydrolysis of phosphatidylcholine (PC) to form phosphatidic acid (PA) and free choline. Alternatively, in the presence of a short chain alcohol, such as ethanol, a phophatidylalcohol is produced in preference to PA. PA has been implicated as an activator of PKCξ (PKCξ) (Limatola et al., 1994), type II phosphatidylinositol 4-phosphate 5-kinase (PI4P 5-K) (Jenkins et al., 1994), along with a novel form of serine-threonine kinase (Khan et al., 1994). PA can also readily be converted to diacylglycerol (DAG) by the enzyme phosphatidate phosphohydrolase (PAP) and may thus also function to activate other PKC isoforms although there is some evidence that the fatty acid composition of PA derived from PC is not suitable as an activator of PKC (Leach et al., 1991).