Michael Wakelam
Tel. (01223) 496202

• Contact via email


• Recent, selected Publications
• Group Members

Phosphatidic-acid-mediated signalling

The hydrolysis of phosphatidylcholine to phosphatidic acid is catalysed by PLD1 and PLD2. The regulation of cellular PLD activity is complex, and is dependent on stimulatory inputs from both phosphoinositides and cellular proteins, such as protein kinase C isoforms and members of the ARF and RHO families of small GTPases.

PLD isoforms contain adjacent PX and PH domains that have proven phosphoinositides-binding capability, and are important for proper cellular localisation and catalytic activity. In addition, PLD1 and PLD2 contain a polybasic motif that mediates phosphatidylinositol (4,5) bisphosphate-dependent activation. However despite PLD1 and PLD2 having identical domain structures, each PLD isoform exhibits cell-specific differences in cellular localisation and regulation. PLD1 has been reported to localise to Golgi, endosomes, lysosomes, and the plasma membrane. In addition PLD1 has been shown to localise to specialised vesicles, such as histamine granules in mast cells and GLUT4-containing vesicles in adipocytes. PLD2 predominantly localises to the plasma membrane, but has also been shown to localise to endosomes.

The regulation of PLD1 and PLD2 catalytic activity also differs dramatically. PLD1 is regulated primarily by cell-surface receptors through the actions of phosphoinositides, GTP-binding proteins and protein kinases. In contrast the regulation of PLD2 activity is poorly understood, but is thought to involve phosphoinositides and inhibitory interacting proteins. Our lab seeks to understand how each PLD isoform is regulated, through the identification of the molecular mechanisms that determine cellular localisation and catalytic activity.

In order to fully understand the physiological importance of PLD1 and PLD2, we are currently generating mice that no longer express one or both PLD genes. In addition we are generating mice in which the expression of either, or both PLD genes is ablated in specific organs and tissue. From the study of these animals we will obtain cell lines that no longer express PLD activity. These studies, in combination with of lentiviral shRNA methodology, will further our understanding of PLD signalling and function, and will allow us to understand how PLD function relates to other signalling pathways, such as phosphoinositide-mediated signalling.

Our laboratory also utilises the model organisms Saccharomyces cerevisae (budding yeast) and Dictyostelium discoideum (a social amoeba) to investigate the cellular functions of PLD.

Yeast that no longer express PLD are unable to form viable progeny after meiosis. First discovered a decade ago, this PLD phenotype is currently the best defined. The failure of PLD mutants to form viable progeny is due to a complete inability to synthesise intracellular membranes needed to package the haploid nuclei. Consequently, the study of yeast PLD will further our understanding of the precise role(s) of PLD in membrane trafficking and secretion.

The social amoeba, Dictyostelium discoideum, is a powerful model organism to study the processes of cell adhesion and motility. In collaboration with Dr Robert Insall (The Beatson Institute for Cancer Studies, UK), we have discovered that PLD activity in Dictyostelium discoideum is essential for cell motility. Further work will define the molecular mechanisms that drive phosphatidic-acid signalling required for cell motility.

The relative ease and speed in which these organisms can be genetically manipulated provides us with an ideal complement to our mouse work.

PLD superfamily

The completion of genomic sequencing in a host of organisms, has facilitated the identification of evolutionary conserved new members of the PLD family, i.e. PLD3, PLD4, and PLD5. These PLD-like proteins are poorly understood, but members of this family have been implicated in a variety of diverse processes, such as brain development and aging, neurotransmission and sperm maturation. In addition virus orthologues exist within the Orthopoxvirus genus, for these PLD-like proteins, that are required for virus maturation and release from the host cell. This suggests that the human PLD-like proteins may function in membrane trafficking pathways. However, since no PC-PLD catalytic activity has been demonstrated for these proteins, it is conceivable that the PLD-like proteins utilize other phospholipids as substrates or instead, exhibit novel catalytic and/or binding activities.

Our laboratory is undertaking the task of understanding the function of these PLD-like proteins in cells, and to determine how they function in relationship to PLD1 and PLD2. To achieve this aim we are using both the mouse model, and the fruit fly (in collaboration with Dr Raghu Padinjat).

Phosphatidic effector proteins

In stark contrast to the identification of phosphoinositide binding domains within proteins i.e. PH, PX, FYVE etc, no consensus PA binding domain has been identified. This has severely hampered the bioinformatic identification of putative PA-binding proteins from the analysis of genome sequences. To address this problem, we are currently determining the structures of PA-binding sites of known PA-binding proteins using X-ray crystallography and NMR.

Lyso-phosphatidic-acid-mediated signalling

Autotaxin (ATX), also known as ectonucleotide pyrophosphate-phospodiesterase 2 (ENPP2), is synthesised as a pre-pro-protein, which is proteolytically processed and secreted through the classical secretory pathway. Autotaxin was initially isolated as an autocrine motility factor for melanoma cells. Subsequently, it was shown to promote metastasis and tumour vascularization in nude mice and has been identified as an angiogenic factor. ATX expression is up-regulated in both invasive and metastatic cancers. This has led to the hypothesis that ATX facilitates tumour progression by stimulating the formation of an invasive and/or angiogenic microenvironment for both malignant and stromal cells.

ATX is a lysophospholipase D (lysoPLD) that hydrolyses lysophosphatidylcholine (LPC) to lysophosphatidic acid (LPA). LPA produced by ATX binds to specific cell surface receptors and regulates vascular function, cell proliferation, survival and migration. LPA and ATX are therefore important in the development and progression of human cancer.

Recent data from the lab has demonstrated a co-operative relationship between PLD3 and ATX, suggesting that PLD3 has a role in regulating the release of ATX from cells. Thus there exists a complex relationship between these phospholipases, which we seek to understand.

Lipidomics

Analysis of the cellular lipidome by mass spectrometry is now possible. Our work has pioneered a number of these methodologies, in particular the analysis of phosphoinositides. These studies have demonstrated the key role for the acyl chain structure of lipids in defining their cellular role. For instance polyunsaturated diacylglycerol species and monounsaturated phosphatidic acids are intracellular signaling lipids, other structures of these lipids may function as signals in vitro but play a metabolic role in intact cells. Analysis of lipid changes in intact cells have also identified signaling pathways operating in disease states e.g. in vasculitis. Work in the lab supported by Cancer Research UK is defining changes in lipids in cancer cells and tissues with the aim of identifying novel cancer-associated signaling pathways and thus potential therapeutic targets.

Recent, selected publications

Williams JM, Pettitt TR, Powell W, Grove J, Savage COS, Wakelam MJO (2007) Antineutrophil cytoplasm antibody–stimulated neutrophil adhesion depends on diacylglycerol kinase–catalyzed phosphatidic acid formation.
Journal of the American Society of Nephrology 18 1112-1120
http://dx.doi.org/10.1681/ASN.2006090973

Garcia-Murillas I, Pettit T, Macdonald E, Okkenhaug H, Georgiev P, Trivedi D, Hassan B, Wakelam MJO, Padinjat R (2006) Iazaro encodes a lipid phosphate phosphohydrolase that regulates phosphatidylinositol turnover during Drosophila phototransduction.
Neuron 49 533-546
http://dx.doi.org/10.1016/j.neuron.2006.02.001

Pettitt TR, Dove SK, Lubben A, Calaminus SDJ, Wakelam MJO (2006) Analysis of intact phosphoinositides in biological samples.
Journal of Lipid Research 47 1588-1596
http://dx.doi.org/10.1194/jlr.D600004-JLR200

Powner DJ, Payne RM, Pettitt TR, Giudici ML, Irvine RF, Wakelam MJO (2005) Phospholipase D2 stimulates integrin-mediated adhesion via phosphatidylinositol 4-phosphate 5-kinase Iγb.
Journal of Cell Science 118 2975-2986
http://dx.doi.org/10.1242/10.1242/jcs.02432

Powner DJ, Hodgkin MN, Wakelam MJO (2002) Antigen-stimulated activation of phospholipase D1b by Rac1, ARF6, and PKCα in RBL-2H3 cells.
Molecular Biology of the Cell 13 1252-1262
http://dx.doi.org/10.1091/mbc.01-05-0235

Taylor PM, Woodfield RJ, Hodgkin MN, Pettitt TR, Martin A, Kerr DJ, Wakelam MJO (2002) Breast cancer cell-derived EMMPRIN stimulates fibroblast MMP2 release through a phospholipase A2 and 5-lipoxygenase catalyzed pathway.
Oncogene 21 5765-5772
http://dx.doi.org/10.1038/sj.onc.1205702

Members:

Professor Michael Wakelam - Group leader and Institute Director - Contact by email
Dr. Simon Rudge – Snr post-doc - Contact by email
Dr. Qifeng Zhang – Post-doc - Contact by email
Dr. Yan-feng Dai – Post-doc - Contact by email
Kathy Hadfield-Moorhouse – Research Assistant - Contact by email
Chloe-Jane Wilde – Research Assistant
Gavin McNee – Graduate Student - Contact by email