Phosphoinositides have been in the limelight for over two decades following their identification as sources of the second messengers, diacylglycerol (DG), inositol(1,4,5) trisphosphate (IP3) and phosphatidylinositol(3,4,5)trisphosphate (PI(3,4,5)P3). The last 10 years have witnessed an explosion of experimental results that demonstrate inositol lipids themselves, in particularly PI(4,5)P2, should be considered as second messengers in their own right. The inositol headgroup of phosphatidylinositol (PtdIns) can be phosphorylated at single or multiple sites to give rise to seven different phosphoinositides.
Phosphoinositides can control many biological processes via their ability to interact with specific protein domains. Many domains including the PH, PX, ENTH domains can bind to specific species of phosphoinositides. This recruitment can be transient depending on the life-time of the phosphoinositides, which is governed by the kinases and the phosphatases which will regulate their levels in specific locations.
Proteins recruited in this manner can thus regulate membrane trafficking events including endocytosis, exocytosis, vesicle budding at the Golgi, actin assembly and other biological processes.
Our Research
Molecular and Cell Biology of Phosphatidylinositol transfer proteins
PITP α, a 35 kDa soluble protein, was first identified by its ability to mediate the transfer of PI or PC between membrane compartments. A second highly related phosphatidylinositol transfer protein (PITP β) has also been described. PITPα and PITPβ are the focus of our studies. These proteins are found in all mammalian cells and play essential roles in phosphoinositide-dependent signalling pathways. PITPs are known to be required for:
- Hydrolysis of PI(4,5)P2 by G-protein-regulated phospholipase Cβ, Ca2+ - regulated phospholipase Cδ, and by tyrosine-kinase regulated Phospholipase Cγ.
- Production of PI(3,4,5)P3, a lipid second messenger that recruits PH-domain-containing proteins
- Membrane trafficking events including vesicle formation and exocytosis.
PITPs are lipid binding proteins that allow for compartmentalized phosphoinositide signalling in cells. We are currently examining the molecular basis for PITP function using biochemical, molecular and cell biological techniques.
Regulation and Signalling Functions of Receptor-regulated Phospholipase D
Phospholipase D (PLD) enzymes are widely distributed in mammalian cells and are regulated by hormones, neurotransmitters, immunoreceptors, growth factors and cytokines. PLD catalyses the hydrolysis of the major membrane phospholipid, phosphatidylcholine (PC), to produce phosphatidic acid (PA) and choline. Two mammalian PLDs have been cloned, PLD1 and PLD2.
Regulation of PLD enzymes is complex and is in part mediated by the ARF and Rho families of small GTPases and by protein kinase C. We focus on two major questions: Receptor regulation of phospholipase D enzymes and the role of PA in cellular signalling.
The ARF family and their downstream effectors, phospholipase D and PIP 5-kinase
ARFs are small GTPases which belong to the Ras superfamily. There are 6 mammalian ARFs and they have multiple downstream effectors. ARF1 and ARF6 are the best-studied ARF proteins and although they show distinct localizations in cells, in vitro they can interact with identical downstream effectors. These include PLD, PIP 5-kinase α, arfophilin, arfaptin/POR, COPI (Coatomer protein), AP-1, AP-3, AP-4 (Adaptor Protein) , GGA1-3 (Golgi-localizing, gamma-adaptin ear homology domain, ARF binding proteins), cholera Toxin A1 and LTA1.
Ongoing studies include the identification of the effector domain in ARFs that is responsible for activating the lipid metabolizing enzymes, PLDs, PI4Kβ and PIP5Kα. ARFs stimulate PIP2 synthesis and localised production in living cells will be governed by the spatial juxtaposition of the appropriate regulators (ARFs) and their effectors (lipid kinases and PLD).
We are therefore engaged in co-localisation studies between ARF proteins (ARF1-6), phospholipase Ds (PLD1 and PLD2), and the specific lipid kinases using confocal microscopy in live cells. Other important questions include the effector proteins that are regulated by PA. The PLD pathway is required for exocytosis from neutrophils and mast cells and we are engaged in identifying the function of the ARF-regulated PLD pathway in exocytosis.
Biology of Phosphoinositides
Cockcroft, S. (Editor) (2000)
Biology of Phosphoinositides.
Frontiers in Molecular Biology 1-338 Oxford University Press
"Biology of Phosphoinositides is a review of current lipid signalling research with emphasis on the integration of the use of lipid signals in signal transduction and membrane trafficking. These two areas have traditionally been seen as separate but now anyone ignoring one area at the expense of the other does so at their peril."
"The regulation of phospholipase C (and its isozymes), phospholipase D, the phosphoinositide 3-kinases, chloride channel conductance by inositol (3,4,5,6) tetraphosphate, and of cytoskeletal protein activity by inositol lipids are all covered in depth. There is specific discussion of the PH and FVYE lipid binding domains that allow lipids to control the movement, location, and activation-state of membrane proteins. The central issue of the control of synthesis, translocation, and degradation of phosphoinositides is also given due coverage."
"The central role of lipids in cell function is now widely acknowledged. Biology of phosphoinositides serves both as an intriguing introduction to what these molecules can achieve, as well as update on current research finding for existing afficionados."
Edited by Shamshad Cockcroft
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