Phosphatidic Acid & Related Lipids

1.  Phosphatidic Acid – Occurrence and Biosynthesis

Phosphatidic acid or 1,2-diacyl-sn-glycero-3-phosphate is not an abundant lipid constituent of any living organism to my knowledge, but it is extremely important as an intermediate in the biosynthesis of triacylglycerols and phospholipids and as a signalling molecule. Indeed, it is often overestimated in tissues as it can arise by inadvertent enzymatic hydrolysis during inappropriate storage or extraction conditions during analysis. It is the simplest diacyl-glycerophospholipid, and the only one with a phosphomonoester as the head group. The molecule is acidic and carries a negative charge, i.e. it is an anionic lipid.

Structural formula of phosphatidic acid

The main biosynthetic route in plant and animal tissues is discussed in greater detail in in another page on this site, but in brief it involves sequential acylation of α-glycerophosphate, derived from catabolism of glucose, by acyl-CoA derivatives of fatty acids as illustrated. Specific acyltransferases catalyse first the acylation of position sn-1 to form lysophosphatidic acid (1-acyl-sn-glycerol-3-phosphate) and then of position sn-2 to yield phosphatidic acid.

Biosynthesis of phosphatidic acid

In mammals, the glycerol-3-phosphate acyltransferase that catalyses the first step exists in four isoforms, two in mitochondria (designated GPAT1 and 2) and two in the endoplasmic reticulum (GPAT3 and 4). The activity in the endoplasmic reticulum predominates in adipose tissue, but the mitochondrial forms are responsible for half the activity in liver. All are membrane-bound enzymes, which are believed to span the membranes, but many questions remain regarding the regulation and function of the different isoforms. Similarly, at least two acyl-CoA:lysophosphatidic acid acyltransferases (LPAAT1 and LPAAT2) are known that catalyse the second step. Human LPAAT1 showed higher activity with 14:0-, 16:0- and 18:2-CoAs, while LPAAT2 prefers 20:4-CoA.

A second biosynthetic pathway in animals utilizes dihydroxyacetone phosphate (DHAP) as the primary precursor and the peroxisomal enzyme, DHAP acyltransferase, which produces acyl-DHAP. This intermediate is converted to lysophosphatidic acid in a NADPH-dependent reaction catalysed by acyl-DHAP reductase, and this is in turn acylated to form phosphatidic acid. This pathway is of particular importance in the biosynthesis of ether lipids.


Figure 10

In bacteria, two families of enzymes are responsible for acylation of position sn-1 of glycerol-3-phosphate. One present in Escherichia coli, for example, uses the acyl-acyl carrier protein products of fatty acid synthesis as acyl donors as well as acyl-CoA derived from exogenous fatty acids. The second set of enzymes makes use of the unique acyl donor, acyl-phosphate derived from acyl-ACP, and is present in a wider range of bacteria. Acylation of position sn-2 is performed by a further family of enzymes that uses acyl-acyl carrier protein as the acyl donor, although some bacterial species may use acyl-CoA also.

Under some conditions, phosphatidic acid can be generated from 1,2-diacyl-sn-glycerols by the action of diacylglycerol kinases (see our web page on diacylglycerols). Such enzymes appear to be ubiquitous in nature, although those in bacteria and yeast are structurally different from the mammalian enzymes. The latter, at least ten isoforms of which exist with different subcellular locations and functions, use ATP as the phosphate donor. Aside from producing phosphatidic acid for phospholipid production or signalling, these enzymes may attenuate the signalling effects of diacylglycerols.


Biosynthesis of phosphatidic acid by diacylglycerol kinases

However, a more important route in quantitative terms is via hydrolysis of other phospholipids, but especially phosphatidylcholine, by the enzyme phospholipase D (or by a family or related enzymes of this kind). Such enzymes are present in most animal cell types and they are especially important in plants. The activity of the animal enzyme is regulated by phosphatidylinositol-4,5-bisphosphate, which binds to it and is an essential cofactor, together with a number of proteins including protein kinase C.

Generation of phosphatidic acid by the action of phospholipase D

phospholipid biosynthetic pathwaysThe subsequent steps in the utilization of phosphatidic acid in the biosynthesis of triacylglycerols and phospholipids are described in separate documents in this section of the web site or better in the separation sections dealing with animal and plant biochemistry.

In brief, hydrolysis of phosphatidic acid by the enzyme phosphatidate phosphatase is the source of sn-1,2-diacylglycerols (DAG), which are the precursors for the biosynthesis of triacylglycerols (TAG), phosphatidylcholine (PC) and phosphatidylethanolamine (PE) via the so-called Kennedy pathway (also of monogalactosyldiacylglycerols in plants). Via reaction with cytidine triphosphate, phosphatidic acid is the precursor of cytidine diphosphate diacylglycerol, which is the key intermediate in the synthesis of phosphatidylglycerol (PG), and thence of cardiolipin, and of phosphatidylinositol (PI) and phosphatidylserine (PS). Depending on the organism and other factors, phosphatidylserine can be a precursor for phosphatidylethanolamine. Similarly, the latter can give rise to phosphatidylcholine by way of mono- and dimethyl-phosphatidylethanolamine intermediates.

While the fatty acid composition of phosphatidic acid can resemble that of the eventual products, the latter are generally much altered by remodelling after synthesis via deacylation-reacylation reactions (the 'Lands cycle’).


2.  Phosphatidic Acid - Biological Functions in Animals

In addition to its role as an intermediate in lipid biosynthesis, the phosphatidic acid generated by the action of phospholipase D and by diacylglycerol kinases may have signalling functions as a second messenger, although it is not certain whether all the activities suggested by studies in vitro operate in vivo. Nonetheless, phosphatidic acid has been implicated in many aspects of animal cell biochemistry and physiology, including cell proliferation and differentiation, cell transformation, tumor progression and survival signalling. It appears to regulate some membrane trafficking events, and it is involved in activation of the enzyme NADPH oxidase, which operates as part of the defence mechanism against infection and tissue damage during inflammation. It may have a role in promoting phospholipase A2 activity, and it appears to function in vesicle formation and transport within the cell. By binding to targeted proteins, including protein kinases, protein phosphatases and G-proteins, it may increase or inhibit their activities. For example in yeast, phosphatidic acid in the endoplasmic reticulum binds directly to a specific transcriptional repressor to keep it inactive outside the nucleus; when the lipid precursor inositol is added, this phosphatidic acid is rapidly depleted, releasing the transcriptional factor so that it can be translocated to the nucleus where it is able to repress target genes. The overall effect is a mechanism to control phospholipid synthesis. In addition, the murine phosphatidylinositol 4-phosphate 5-kinase does not appear to function unless phosphatidic acid is bound to it.

In relation to signalling activities, it should be noted that phosphatidic acid can be metabolized to sn-1,2-diacylglycerols or to lysophosphatidic acid (see next section), both of which have distinctive signalling functions in their own right. Conversely, both of these compounds can be in effect be deactivated by conversion back to phosphatidic acid. In many cell types, vesicle trafficking, secretion and endocytosis may also require phosphatidic acid derived by the action of phospholipase D.

Some of these effects may be explained simply by the physical properties of phosphatidic acid, which has a propensity to form a hexagonal II phase, especially in the presence of calcium ions. Thus, hydrolysis of phosphatidylcholine, a cylindrical nonfusogenic lipid, converts it into cone-shaped fusogenic phosphatidic acid, which promotes negative membrane curvature. It differs from other anionic phospholipids in that its small anionic phosphomonoester head group lies very close to the hydrophobic interior of the lipid bilayer. Phosphatidic acid can effect membrane fusion in model systems, probably because of its ability to form non-bilayer phases.

Also of relevance in this context is its overall negative charge, and it is not always clear whether some of the observed biological effects are specific to phosphatidic acid or simply to negatively charged phospholipids in general. However, it has been demonstrated that the positively charged lysine and arginine residues on proteins can bind with some specificity to phosphatidic acid through hydrogen bonding with the phosphate group, thus distinguishing it from other phospholipids. An ‘electrostatic-hydrogen bond switch model’ has been proposed in which the head group of phosphatidic acid forms a hydrogen bond to a basic amino acid residue, leading to de-protonation of the head group, increasing its negative charge from -1 to -2 and thus enabling stronger interactions with further basic residues and tight docking with the membrane interacting protein.



3.  Phosphatidic Acid – Biological Functions in Plants

The diacylglycerol signalling pathway is believed to be relatively insignificant in plants. Instead, phosphatidic acid is the key plant lipid second messenger, which is rapidly and transiently generated in response to many different biotic and abiotic stresses. The main source of phosphatidic acid for this purpose is the action of phospholipase D on membrane phospholipids, such as phosphatidylcholine and phosphatidylethanolamine. Plants contain a large number of related enzymes of this type, 12 in Arabidopsis and 17 in rice, in comparison with two in humans and one in yeast. In plants, they can be classified into two groups, depending on their lipid-binding domains, with some homologous with the human and yeast enzymes, but most belonging to a second class that contains a characteristic ‘C2’ (calcium- and lipid-binding) domain. Phosphatidic acid can also be produced by the sequential action of phospholipase C and diacylglycerol kinase on membrane phospholipids, with diacylglycerols as an intermediate (there are 7 isoenzymes in Arabidopsis thaliana). Whether phosphatidic acid molecules from the two pathways elicit different responses is an open question.

Phospholipase D activity and the phosphatidic acid produced have long been recognized as of importance during germination and senescence, and they have an important role in response to stress damage and pathogen attack, both in higher plants and in green algae. A high content of phosphatidic acid induced by phospholipase D action during wounding or senescence brings about a loss of the membrane bilayer phase, as a consequence of the conical shape of this phospholipid in comparison to the cylindrical shape of structural phospholipids. As a result, cells lose their viability. The phosphatidic acid generated in this way is broken down further by phosphatases, acyl-hydrolases and lipoxygenases into fatty acids and other small molecules, which are subsequently absorbed and recycled. In addition, phosphatidic acid is important in the response to other forms of stress, including osmotic stress (salinity or drought), cold and oxidation. Although much remains to be learned of the mechanism by which it exerts its effects, it is believed to promote the response to the hormone abscisic acid.

Phosphatidic acid is of considerable importance in cellular signalling in plants, often acting in concert with phosphatidylinositol 4,5-bisphosphate. As in mammalian cells, targets for such signalling include protein kinases and phosphatases, in addition to proteins involved in membrane trafficking and the organization of the cytoskeleton, and it can both activate or inhibit enzymes. It is involved in promoting the growth of pollen-tubes and root hairs, decreasing peroxide-induced cell death, and mediating the signalling processes that lead to responses to ethylene and again to the plant hormone abscisic acid. Thus in the 'model' plant Arabidopsis,  phosphatidic acid interacts with a protein phosphatase to signal the closure of stomata promoted by abscisic acid; it interacts also with a further enzyme to mediate the inhibition of stomatal opening effected by abscisic acid. Together these reactions constitute a signalling pathway that regulates water loss from plants. A further difference from animal metabolism is that diacylglycerol pyrophosphate can be synthesised from phosphatidic acid in plants (see below).

The signalling functions are terminated mainly by enzymes that dephosphorylate phosphatidic acid, such as lipid phosphate phosphatases and phosphatidic acid hydrolases (lipins).


4.  Lysophosphatidic Acid

Structural formula of lysophosphatidic acidLysophosphatidic acid or 1-acyl-sn-glycerol-3-phosphate differs from phosphatidic acid in having only one mole of fatty acid per mole of lipid. As such, it is the simplest possible glycerophospholipid. Molecular species with both saturated and unsaturated fatty acid constituents occur in different tissues. Although it is present at very low levels only in animal tissues, it is extremely important biologically, influencing many biochemical processes. These activities seem to be shared by the 1-alkyl- and alkenyl-ether forms. The additional hydroxyl group in comparison to phosphatidic acid enables hydrogen bonding within membranes, which may be relevant to its function. Lysophosphatidic acid is important as the biosynthetic precursor of phosphatidic acid, but there is particular interest in its role as an intercellular lipid mediator with growth factor-like activities. For example, it is rapidly produced and released from activated platelets to influence target cells.

The most important source is the activity of a specific lysophospholipase D known as ‘autotaxin’ on lysophosphatidylcholine (200 μM in plasma) to yield lysophosphatidic acid in an albumin-bound form. This is more abundant in serum (1-5 μM) than in plasma, where it accounts for much of the biological activity. A similar route operates in adipocytes. Autotaxin is a member of the nucleotide pyrophosphatase/phosphodiesterase family and is also present in cerebrospinal and seminal fluids.

Biosynthesis of lysophosphatidic acid

It is now established that lysophosphatidic acid is produced intracellularly by a wide variety of cell types by various mechanisms with phosphatidic acid as the primary precursor. Hydrolysis of phosphatidic acid by a lysophospholipase A2 is the main mechanism in platelets, but other cellular enzymes include a phosphatidic acid-selective lysophospholipase A1 producing an sn-2-acyl-lysophosphatidic acid, monoacylglycerol kinase and glycerol-3-phosphate acyltransferase. A surprising recent finding is that the activity of the phosphatidic acid-selective lysophospholipase A1 is essential for normal hair growth in humans. On the other hand, it is possible that most of the lysophosphatidic acid produced intracellularly is used for synthesis of other phospholipids rather than for signalling purposes. Autotaxin is the primary source of extracellular lysophosphatidic acid.

Although lysophospholipids are relatively small molecules, they carry a high content of information through the nature of the phosphate head group, the positional distribution of the fatty acids on the glycerol moiety, the presence of ether or ester linkages to the glycerol backbone, and the chain length and degree and position of saturation of the fatty acyl chains. This informational content leads to selectivity in the functional relationship with cell receptors. Most mammalian cells express receptors for lysophosphatidic acid, and lysophosphatidic acid may initiate signalling in the cells in which it is produced, as well as affecting neighbouring cells. In the last few years, the characterization of cloned lysophosphatidic acid receptors in combination with strategies of molecular genetics has allowed determination of both signalling and biological effects that are dependent on receptor mechanisms. At least six G-protein-coupled receptors that are specific for lysophosphatidic acid have now been identified, each found in particular organs. They are only present in vertebrates. Experimental activation of these receptors has shown that a range of downstream signalling cascades mediate lysophosphatidic acid signalling. These include activation of protein kinases, adenyl cyclase and phospholipase C, release of arachidonic acid, and much more. There is evidence that lysophosphatidic acid is involved in cell survival in some circumstances, and in programmed cell death in others. In some instances, molecular species with specific fatty acid components may be involved.

Lysophosphatidic acid signalling has regulatory functions in the mammalian reproductive system, both male and female, facilitating oocyte maturation and spermatogenesis, for example. There is also evidence that the lipid is involved in brain development, through its activity in neural progenitor cells, neurons and glia, and in vascular remodelling.

There is particular interest in the activity of lysophosphatidic acid in various disease states, where intervention in its metabolism has the potential for beneficial health effects. For example, a finding that lysophosphatidic acid is markedly elevated in the plasma of ovarian cancer patients, compared to healthy controls may be especially significant. In particular, elevated plasma levels were found in patients in the first stage of ovarian cancer, suggesting that it may represent a useful marker for the early detection of the disease. Lysophosphatidic acid is believed to stimulate DNA synthesis and the proliferation of ovarian cancer cells, it may induce cell migration, and there is evidence that signalling by lysophosphatidic acid is causally linked to hyperactive lipogenesis. Increased autotaxin expression has been demonstrated in many different cancer cell lines, and the expression of many of the surface receptors for lysophosphatidic acid in cancer cells is aberrant. Therefore, lysophosphatidic acid metabolism is a target of the pharmaceutical industry for cancer therapy.

In addition, lysophosphatidic acid generated by the action of a lysophospholipase D is believed to play an important role in reproductive biology. Under certain conditions, it can become athero- and thrombogenic and might aggravate cardiovascular disease. As oxidized low-density lipoproteins promote the production of lysophosphatidic acid, its content in atherosclerotic plaques is high, suggesting that it might serve as a biomarker for cardiovascular disease. Indeed, lysophosphatidic acid promotes pro-inflammatory events that lead to the development of atheroma as well encourage progression of the disease. By mediating platelet aggregation, it could lead to arterial thrombus formation. There is evidence that it is involved in such inflammatory diseases as rheumatoid arthritis and multiple sclerosis.

Lysophosphatidic acid has been found in saliva in significant amounts, and it has been suggested that it is involved in wound healing in the upper digestive organs such as the mouth, pharynx, and oesophagus. It has similar effects when applied topically to skin wounds, probably by stimulating proliferation of new cells to seal the wound.

Other lysophospholipids and especially the sphingolipid analogue, sphingosine-1-phosphate, show a related range of activities.

Catabolic deactivation of lysophosphatidic acid is accomplished by dephosphorylation to monoacylglycerol by a family of three lipid phosphate phosphatases, which also dephosphorylate sphingosine-1-phosphate, phosphatidic acid and ceramide 1-phosphate in a nonspecific manner. It can be converted back to phosphatidic acid by a membrane-bound O-acyltransferase (MBOAT2) specific for lysophosphatidic acid (and lysophosphatidylethanolamine) with a preference for oleoyl-CoA as substrate.


5.  Cyclic Phosphatidic Acid

Structural formula of cyclic phosphatidic acidCyclic phosphatidic acid (sometimes termed ‘cyclic lysophosphatidic acid’) was isolated originally from a slime mould, but has now been detected in a wide range of organisms including humans, especially in the brain but also bound to albumin in serum (at a concentration of 10-7M, or a tenth that of lysophosphatidic acid). It has a cyclic phosphate at the sn-2 and sn-3 positions of the glycerol carbons, and this structure is absolutely necessary for its biological activity. It is most abundant in tissues subject to injury. In human serum, the main molecular species contains palmitic acid.

Studies of the biosynthesis of cyclic phosphatidic acid in fetal bovine serum suggest that it is the product of an enzyme related to the human enzyme autotaxin, the serum lysophospholipase D that produces lysophosphatidic acid (see above), or to phospholipase D2. This enzyme appears to produce cyclic phosphatidic acid in serum by an intramolecular transphosphatidylation reaction. However, it can also be formed artefactually by the addition of strong acid to serum.

While cyclic phosphatidic acid may have some similar signalling functions to lysophosphatidic acid per se in that it binds to some of the same receptors, it also has some quite distinct activities in animal tissues. For example, cyclic phosphatidic acid is known to be a specific inhibitor of DNA polymerase alpha. It has an appreciable effect on the inhibition of cancer cell invasion and metastasis, a finding that is currently attracting great pharmacological interest. In addition, it inhibits the platelet aggregation induced by lysophosphatidic acid, and it inhibits the nuclear hormone receptor PPARγ with high specificity.


6.  Pyrophosphatidic Acid

Structural formula of pyrophosphatidic acidPyrophosphatidic acid or sn-1,2-diacylglycero-3-pyrophosphate is an unusual and little-known phospholipid that was first identified as a minor component in yeasts, and is also know to be present in mushrooms and higher plants as a product of the enzyme phosphatidic acid kinase, which is present in all plant tissues but especially the plasma membrane.

It is rapidly metabolized back to phosphatidic acid by a specific phosphatase and thence to diacylglycerols, and it may have a function in the phospholipase D signalling cascade in plants, perhaps by attenuating the effects of phosphatidic acid. Pyrophosphatidic acid is barely detectable in nonstimulated plant cells but its concentration increases very rapidly in response to stress situations, including osmotic stress and attack by pathogens. Such findings add to the belief that it is an important signalling molecule in plants under stress. In yeasts, it may have a role in the regulation of the synthesis and metabolism of phospholipids, especially phosphatidylserine.


7.  Analysis

Phosphatidic acid and related lipids are not the easiest to analyse. On adsorption chromatography, retention times tend to be variable and may be dependent to some extent on the nature of the cations associated with the acidic lipids. However, two-dimensional TLC can give good results. Phosphatidic acid, lysobisphosphatidic acid and pyrophosphatidic acid are never easy to distinguish, but modern liquid chromatography-mass spectrometric methods appear to be the answer..


Recommended Reading

  • Blaho, V.A. and Hla, T. Regulation of mammalian physiology, development, and disease by the sphingosine 1-phosphate and lysophosphatidic acid receptors. Chem. Rev., 111, 6299–6320 (2011) (DOI: 10.1021/cr200273u).
  • Christie, W.W. and Han, X. Lipid Analysis - Isolation, Separation, Identification and Lipidomic Analysis (4th edition), 446 pages (Oily Press, Bridgwater, U.K. and Woodhead Publishing Ltd, Cambridge, U.K.) (2010) - Woodhead Publishing Ltd.
  • Fujiwara, F. Cyclic phosphatidic acid - a unique bioactive phospholipid. Biochim. Biophys. Acta, 1781, 519-524 (2008) (DOI: 10.1016/j.bbalip.2008.05.002).
  • Haucke, V. and Di Paolo, G. Lipids and lipid modifications in the regulation of membrane traffic. Curr. Opin. Cell Biol., 19, 426-435 (2007) (DOI: 10.1016/
  • Kooijman, E.E. and Burger, K.N.J. Biophysics and function of phosphatidic acid: A molecular perspective. Biochim. Biophys. Acta, 1791, 881-888 (2009) (DOI: 10.1016/j.bbalip.2009.04.001).
  • Nakanaga, K., Hama, K. and Aoki, J. Autotaxin - an LPA producing enzyme with diverse functions. J. Biochem. (Tokyo), 148, 13-24 (2010) (DOI: 10.1093/jb/mvq052).
  • Pyne, S. Lysolipids: sphingosine-1-phosphate and lysophosphatidic acid. In: Bioactive Lipids. pp. 37-61. (edited by A. Nicolaou and G. Kokotos, The Oily Press, Bridgwater) (2004).
  • Selvy, P.E., Lavieri, R.R., Lindsley, C.W. and Brown, H.A. Phospholipase D: enzymology, functionality, and chemical modulation. Chem. Rev., 111, 6064–6119 (2011). (DOI: 10.1021/cr200296t)
  • Sevastou, I., Kaffe, E., Mouratis, M.A. and Aidinis, V. Lysoglycerophospholipids in chronic inflammatory disorders: The PLA2/LPC and ATX/LPA axes. Biochim. Biophys. Acta, 131, 42-60 (2013) (DOI: 10.1016/j.bbalip.2012.07.019).
  • Shindou, H. and Shimizu, T. Acyl-CoA:lysophospholipid acyltransferases. J. Biol. Chem., 284, 1-5 (2009) (DOI: 10.1074/jbc.R800046200).
  • Stace, C.L. and Ktistakis, N.T. Phosphatidic acid- and phosphatidylserine-binding proteins. Biochim. Biophys. Acta, 1761, 913-926 (2006) (DOI: 10.1016/j.bbalip.2006.03.006).
  • Testerink, C. and Munnik, T. Molecular, cellular, and physiological responses to phosphatidic acid formation in plants. J. Exp. Botany, 62, 2349-2361 (2011) (DOI: 10.1093/jxb/err079).
  • Tokumura, A. Physiological and pathophysiological roles of lysophosphatidic acids produced by secretory lysophospholipase D in body fluids. Biochim. Biophys. Acta, 1582, 18-25 (2002) - and various other articles in this volume.
  • Vance, D.E. and Vance, J. (Editors) Biochemistry of Lipids, Lipoproteins and Membranes (5th Edition). (Elsevier Science, Amsterdam) (2008) - several chapters.
  • van Schooten, B., Testerink, C. and Munnik, T. Signalling diacylglycerol pyrophosphate, a new phosphatidic acid metabolite. Biochim. Biophys. Acta, 1761, 151-159 (2006) (DOI: 10.1016/j.bbalip.2005.12.010).
  • Wang, X., Devaiah, S.P., Zhang, W. and Welti, R. Signaling functions of phosphatidic acid. Prog. Lipid Res., 45, 250-278 (2006) (DOI: 10.1016/j.plipres.2006.01.005).
  • Yao, J.W. and Rock, C.O. Phosphatidic acid synthesis in bacteria. Biochim. Biophys. Acta, 1831, 495-502 (2013) (DOI: 10.1016/j.bbalip.2012.08.018).


 Updated: May 1st, 2014