Inositol Phospholipids: Properties & Biological Function

Article By Karol S. Bruzik

One of the most significant discoveries of the last two decades of the 20^th century was the finding that multiple cellular signal transduction pathways are mediated by inositol phospholipids and inositol phosphates (IP_n) as intracellular second messengers.¹ Once deemed obscure minor components of biological membranes, phosphatidylinositols (PtdIns) are now known to be involved in the transduction of the vast array of extracellular signals ranging from neurotransmitters to growth hormones.^1,2 Due to the broad scope of these signaling events, several thousand research publications related to phosphatidylinositol phosphates (PIP_n) are published every year.³ Much of this research activity is made possible by the availability of synthetic phosphoinositides, such as those listed below.^4,5

Of the two possible enantiomers of the unsymmetrically substituted inositol residue, only the molecules in which the phosphatidate residue is attached to the D-1 position of myo-inositol are physiologically active. Most naturally occurring PIP_n that participate in signal transduction pathways in mammalian cells feature an inositol ring phosphorylated at the 3, 4, and/or 5 positions (see Fig. 1). Rapid generation of such an array of molecules and facile interconversions between its members may be the basis for the omnipresence of PIP_n in the signaling phenomena. The molecules that are nonphosphorylated at the 3, 4 and 5 positions can also be glycosylated at the 6 position forming glycosylphosphatidylinositol (GPI).⁸ GPIs play a multitude of other roles, such as protein and protozoan VSG anchoring⁹ and serving as precursors of insulin mediators.¹⁰

In addition, the mammalian PIP_n carry stearic and arachidonic acid esters at the glycerol sn-1 and sn-2 positions, respectively.¹¹ The composition of PIP_n isolated from plants vary from those present in mammalian tissues in that they contain a linoleoyl residue at the sn-2 position and a palmitoyl residue at the sn-1 position. Due to the synthetic expediency, the majority of commercially available PIP_n are supplied as unnatural analogs containing saturated fatty acid chains, with either long (dipalmitoyl) or shorter chains (dioctanoyl or dihexanoyl). These saturated analogs are not only easier to synthesize, but are also more stable chemically, since they do not undergo peroxidation reactions common to unsaturated fatty acids. While more "user friendly", these compounds may, however, not reproduce all biological functions of natural PIP_n.

Biosynthesis of Phosphatidylinositols

Phosphatidylinositol (Compound 1 in Figures 1 and 2) is the most abundant member of the PIP_n family and is a precursor to all known PIP_n. This compound is biosynthesized from myo-inositol and CDP-diacylglycerol by phosphatidylinositol synthetase localized in the endoplasmic reticulum.¹² Further PtdIns metabolism involves sequential phosphorylations at the 3,¹³ 4,¹⁴ and 5 positions¹⁵ to provide increasingly polar compounds (see Fig. 1 and Fig. 2). In addition, PtdIns is subject to degradation by phospholipases C (PLCs), as well as PLA₂s. Although most mammalian PLCs prefer phosphatidylinositol 4,5 phosphate (PtdIns-4,5-P₂) and PtdIns-4-P over PtdIns,¹⁶ there are isolated reports of PtdIns-specific PLs in some mammalian tissues.¹⁷ In contrast to mammalian PtdIns-PLC, the bacterial enzymes display strict specificity for unphosphorylated PtdIns.¹⁶ PIP_n that bear a phosphate group at the inositol 3-position are generally regarded as resistant to PLC cleavage.¹⁸ PtdIns also seems refractive to hydrolysis by two major isozymes of mammalian PLD.¹⁹ PtdIns is also subject to complete deacylation by sequential action of PLA and lysophospholipase (LPA) to generate glycerophosphoinositols,²⁰ recently shown to inhibit invasion of cancer cells.²¹

PtdIns-3-P (Compound 3, Fig. 2) is generated from PtdIns by the action of class II and III PtdIns-3 kinase (PI3K)^22-24 and via hydrolysis of the 5-phosphate from PtdIns-3,5-P₂ by phosphatidylinositol phosphate 5-phosphatases SHIP.²⁵

PtdIns-4-P (Compound 4, Fig. 2) is the product of phosphorylation by four different species of PI4K.¹⁴ PtdIns-4-P is the second most abundant phospholipid (after PtdIns) in biological membranes, and is a precursor to PtdIns-4,5-P₂ and PtdIns-3,4,5-P₃, the two inositol lipids whose biological roles are understood best. The biological function of PtdIns-4-P is not as well known, however, studies in yeast indicate its role extends well beyond that of a simple substrate for PI5K.¹⁴ PtdIns-4-P is metabolized by the subsequent phosphorylation to PtdIns-4,5-P₂ or dephosphorylation to PtdIns by ER-localized phosphatase.¹⁴

PtdIns-5-P (Compound 2, Fig. 2) is formed from PtdIns¹⁵ or by the hydrolysis of 3-phosphate from PtdIns-3,5-P₂ by myotubularin, MTM1.²⁶ PtdIns-5-P can be further elaborated either by phosphorylation into PtdIns-4,5-P₂ by the β-form of PI4K,²⁷ or by dephosphorylation to PtdIns by the novel form of PTEN-like proteins with 5-phosphate selectivity.²⁸ Recent studies suggest a role for PtdIns-5-P in a variety of cellular events, such as tumor suppression and response to bacterial invasion.²⁸ This phospholipid has also recently been shown to function as a second messenger, binding to an Arapidopsis homolog of trithorax, thus suggesting that it may have a regulatory function connecting lipid-signaling with nuclear functions.²⁹

PtdIns-3,5-P₂ (Compound 5, Fig. 2) is the low-abundance, newest member of PIP_n family.²⁵ It is involved in mediation of a number of cellular processes such as vacuolar homeostasis, membrane trafficking, and vesicular protein sorting.^25,30 The recently discovered PtdIns-3,5-P₂ effectors include a family of β-propeller, epsin, and CHMP protein families.³¹ The importance of PtdIns-3,5-P₂ in human physiology is demonstrated by its role in insulin signaling, myotubular myopathy, and corneal dystrophy.³¹

PtdIns-3,4-P₂ (Compound 6, Fig. 2) is biosynthesized by phosphorylation of PtdIns-4-P at the 3-position,^2a by dephosphorylation of PtdIns-3,4,5-P₃ at the 5-position by the SHIP phosphatase,³² and phosphorylation of PtdIns-3-P by a type II 4-kinase.³³ The significance of this lipid is underscored by the association between SHIP₂ gene polymorphism and type 2 diabetes mellitus. SHIP₂ therefore represents an important target for treatment of both type 2 diabetes and obesity.³²

PtdIns-4,5-P₂ (Compound 7, Fig. 2) is the third most abundant inositol phospholipid in biological membranes. The predominant biosynthetic pathway is via phosphorylation of PtdIns-4-P by a 5-kinase.^1h This inositol lipid is best known for its participation in receptor-mediated cleavage by mammalian PLC-β, -γ, -δ, and -ε to produce the second messenger inositol 1,4,5-trisphosphate (1,4,5-IP₃),^34,35 thus initiating an extremely complex network of inositol phosphates, including formation of several second messengers, such as 1,3,4,5-IP₄, 1,3,4-IP₃, and 3,4,5,6-IP₄.^1e,36 The biological role of PtdIns-4,5-P₂ extends beyond generation of 1,4,5-IP₃, since it is also a substrate for type I 3-kinases ultimately providing PtdIns-3,4,5-P₃, another important signaling molecule.³⁷ In addition, PtdIns-4,5-P₂ is specifically recognized by pleckstrin homology domains of many important proteins (see Table 1, pg. 2), including a number of enzymes (e.g., PLD),³⁸ thus providing an interesting cross-talk between the two lipolytic activities. Furthermore, association of certain transmembrane receptors with PtdIns-4,5-P₂ affects their functional activity. For example, interaction of vanilloid receptors with PtdIns-4,5-P₂ is necessary for receptor desensitization.³⁹ PtdIns-4,5-P₂ therefore plays and important role in both human health and disease.⁴⁰

PtdIns-3,4,5-P₃ (Compound 8, Fig. 2) is the most phosphorylated among inositol phospholipids and a key factor linked to cell growth, survival, and differentiation.⁴¹ The metabolic levels of PtdIns-3,4,5-P₃ are maintained in a major part by phosphorylation of PtdIns-4,5-P₂ by the type-I PI3K, and its hydrolysis by two 3-phosphoinositide phosphatases, PTEN and SHIP, that leads to the regeneration of PtdIns-4,5-P₂ and formation of one more second messenger, PtdIns-3,4-P₂, respectively.³⁷ Strong evidence suggests that PtdIns-3,4,5-P₃ is an important player of signaling pathways in the nucleus. Recent results also indicate that nuclear translocation of cell surface receptors could activate nuclear PI3K suggesting a new pathway of signal transduction.³⁷ The most important cellular function of PtdIns-3,4,5-P₃ at the moment seems to be its strong interaction with the pleckstrin homology (PH) domain of Akt causing translocation of Akt to the plasma membrane where it becomes phosphorylated and activated. Activated Akt then phosphorylates downstream cellular proteins that promote cell proliferation and survival ultimately resulting in tumorigenesis. The proper concentration of PtdIns-3,4,5-P₃ is therefore pivotal to cell homeostasis and its elevated levels promote carcinogenesis.⁴²

Phosphoinositide-binding Protein Domains

A major advance in understanding phosphoinositide signaling has been the discovery of hundreds of highly conserved modular protein domains that bind various PtdIns phosphates. These domains are tethered ("pasted") into a diverse array of multidomain proteins in an effort to translocate the host protein to specific regions of membranes via their binding to inositol phopsholipids.⁴³ Due to the presence of these highly specific, high-affinity phosphoinositide-binding domains in proteins, phosphoinositides can act as signal mediators in a time- and space-resolved manner. The change in concentration of a specific inositol phospholipid results in membrane sequestration of cytosolic proteins equipped with the corresponding specific phosphoinositol-binding module. The sequestration of such proteins to the membrane is frequently a starting point of complex intracellular events such as cytoskeletal rearrangement and membrane trafficking. The PH domain was the first identified phosphoinositide-binding domain, represented in a number of proteins that form signaling complexes on the plasma membrane. The discovery of new modules that bind phosphoinositides have increased dramatically in recent years, and they are universally found in proteins involved in intracellular trafficking and cytoskeletal remodeling. Recently discovered phosphoinositide-binding motifs include FYVE, PX, and ENTH, indicating functional versatility of phosphoinositides in signaling events.⁴⁴ Analysis of phosphoinositide binding by these domains has provided significant insight into the mechanism of protein recruitment by the membranes enriched with different phosphoinositides. For example, domains that interact with minor inositol phospholipids such as 3-phosphoinositols must display high affinity and structural specificity. This could be achieved by headgroup interactions alone as is the case of certain PH domains that bind PtdIns-3,4,5-P₃ and/or PtdIns-3,4-P₂, or by combined headgroup interactions with membrane-insertion, such as in the case of the PX and FYVE domains that bind PtdIns-3-P. In contrast, the domains that target a much more abundant membrane constituent, PtdIns-4,5-P₂, do not require as high affinity or specificity, and tend to be structurally more diverse.^45,46 Specificity of the phosphoinositide binding domains is summarized in Table 1.

In conclusion, the field of cellular signaling via inositol phospholipids has been rapidly revealing daunting complexity of signaling events, their interdependence and redundancy. Given the history of the field, we can be all but assured that new unexpected findings are just around the corner.

References

1. Seminal reviews on phosphoinositide metabolism:
   1. Berridge, M. J., Irvine, R. F., Nature 312, 315-321 (1984).
   2. Hokin, R. H., Ann. Rev. Biochem. 54, 205-235 (1985).
   3. Michell, R. H., Nature 19, 176-177 (1986).
   4. Majerus, P. W., Conolly, T. M., Bansal, V. S., et al J. Biol. Chem. 263, 3051-3054 (1988).
   5. Berridge, M. J., Ann. Rev. Biochem. 56, 159-193 (1987).
   6. Berridge, M. J., Irvine, R. F., Nature 341, 197-205 (1989).
   7. Clark, E. A., Brugge, J. S., Science 268, 233-239 (1995).
   8. Duckworth, B. C., Cantley, L. C. in Handbook of Lipid Research, Bell, R. M., Exton, J. H., Prescott, S. M., editors, Plenum Press, New York 8, 125 (1996).
   9. Tolias, K. F., Cantley, L. C., Chem. Phys. Lipids 98, 69-77 (1999).
2. Specific roles of phosphoinositides:
   1. Rameh, L. E., Cantley, L. C., J. Biol. Chem. 274, 8347-50 (1999).
   2. Hinchliffe, K. A., Ciruela, A., Irvine, R. F., Biochim. Biophys. Acta 1436, 87-104 (1998).
   3. Brown, F. D., Rozelle, A. L., Yin, H. L., et al J. Cell. Biol. 154, 1007-17 (2001).
   4. Takenawa. T., Itoh, T. Biochim., Biophys. Acta. 1533, 190-206 (2001).
   5. Exton, J. H., Annu. Rev. Pharmacol. Toxicol. 36, 481-509 (1996).
   6. Cremona, O., De Camilli, P., J. Cell. Sci. 114, 1041-52 (2001).
   7. Jones, D. R., Varela-Nieto, I., Mol. Med. 5, 505-14 (1999).
3. The 2005 search of Chemical Abstract Service database under the "inositol" keyword resulted in 1500 to 3200 hits during each year between 1986 and 2004.
4. Reviews on synthesis of phosphoinositides:
   1. Potter, B. L. V., Nat. Prod. Rep. 1-24, (1990).
   2. Billington, D. C., Chem. Soc. Rev. 18, 83-122 (1989).
   3. Potter, B. V. L., Lampe, D., Angew. Chem. Int. Ed. Engl. 34, 1933-1972 (1995).
   4. Billington, D. C., "The Inositol Phosphates: Chemical Synthesis and Biological Significance" , VCH, Weinheim (1993).
   5. Prestwich, G. D., Acc. Chem. Res. 29, 503-513 (1996).
5. 1. Inositol Phosphates and Derivatives. Synthesis, Biochemistry, and Therapeutic Potential, Reitz, A. B. Ed., ACS Symp. Ser. 463 (1991).
   2. "Phosphoinositides: Chemistry, Biochemistry and Biomedical Applications", Bruzik, K. S. Ed., ACS Symp. Ser. 718 (1998).
6. IUPAC-IUB Commission on Biochemical Nomenclature (CBN), Chem. Phys. Lipids 2, 156-67 (1968).
7. Parthasarathy, R., Eisenberg, F. Jr., Biochem. J. 235, 313-2 (1986).
8. Ferguson, M. A., J. Cell. Sci. 112, 2799-809 (1999).
9. Zacks, M. A., Garg, N., Mol. Membr. Biol. 23, 209-25 (1999).
10. Larner, J., Int. J. Exp. Diabetes Res. 3, 47-60 (2002).
11. Allan, D., Cockroft, S., Biochem. J. 213, 555-7 (1983).
12. Gardocki, M. E., Jani, N., Lopes, J. M., Biochim. Biophys. Acta 1735, 89-100 (2005).
13. Cantley, L. C., Science 296, 1655-1657 (2002).
14. Balla, A., Bala, T., Trends Cell Biol. 16, 351-361 (2006).
15. Giudici, M. L., Hinchliffe, K. A., Irvine, R. F., J. Endocrinol. Inv. 27, 137-42 (2004).
16. Bruzik, K. S., Tsai, M.-D., Bioorg. Med. Chem. 2, 49-72 (1994).
17. Perrella, F. W., Jankiewicz, R., Dandrow, E. A., Biochim. Biophys. Acta 1076, 209-14 (1991).
18. Serunian, L. A., Haber, M. T., Fukui, T., Kim, et al J. Biol. Chem. 264, 17809-15 (1989).
19. Pettitt, T. R., McDermott, M., Saqib, K. M., et al Biochem. J. 360, 707-15 (2001).
20. Falasca, M., Marino, M., Carvelli, A., et al Eur. J. Biochem. 241, 386-92 (2001).
21. Buccione, R., Baldasarre, M., Trapani, V., et al Eur. J. Cancer 41, 470-6 (2005).
22. Traer, C. J., Foster, F. M., Abraham, F. M., et al Bull Cancer 93, E53-8 (2006).
23. Erneux, C., Govaerts, C., Communi, D., Pesesse, X., Biochim. Biophys. Acta 1436, 185-99 (1998).
24. Wymann, M. P., Pirola, L., Biochim. Biophys. Acta 1436, 127-50 (1998).
25. Cook, F. T., Arch. Bioch. Biophys. 407, 143-151 (2002).
26. Tronchere, H., Laporte, J., Pendaries, C., et al J. Biol. Chem. 279, 7304-12 (2004).
27. Doughman, R. R., Firestone, A. J., Anderson, R. A., J. Membr. Biol 194, 77-89 (2003).
28. Pagliarini, D. J., Worby, C.A., Dixon, J. E., J. Biol. Chem. 279, 38950-6 (2004).
29. Alvarez-Venegas, R., Sadder, M., Hlavacka, A., et al Proc. Natl. Acad. Sci. USA 103, 6049-54 (2006).
30. Dove, S. K., Cooke, F. T., Douglass, M. R., et al Nature 390 187-92, 1997 ($year).
31. Michell, R. H., Heath, V. L., Lemmon, M. A., et al Trends Biochem. Sci. 31, 52-63 (2006).
32. Dyson, J. M., Kong, A. M., Wiradjaja, F., et al Int. J. Biochem. Cell Biol. 37, 2260-5 (2005).
33. Banfic, H., Tang, X., Batty, I. H., et al J. Biol. Chem. 273, 13-16 (1998).
34. Rebecchi, M. J., Pentyala, S. N., Physiol. Rev. 80, 1291-335 (2000).
35. Rhee, S. G., Annu. Rev. Biochem. 70, 281-312 (2001).
36. Shears, S. B., Biochim. Biophys. Acta 1436, 49-67 (1998).
37. Deleris, P., Gayral, S., Breton-Douillon, M., J. Cell. Biochem. 98, 469-85 (2006).
38. Henage, L. G., Exton, J. H., Brown, H. A., J. Biol. Chem. 281, 3408-17 (2006).
39. Liu, B., Zhang, C., Qin, F., J. Neurosci. 25, 4835-43 (2005).
40. Halstead, J. R., Jalink, K., Divecha, N., Trends Pharmacol. Sci. 26, 654-660 (2005).
41. Wymann, M. P., Marone, R., Curr. Opin. Cell Biol. 17, 141-9 (2005).
42. Robertson, G. P., Cancer Metastasis Rev. 24, 273-85 (2005).
43. Cullen, P. J., Cozier, G. E., Banting, G., Curr. Biol. 11, R882-93 (2001).
44. Itoh, T, Takenawa, T., Cell Signal. 14, 733-43 (2002).
45. Lemmon, M. A., Traffic 4, 201-13 (2003).
46. Cho, W., Stahelin, R. V., Biochim. Biophys. Acta Epub. , July 26 (2006).