How is PtdIns(5)P Made?

For most phosphatidylinositides, the routes of synthesis and degradation have been largely elucidated.  However, due to difficulty in detecting PtdIns(5)P, only recently have investigators been able to assess the synthesis and degradation of this phospholipid.  The major stumbling block has been the separation of PtdIns(5)P from PtdIns(4)P, which migrate quite closely on HPLC/TLC based separations.  The two major advances in the field have been improved separation of these monophosphorylated lipids (for example see Sarkes and Rameh 2010 and Zolov et al. 2012) and separation-independent identification of PtdIns(5)P by an enzyme based phosphorylation assay. (see Jones et al., 2012).  I am a co-author on the Zolov paper and work closely with that group.

Which enzymes are involved?

Potential Routes for PtdIns(5)P Synthesis.

The simplest mechanism is through phosphorylation of PI directly by a PtdIns-5-Kinase.  There are two known classes of Ptdins-5-Kinases in mammalian cells, Pikfyve and three isoforms in the PtdIns(4)P-5-Kinase family (Pip5k1a, Pip5k1b and Pip5k1c).  Classically, Pikfyve is thought to convert PtdIns(3)P into PtdIns(3,5)P₂ wheras the other classes phosphorylate PtdIns(4)P into PtdIns(4,5)P₂.  I think that the strongest evidence is that Pifkve is essential for PtdIns(5)P levels in the cell, either directly or indirectly.

Biochemically, there seems to be three potential ways by which PtdIns(5)P could be made, through direct phosphorylation of PtdIns, or through dephosphorylation of either PtdIns(3,5)P₂ or PtdIns(4,5)P₂.  Of course, it is possible that in different contexts, each of these pathways could be involved.

Route 1: Direct Phosphorylation of PtdIns

Although there is limited evidence that the PtdIns(4)P-5-Kinases can phosphorylate PI, there is substantial evidence that PI(5)P can be generated by Pikfyve, in vitro (Sbrissa et. al, 1999).  Inside cells, it is less clear whether this is the case.  There is rapid and tightly correlated turnover of both PtdIns(3,5)P₂ and PtdIns(5)P in most cells (Zolov et. al, 2012, Sbrissa et al., 2012) to the point that it is difficult to tell if changes in PtdIns(3,5)P₂ preceed changes in PtdIns(5)P or correlate with them independently.  

Route 2: De-Phosphorylation of PtdIns(3,5)P₂

Two main lines of evidence support the possibility that PtdIns(3,5)P₂ could be the source of some or all of the PtdIns(5)P in the cell:  

1. Myotubularins, which are 3-phoshphatses leads to increased PtdIns(5)P and their deletion may lead to reductions in PtdIns(5)P (Vaccari, et al., 2011, Oppelt et al., 2012).
2. The kinetics of acute PtdIns(5)P synthesis or degradation may lag slightly behind the synthesis or degradation of PtdIns(5)P.  In any case, the levels of PtdIns(5)P and PtdIns(3,5)P₂ are very tightly correlated (Zolov et al., 2012).

The killer experiment here would be to test whether ablation of PtdIns(3)P levels would have direct effects on PtdIns(5)P levels, but since it is not clear whether PI3K inhibitors such as Wortmannin would affect Pikfyve in vivo that experiment may not be interpretable without ruling out direct effects first.

Route 3: De-Phosphorylation of PtdIns(4,5)P₂

An alternate theory has suggested that some or all of PtdIns(5)P is derived by the activity of a 4-Phosphatase which convertes PtdIns(4,5)P₂ into PtdIns(5)P.  The exact identity of this 4-phosphatase is not yet known.  Jones et al. show that peroxide increases PtdIns(5)P levels, and propose a role for PtdIns(4,5)P₂ dephosphorylation in that process.  However, in contrast to our findings (Zolov et al., 2012), this paper finds no role for Pikfyve in the synthesis of PtdIns(5)P, using similar approaches but a different assay to measure PtdIns(5)P (see below).

What is the Best Way to Measure PtdIns(5)P?

Regarding the role of Pikfyve, there seems to be a controversy here.  I've summarized the assays and their results in the table below.

Assay	Inositol Labelling	Mass Assay
Summary	Cells are grown in inositol depleted media with radioactive inositol. Cells are lysed and lipid headgroups are separated by HPLC based on charge.	Cells are grown in any condition, lipids are extracted and phosphorylated with PIP4K and radioactive ATP. Only PtdIns(5)P can be phosphorylated by this enzyme, so all hot PIP2 (based on TLC and counting) is derived from PtdIns(5)P.
Normalization	Total phosphatidylinosotol	Total cellular phospholipids
Result	Pikfyve knockdown/inhibition nearly completely decreases PtdIns(5)P levels.	Pikfyve knockdown/inhibition does not affect PtdIns(5)P levels.

Setting aside the role of peroxide in PtdIns(5)P as potentially a special case, you could make arguments for both methods.  Hopefully this can be resolved quickly since knowing where this lipid comes from is the first step in figuring out what it does.

References

Jones, D., Foulger, R., Keune, W., Bultsma, Y., & Divecha, N. (2012). PtdIns5P is an oxidative stress-induced second messenger that regulates PKB activation The FASEB Journal DOI: 10.1096/fj.12-218842

Oppelt, A., Lobert, V. H., Haglund, K., Mackey, A. M., Rameh, L. E., Liestøl, K., Oliver Schink, K., et al. (2012). Production of phosphatidylinositol 5-phosphate via PIKfyve and MTMR3 regulates cell migration. EMBO reports. doi:10.1038/embor.2012.183

Sarkes, D., & Rameh, L. E. (2010). A Novel HPLC-Based Approach Makes Possible the Spacial Characterization of Cellular PtdIns5P and Other Phosphoinositides.The Biochemical journal384, 375–384. doi:10.1042/BJ20100129

Sbrissa, D., Ikonomov, O. C., & Shisheva, A. (1999). PIKfyve, a mammalian ortholog of yeast Fab1p lipid kinase, synthesizes 5-phosphoinositides. Effect of insulin. J Biol Chem, 274(31), 21589–21597. pmid:10419465

Sbrissa, D., Ikonomov, O. C., Filios, C., Delvecchio, K., & Shisheva, A. (2012). Functional dissociation between PIKfyve-synthesized PtdIns5P and PtdIns(3,5)P2 by means of the PIKfyve inhibitor YM201636. American journal of physiology. Cell physiology, (313). doi:10.1152/ajpcell.00105.2012

Vaccari, I., Dina, G., Tronchère, H., Kaufman, E., Chicanne, G., Cerri, F., Wrabetz, L., et al. (2011). Genetic interaction between MTMR2 and FIG4 phospholipid phosphatases involved in Charcot-Marie-Tooth neuropathies. PLoS genetics, 7(10), e1002319. doi:10.1371/journal.pgen.1002319

Zolov, S. N., Bridges, D., Zhang, Y., Lee, W., Riehle, E., Verma, R., Lenk, G. M., et al. (2012). In vivo, Pikfyve generates PI(3,5)P2, which serves as both a signaling lipid and the major precursor for PI5P. Proceedings of the National Academy of Sciences of the United States of America, 109(43), 17472–7. doi:10.1073/pnas.1203106109


How is PtdIns(5)P Made? by Dave Bridges is licensed under a Creative Commons Attribution 3.0 Unported License.

PI(3)P and Exocytosis

The classic opinion regarding PI(3)P’s role in intracellular trafficking is that it is synthesized and functions primarily on early endosomes. Several recent publications have highlighted a potential second role for this lipid in exocytosis. Extending previous work by this group and others on the positive role of PI3K-C2a in neurosecretory pathways (1,2) and GLUT4 exoctyosis (3,4) this paper from Tania Maffucci's group interrogates the role of this lipid kinase in insulin secretion in INS1 cells. Combined these results suggest a general role for PI3K-C2a and its product, PI(3)P in exocytotic events.

This group has been investigating the role of the class II PI3K isoforms in exocytosis in a variety of systems. In this paper they investigate the role of PI3K-C2a in insulin secretion using INS1 rat insulinoma cells as a model. Stable knockdown cells did not show any defects in proliferation, calcium signaling, intracellular insulin levels or the expression levels and sub-cellular localization of exocytotic proteins. The PI3K-C2a knockdown cells did however show significant defects in insulin secretion, stimulated by either a secretagogue cocktail or potassium chloride.

Mechanistically, the authors show that there is no defect in insulin granules proximal to the plasma membrane at the resting state. They do detect a decrease in the amount of SNAP25 hydrolysis induced by the secretagogue cocktail. SNAP25 hydrolysis has been proposed to be an important step in the fusion of exocytic vesicles (5). This proteolytic event has not been established as a major mechanism in exocytosis, and it may only correlate with defects in PI3K-C2a signaling. However if reduced SNAP25 proteolysis is the mechanistic defect resulting from PI3K-C2a knockdown, then this suggests a role in protease regulation by PI(3)P or another PI(3)P-derived molecule as a key part of the general exocytotic machinery. It is also possible that there is another, as of yet unstudied role of PI3K-C2a and PI(3)P in exocytosis.



Dominguez, V., Raimondi, C., Somanath, S., Bugliani, M., Loder, M., Edling, C., Divecha, N., da Silva-Xavier, G., Marselli, L., Persaud, S., Turner, M., Rutter, G., Marchetti, P., Falasca, M., & Maffucci, T. (2010). Class II Phosphoinositide 3-Kinase Regulates Exocytosis of Insulin Granules in Pancreatic Cells Journal of Biological Chemistry, 286 (6), 4216-4225 DOI: 10.1074/jbc.M110.200295

References

(1) Meunier, Frederic, Shona Osborne, Gerald Hammond, Frank Cooke, Peter Parker, Jan Domin, and Giampietro Schiavo. “Phosphatidylinositol 3-Kinase C2{alpha} Is Essential for ATP-dependent Priming of Neurosecretory Granule Exocytosis.” Molecular Biology of the Cell 16, no. 10 (2005): 4841-4851. PubMed, DOI.

(2) Wen, Peter J, Shona L Osborne, Isabel C Morrow, Robert G Parton, Jan Domin, and Frederic A Meunier. “Ca2+-regulated pool of phosphatidylinositol-3-phosphate produced by phosphatidylinositol 3-kinase C2alpha on neurosecretory vesicles.” Molecular biology of the cell 19, no. 12 (December 2008): 5593-603. PMC, PubMed, DOI.

(3) Maffucci, Tania, Anna Brancaccio, Enza Piccolo, Robert C Stein, and Marco Falasca. “Insulin induces phosphatidylinositol-3-phosphate formation through TC10 activation.” The EMBO journal 22, no. 16 (August 15, 2003): 4178-89. PubMed, DOI.

(4) Falasca, Marco, William E Hughes, Veronica Dominguez, Gianluca Sala, Florentia Fostira, Michelle Q Fang, Rosanna Cazzolli, Peter R Shepherd, David E James, and Tania Maffucci. “The role of phosphoinositide 3-kinase C2alpha in insulin signaling.” The Journal of biological chemistry 282, no. 38 (September 21, 2007): 28226-36. PubMed, DOI.