Sunday, December 30, 2012

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.  ResearchBlogging.orgThe 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 (Pip5k1aPip5k1b 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

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

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