Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2) is one of the seven phosphoinositides found in eukaryotic cell membranes.Di Paolo G, De Camilli P. Phosphoinositides in cell regulation and membrane dynamics. Nature. 2006 Oct 12;443(7112):651-7. In quiescent cells, the PtdIns(3,5)P2 levels, typically quantified by HPLC, are the lowest amongst the constitutively present phosphoinositides. They are approximately 3 to 5-fold lower as compared to PtdIns3P and PtdIns5P (Phosphatidylinositol 5-phosphate) levels, and more than 100-fold lower than the abundant PtdIns4P (Phosphatidylinositol 4-phosphate) and PtdIns(4,5)P2.Shisheva A. Regulating Glut4 vesicle dynamics by phosphoinositide kinases and phosphoinositide phosphatases. Front Biosci. 2003 Sep 1;8:s945-56. Review. PtdIns(3,5)P2 was first reported to occur in mouse fibroblasts and budding yeast S. cerevisiae in 1997.Whiteford CC, Brearley CA, Ulug ET. Phosphatidylinositol 3,5-bisphosphate defines a novel PI 3-kinase pathway in resting mouse fibroblasts. Biochem J. 1997 May 1;323 ( Pt 3):597-601. In S. cerevisiae PtdIns(3,5)P2 levels increase dramatically during hyperosmotic shock.Dove SK, Cooke FT, Douglas MR, Sayers LG, Parker PJ, Michell RH. Osmotic stress activates phosphatidylinositol-3,5-bisphosphate synthesis. Nature. 1997 Nov 13;390(6656):187-92. The response to hyperosmotic challenge is not conserved in most tested mammalian cells except for differentiated 3T3L1 adipocytes.
Sbrissa D, Shisheva A. Acquisition of unprecedented phosphatidylinositol 3,5-bisphosphate rise in hyperosmotically stressed 3T3-L1 adipocytes, mediated by ArPIKfyve-PIKfyve pathway. J Biol Chem. 2005 Mar 4;280(9):7883-9. Epub 2004 Nov 16.
Metabolism
The only currently known pathway for PtdIns(3,5)P2 production is through synthesis catalyzed by the phosphoinositide kinase PIKfyve. Pulse-chase experiments in mouse fibroblasts reveal that PtdIns(3,5)P2 is reverted to PtdIns3P soon after its synthesis. In mammalian cells, PtdIns(3,5)P2 is synthesized from and turned over to PtdIns3P by a unique protein complex containing two enzymes with opposite activities: the phosphoinositide kinase PIKfyve and the Sac1 domain-containing PtdIns(3,5)P2 5-phosphatase, Sac3/Fig4.Sbrissa D, Ikonomov OC, Fu Z, Ijuin T, Gruenberg J, Takenawa T, Shisheva A. Core protein machinery for mammalian phosphatidylinositol 3,5-bisphosphate synthesis and turnover that regulates the progression of endosomal transport. Novel Sac phosphatase joins the ArPIKfyve-PIKfyve complex. J Biol Chem. 2007 Aug 17;282(33):23878-91. Epub 2007 Jun 7. The two enzymes do not interact directly. Rather, they are brought together by an associated regulator of PIKfyve, called ArPIKfyve/VAC14, that scaffolds a ternary regulatory complex, known as the PAS complex (from the first letters of PIKfyve/ArPIKfyve/Sac3).Sbrissa D, Ikonomov OC, Fenner H, Shisheva A. ArPIKfyve homomeric and heteromeric interactions scaffold PIKfyve and Sac3 in a complex to promote PIKfyve activity and functionality. J Mol Biol. 2008 Dec 26;384(4):766-79. Epub 2008 Oct 11. PIKfyve attaches the PAS complex onto Rab5GTP/PtdIns3P-enriched endosomal microdomains via its FYVE finger domain that selectively binds PtdIns3P. Ikonomov OC, Sbrissa D, Shisheva A. Localized PtdIns 3,5-P2 synthesis to regulate early endosome dynamics and fusion. Am J Physiol Cell Physiol. 2006 Aug;291(2):C393-404. Epub 2006 Mar 1. Shisheva A, Sbrissa D, Ikonomov O. Cloning, characterization, and expression of a novel Zn2+-binding FYVE finger-containing phosphoinositide kinase in insulin-sensitive cells. Mol Cell Biol. 1999 Jan; 19(1):623-34. Sbrissa D, Ikonomov OC, Shisheva A. Phosphatidylinositol 3-phosphate-interacting domains in PIKfyve. Binding specificity and role in PIKfyve. Endomenbrane localization. J Biol Chem. 2002 Feb 22;277(8):6073-9. Epub 2001 Nov 12. The essential role of the PAS complex in PtdIns(3,5)P2 synthesis and turnover is supported by data from siRNA-mediated protein silencing and heterologous expression of the PAS complex components in various cell types as well as by data from genetic knockout of the PAS complex proteins.
Ikonomov OC, Sbrissa D, Dondapati R, Shisheva A. ArPIKfyve-PIKfyve interaction and role in insulin-regulated GLUT4 translocation and glucose transport in 3T3-L1 adipocytes. Exp Cell Res. 2007 Jul 1;313(11):2404-16. Epub 2007 Mar 30. Ikonomov OC, Sbrissa D, Fenner H, Shisheva A. PIKfyve-ArPIKfyve-Sac3 core complex: contact sites and their consequence for Sac3 phosphatase activity and endocytic membrane homeostasis. J Biol Chem. 2009 Dec 18;284(51):35794-806. Epub . Ikonomov OC, Sbrissa D, Delvecchio K, Xie Y, Jin JP, Rappolee D, Shisheva A. The phosphoinositide kinase PIKfyve is vital in early embryonic development: preimplantation lethality of PIKfyve-/- embryos but normality of PIKfyve+/- mice. J Biol Chem. 2011 Apr 15;286(15):13404-13. Epub 2011 Feb 24. Zhang Y, Zolov SN, Chow CY, Slutsky SG, Richardson SC, Piper RC, Yang B, Nau JJ, Westrick RJ, Morrison SJ, Meisler MH, Weisman LS. Loss of Vac14, a regulator of the signaling lipid phosphatidylinositol 3,5-bisphosphate, results in neurodegeneration in mice. Proc Natl Acad Sci U S A. 2007 Oct 30;104(44):17518-23. Epub 2007 Oct 23. Chow CY, Zhang Y, Dowling JJ, Jin N, Adamska M, Shiga K, Szigeti K, Shy ME, Li J, Zhang X, Lupski JR, Weisman LS, Meisler MH. Mutation of FIG4 causes neurodegeneration in the pale tremor mouse and patients with CMT4J. Nature. 2007 Jul 5;448(7149):68-72. Epub 2007 Jun 17.
An additional pathway for PtdIns(3,5)P2 turnover involves the myotubularin family of phosphatases. Myotubularin 1 and MTMR2 dephosphorylate the 3-position of PtdIns(3,5)P2; therefore, the product of this hydrolysis is PtdIns5P, rather than PtdIns3P. Shisheva A. PIKfyve: Partners, significance, debates and paradoxes. Cell Biol Int. 2008 Jun;32(6):591-604. Epub 2008 Jan 25. Review.
The PAS complex proteins are evolutionarily conserved with orthologs found in S. cerevisiae (i.e., Fab1p, Vac14p, and Fig4p proteins) as well as in all eukaryotes with sequenced genomes. Therefore, it is believed that PtdIns(3,5)P2 is present in all eukaryotes where it regulates similar cellular functions. Yeast Fab1p, Vac14p, and Fig4p also form a complex, called the Fab1 complex. Botelho RJ, Efe JA, Teis D, Emr SD. Assembly of a Fab1 phosphoinositide kinase signaling complex requires the Fig4 phosphoinositide phosphatase. Mol Biol Cell. 2008 Oct;19(10):4273-86. Epub 2008 Jul 23. However, the Fab1 complex contains additional proteins, Jin N, Chow CY, Liu L, Zolov SN, Bronson R, Davisson M, Petersen JL, Zhang Y, Park S, Duex JE, Goldowitz D, Meisler MH, Weisman LS. VAC14 nucleates a protein complex essential for the acute interconversion of PI3P and PI(3,5)P(2) in yeast and mouse. EMBO J. 2008 Dec 17;27(24):3221-34. Epub 2008 Nov 27. which might add an additional layer of PtdIns(3,5)P2 regulation in yeast. The composition of the protein complexes regulating PtdIns(3,5)P2 levels in other species is yet to be clarified.
Functions and regulation
PtdIns(3,5)P2 regulates endosomal operations (fission and fusion) that maintain endomembrane homeostasis and proper performance of the trafficking pathways emanating from or traversing endosomes. Decrease of PtdIns(3,5)P2 levels upon perturbations of cellular PIKfyve by heterologous expression of enzymatically inactive PIKfyve point mutants, Ikonomov OC, Sbrissa D, Shisheva A. Mammalian cell morphology and endocytic membrane homeostasis require enzymatically active phosphoinositide 5-kinase PIKfyve. J Biol Chem. 2001 Jul 13;276(28):26141-7. Epub 2001 Apr 2. siRNA-medicated silencing, Rutherford AC, Traer C, Wassmer T, Pattni K, Bujny MV, Carlton JG, Stenmark H, Cullen PJ. The mammalian phosphatidylinositol 3-phosphate 5-kinase (PIKfyve) regulates endosome-to-TGN retrograde transport. J Cell Sci. 2006 Oct 1;119(19):3944-57. Epub 2006 Sep 5. pharmacological inhibition Jefferies HB, Cooke FT, Jat P, Boucheron C, Koizumi T, Hayakawa M, Kaizawa H, Ohishi T, Workman P, Waterfield MD, Parker PJ. A selective PIKfyve inhibitor blocks PtdIns(3,5)P(2) production and disrupts endomembrane transport and retroviral budding. EMBO Rep. 2008 Feb;9(2):164-70. Epub 2008 Jan 11. and PIKFYVE knockout
all cause formation of multiple cytosolic vacuoles, which become larger over time. Importantly, the vacuolation induced by PIKfyve dysfunction and PtdIns(3,5)P2 depletion is reversible and could be selectively rescued by cytosolic microinjection of PtdIns(3,5)P2, Ikonomov OC, Sbrissa D, Mlak K, Kanzaki M, Pessin J, Shisheva A. Functional dissection of lipid and protein kinase signals of PIKfyve reveals the role of PtdIns 3,5-P2 production for endomembrane integrity. J Biol Chem. 2002 Mar 15;277(11):9206-11. Epub 2001 Nov 19. overexpression of PIKfyve
or wash-out of the PIKfyve inhibitor YM201636.
Sac3 phosphatase activity in the PAS complex also plays an important role in regulating PtdIns(3,5)P2 levels and maintaining endomembrane homeostasis. Thus, cytoplasmic vacuolation induced by the dominant-negative PIKfyveK1831E mutant is suppressed upon co-expression of a Sac3 phosphatase-inactive point-mutant along with ArPIKfyve.
In vitro reconstitution assays of endosome fusion and multivesicular body (MVB) formation/detachment (fission) suggest a positive role of PtdIns(3,5)P2 in MVB fission from maturing early endosomes and a negative role in endosome fusion.
PtdIns(3,5)P2 is implicated in the microtubule-dependent retrograde transport from early/late endosomes to the trans Golgi network.
Ikonomov OC, Fligger J, Sbrissa D, Dondapati R, Mlak K, Deeb R, Shisheva A. Kinesin adapter JLP links PIKfyve to microtubule-based endosome-to-trans-Golgi network traffic of furin. J Biol Chem. 2009 Feb 6;284(6):3750-61. Epub 2008 Dec 4. .
Acute insulin treatment increases PtdIns(3,5)P2 levels in 3T3L1 adipocytes, both in isolated membranes and intact cells to promote insulin effect on GLUT4 cell surface translocation and glucose transport.
These cells also show a marked PtdIns(3,5)P2 increase upon hyperosmotic shock.
Other stimuli, including mitogenic signals such as IL-2 and UV light in lymphocytes, Jones DR, González-García A, Díez E, Martinez-A C, Carrera AC, Meŕida I. The identification of phosphatidylinositol 3,5-bisphosphate in T-lymphocytes and its regulation by interleukin-2. J Biol Chem. 1999 Jun 25;274(26):18407-13. activation of protein kinase C by PMA in platelets Banfić H, Downes CP, Rittenhouse SE. Biphasic activation of PKBalpha/Akt in platelets. Evidence for stimulation both by phosphatidylinositol 3,4-bisphosphate, produced via a novel pathway, and by phosphatidylinositol 3,4,5-trisphosphate. J Biol Chem. 1998 May 8;273(19):11630-7. and EGF stimulation of COS cells, Tsujita K, Itoh T, Ijuin T, Yamamoto A, Shisheva A, Laporte J, Takenawa T. Myotubularin regulates the function of the late endosome through the gram domain-phosphatidylinositol 3,5-bisphosphate interaction. J Biol Chem. 2004 Apr 2;279(14):13817-24. Epub 2004 Jan 12. also increase PtdIns(3,5)P2 levels.
PtdIns(3,5)P2 plays a key role in growth and development as evidenced by the preimplantation lethality of the PIKfyve knockout mouse model.
The fact that the heterozygous PIKfyve mice are ostensibly normal and live to late adulthood with only ~60% of the wild-type PtdIns(3,5)P2 levels suggests that PtdIns(3,5)P2 might normally be in excess.
ArPIKfyve/Vac14 or Sac3/Fig4 knockout in mice results in a 30-50% decrease in PtdIns(3,5)P2 levels and cause similar massive central neurodegeneration and peripheral neuropathy.
These studies suggest that reduced PtdIns(3,5)P2 levels, by a yet-to-be identified mechanism, mediate neuronal death. In contrast, MTMR2 phosphatase knockout, which also causes peripheral neuropathy, is accompanied by elevation in PtdIns(3,5)P2.