HGNC Gene name: PIKFYVE – phosphoinositide kinase, FYVE finger containing Homo sapiens

Synonyms: PIP5K3; KIAA0981; MGC40423; p235

Disease relevance of PIKFYVE

• In 8 out of 10 studied families suffering from Francois-Neetens fleck corneal dystrophy, Li et al., 2005 report PIKFYVE missense, frameshift, and/or protein-truncating mutations (1).

Update: Lessons from PIKfyve knockout mouse models

• The consequences of global and haploid PIKFYVE gene knockout in mouse development have been recently reported (28). To knockout PIKFYVE, exon 6 of the mouse gene was flanked with two loxP sequences that were subsequently excised by breeding the “floxed” mice with global Cre-deleter mice. Elimination of exon 6 leads to an early termination (at residue 159) of the PIKfyve protein translation.

• Global PIKFYVE gene deletion in the mouse is embryonically lethal, with nearly all KO/KO embryos dying during the preimplantation stage of early development. Cultured fibroblasts derived from PIKfyve flox/flox embryos were rendered PIKFYVE-null by expression of Cre recombinase. Such fibroblasts displayed multiple cytoplasmic vacuoles and profoundly inhibited DNA synthesis, consistent with impaired cell division causing early embryo lethality (28).

• The heterozygous PIKfyveWT/KO mice were born at the expected Mendelian ratio and developed into adulthood. PIKfyveWT/KO mice were ostensibly normal by several other in vivo, ex vivo and in vitro criteria, despite the fact that their levels of the PIKfyve protein and in vitro enzymatic activity in cells and tissues were 50-55% lower than those of wild-type mice (28).

• Steady-state levels of the PIKfyve products PtdIns(3,5)P₂ and PtdIns5P selectively decreased but this reduction (35-40%) was 10-15% less than that expected based on PIKfyve protein reduction (28). This nonlinear decrease in the levels of the PIKfyve protein vs. lipid products, the potential mechanism(s) being related to reduced functionality of the Sac3 phosphatase (gene symbol FIG4) at the PtdIns(3,5)P₂ membranes, may explain how one functional allele in PIKfyveWT/KO mice is able to support the demands for PtdIns(3,5)P₂ and PtdIns5P synthesis during life (28).

High impact information on PIKFYVE

• PIKFYVE is a single copy gene, located on human chromosome 2 (2q34), containing 41 exons that encode a large protein of 2098 amino acids (2, 3). The mouse gene (ID: 18711) is located on chromosome 1 and is comprised of 42 exons (2).
• PIKFYVE protein harbors 5 evolutionarily-conserved domains. An N-terminal-positioned FYVE finger targets the protein to PtdIns(3)P-enriched endosome membranes (4, 5); next is the functionally uncharacterized DEP domain; the middle part of the molecule is occupied by the chaperonin-like domain and a CHK homology region (conserved in Cys, His, and Lys residues), both responsible for the multiple PIKfyve protein-protein interactions; at the C terminus is the catalytic domain, responsible for PIKFYVE’s enzymatic activities (2).
• PIKFYVE protein is a kinase phosphorylating both lipid and protein substrates. The lipid products of PIKFYVE are phosphatidylinositol 5-phosphate (PtdIns(5)P) and phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2) (6); The protein products are phosphorylated on Ser and include PIKFYVE itself and other proteins (7).
• PIKFYVE physically associates with a subcomplex of two other proteins – ArPIKFYVE (gene name VAC14) (8) and Sac3 (9) – to form a core protein machinery that controls both synthesis and turnover of the low abundance signaling molecule PtdIns(3,5)P2 (9). The core machinery is referred to as the “PAS complex” after the first letters of PIKfyve, ArPIKfyve and Sac3 (10).
• The PAS complex represents a unique association of two enzymes with opposing activities, i.e. for PtdIns(3,5)P2 synthesis and PtdIns(3,5)P2 turnover, the balance of which is essential in maintaining proper endomembrane homeostasis (10,11).
• PIKFYVE pathway mediates unprecedented rise of PtdIns(3,5)P2 in differentiated 3T3L1 adipocytes in response to hyperosmotic stress (12).

Physiological roles of PIKFYVE

• Expression of dominant-negative kinase-inactive PIKFYVE mutants, knockdown of PIKFYVE, and pharmacological inhibition of PIKFYVE enzymatic activity, all lead to endomembrane vacuolation (13, 14, 15). The functional dissection of the lipid and protein kinase signals of PIKFYVE reveals that the PtdIns(3)P 5-kinase activity is essential in cellular endomembrane homeostasis (16).
• Transmission electron microscopy in a HEK293 cell line stably expressing dominant-negative kinase-deficient PIKFYVE^K1831E visualizes dilated multivesicular body (MVB)-like compartments with lower number of intralumenal vesicles and membrane whorls in comparison with the normal MVBs (17).
• Perturbations of the PIKFYVE functionality affect several intracellular trafficking pathways, both constitutive and regulated, that emanate from or traverse the early endosomes, including fluid phase traffic to lysosomes (17), endosome-to-TGN retrieval pathway (14) and insulin-regulated cell surface translocation of GLUT4 (18).
• The PtdIns(3,5)P2-synthesizing activity of PIKFYVE regulates the formation/fission of endosomal transport intermediates from early endosomes thereby regulating the transport of yet unspecified cargo to the lysosomes (9, 19). PIKFYVE is specifically implicated in the regulation of endosome-to-TGN transport as demonstrated for the traffic of chimeric furin (14, 20).
• Microinjection of PIKFYVE’s lipid product PtdIns(5)P, similarly to insulin, rapidly decreases the number and length of filamentous actin fibers in CHO cells stably expressing the insulin receptor. Ectopic expression of PIKFYVE yields similar effect (21).

Other interactions of PIKFYVE

• PIKFYVE associates in vitro with PtdIns(3)P-enriched liposomes (5). In a cell context, the PIKFYVE’s FYVE finger domain targets PtdIns(3)P-enriched endosomal membranes, positive for Rab5 (19).
• PIKFYVE, via its chaperonin-like domain interacts with the Rab9 effector p40 that binds Rab9-GTP to facilitate endosome-to-TGN retrograde transport. The interaction is determined by yeast two-hybrid assays, gluthation S-transferase (GST) pull-down assays, and co-immunoprecipitation in doubly transfected HEK293 cells (22).
• GST-p40 fails to specifically associate with the PIKFYVE lipid products PtdIns (5)P and PtdIns(3,5)P2 in a liposome binding assay but is an in vitro substrate of the PIKFYVE serine kinase activity (22).
• The chaperonin-like domain of PIKFYVE also interacts with the last 75 amino acid residues of the JLP C-terminus. JLP, one of the 3 protein products of the human SPAG9 gene containing a kinesin-binding domain, is a PIKFYVE functional partner in the endosome-to-TGN cargo transport. Using the microtubule-dependent endosome-to-TGN transport of chimeric furin (Tac-Furin) and the microtubule-independent traffic of Tac-TGN38 in CHO cells as a model system, it is established that the association of PIKFYVE with JLP is required in the microtubule-based transport (20).

Analytical, diagnostic and therapeutic context of PIKFYVE

• The role of PIKFYVE and the other two proteins constituting the PAS complex, i.e. ArPIKFYVE and Sac3, is studied in insulin-induced translocation of the glucose transporter GLUT4. The results indicate that the increase of localized PtdIns(3,5)P2 levels improves insulin responsiveness (23, 24, 25).
• Expression of PIKFYVE (WT) in COS and HEK293 cells inhibits vacuolation induced by subsequent intoxication with VacA (the vacuolating agent of Helicobacter pylori). Microinjection of the PIKFYVE lipid product PtdIns (3,5)P2 produces a similar inhibitory effect (26).
• Silencing Rab9 expression dramatically inhibits HIV replication, as does silencing the host genes encoding TIP47, p40, and PIKFYVE, which also facilitate late-endosome-to-trans-Golgi vesicular transport (27).
• Acute treatment of cells with PIKfyve pharmacological inhibitor YM201636 shows that the PIKFYVE pathway is involved in sorting of endosomal transport. Inhibition of PIKFYVE activity leads to the accumulation of a late-endosomal compartment and blockade of retroviral exit (15).

References

1. Mutations in PIP5K3 are associated with François-Neetens mouchetée fleck corneal dystrophy . Li, S., Tiab, L., Jiao, X., Munier, F.L., Zografos, L., Frueh, B.E., Sergeev, Y., Smith, J., Rubin, B., Meallet, M.A., Forster, R.K., Hejtmancik, J.F., Schorderet, D.F. Am. J. Hum. Genet. (2005)

2. PIKfyve: Partners, significance, debates and paradoxes. Shisheva, A. Cell Biol. Internat. (2008)

3. Cloning and subcellular localization of a human phosphatidylinositol 3-phosphate 5-kinase, PIKfyve/Fab1. Cabezas, A., Pattni, K., Stenmark, H. Gene (2006)

4. Cloning, characterization, and expression of a novel Zn2+-binding FYVE finger-containing phosphoinositide kinase in insulin-sensitive cells. Shisheva, A., Sbrissa, D., Ikonomov, O. Mol. Cell. Biol. (1999)

5. PtdIns 3-P-binding domains in PIKfyve: binding specificity and consequences to the protein endomembrane localization. Sbrissa, D., Ikonomov, O.C., Shisheva, A. J. Biol. Chem. (2002)

6. PIKfyve, a mammalian ortholog of yeast Fab1p lipid kinase, synthesizes 5-phosphoinositides. Sbrissa, D., Ikonomov, O.C., Shisheva, A. J. Biol. Chem. (1999)

7. PIKfyve lipid kinase is a protein kinase: downregulation of 5’-phosphoinositide product fortmation by autophosphorylation. Sbrissa, D., Ikonomov, O.C., Shisheva, A. Biochemistry (2000)

8. A mammalian ortholog of Saccharomyces cerevisiae Vac14 that associates with and up-regulates PIKfyve phosphoinositide 5-kinase activity. Sbrissa, D., Ikonomov, O.C., Strakova, J., Dondapati, R., Mlak, K., Deeb, R., Silver, R., Shisheva, A. Mol. Cell. Biol. (2004)

9. 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. Sbrissa, D., Ikonomov, O.C., Fu, Z., Ijuin, T., Gruenberg, J., Takenawa, T., Shisheva, A. J. Biol. Chem. (2007)

10. ArPIKfyve homomeric and heteromeric interactions scaffold PIKfyve and Sac3 in a complex to promote PIKfyve activity and functionality. Sbrissa, D., Ikonomov, O.C., Fenner, H., Shisheva, A. J. Mol. Biol. (2008)

11. PIKfyve-ArPIKfyve-Sac3 core complex. Contact sites and their consequence for Sac3 phosphatase activity and endocytic membrane homeostasis. Ikonomov, O.C., Sbrissa, D., Fenner, H., Shisheva, A. J. Biol. Chem. (2009)

12. Acquisition of unprecedented phosphatidylinositol 3,5-bisphosphate rise in hyperosmotically stressed 3T3-L1 adipocytes, mediated by ArPIKfyve-PIKfyve pathway . Sbrissa, D., Shisheva, A. J. Biol. Chem. (2005)

13. Mammalian cell morphology and endocytic membrane homeostasis require enzymatically active phosphoinositide 5-kinase PIKfyve. Ikonomov, O.C., Sbrissa, D., Shisheva, A. J. Biol. Chem. (2001)

14. The mammalian phosphatidylinositol 3-phosphate 5-kinase (PIKfyve) regulates endosome-to-TGN retrograde transport. Rutherford, A.C., Traer, C., Wassmer, T., Pattni, K., Bujny M.V., Carlton, J.G., et al. J. Cell Sci. (2006)

15. A selective PIKfyve inhibitor blocks PtdIns(3,5)P2 production and disrupts endomembrane transport and retroviral budding. Jefferies, H.B.J., Cooke, F.T., Jat, P., Boucheron, C., Koizumi, T., Hayakawa, M., Kaizawa, H., Ohishi, T., Workman, P., Waterfield, M.D., Parker, P.J. EMBO Rep. (2008)

16. Functional dissection of lipid and protein kinase signals of PIKfyve reveals the role of PtdIns 3,5-P2 production for endomembrane integrity . Ikonomov, O.C., Sbrissa, D., Mlak, K., Kanzaki, M., Pessin, J., Shisheva, A. J. Biol. Chem (2002)

17. PIKfyve controls fluid-phase endocytosis but not recycling/degradation of endocytosed receptors or sorting of procathepsin D by regulating multivesicular body morphogenesis. Ikonomov, O.C., Sbrissa, D., Fligger, J., Foti, M., Carpentier, J-L., Shisheva, A. Mol. Biol. Cell (2003)

18. Phosphoinositides in insulin action on GLUT4 dynamics: not just PtdIns(3,4,5)P3. Shisheva, A. Am. J. Physiol. Endocrinol. Metab. (2008)

19. Localized PtdIns 3,5-P2 synthesis to regulate early endosome dynamics and fusion. Ikonomov, O.C., Sbrissa, D., Shisheva, A. Am. J. Physiol. Cell Physiol. (2006)

20. Kinesin adapter JLP links PIKfyve to microtubule-based endosome-to-trans-golgi network traffic of furin. Ikonomov, O.C., Fligger, J., Sbrissa, D., Dondapati, R., Mlak, K., Deeb, R., Shisheva, A. J. Biol. Chem. (2009)

21. Role for a novel signaling intermediate, phosphatidylinositol 5-phosphate, in insulin-regulated F-actin stress fiber breakdown and GLUT4 translocation. Sbrissa, D., Ikonomov, O.C., Strakova, J., Shisheva, A. Endocrinology (2004)

22. Active PIKfyve associates with and promotes the membrane attachment of the late endosome-to-TGN transport factor Rab9 effector p40. Ikonomov, O.C., Sbrissa, D., Mlak, K., Deeb, R., Fligger, J., Soans, A., et al. J. Biol. Chem. (2003)

23. Requirement for PIKfyve enzymatic activity in acute and long-term insulin cellular effects. Ikonomov, O.C., Sbrissa, D., Mlak, K., Shisheva, A. Endocrinology (2002)

24. ArPIKfyve-PIKfyve interaction and role in insulin-regulated GLUT4 translocation and glucose transport in 3T3-L1 adipocytes. Ikonomov, O.C., Sbrissa, D., Dondapati, R., Shisheva, A. Exp. Cell. Res. (2007)

25. Sac3 is an insulin-regulated phosphatidylinositol 3,5-bisphosphate phosphatase: gain in insulin responsiveness through Sac3 down-regulation in adipopcytes. Ikonomov, O.C., Sbrissa, D., Ijuin, T., Takenawa, T., Shisheva, A. J. Biol. Chem. (2009)

26. PIKfyve kinase and SKD1 AAA ATPase define distinct endocytic compartments. Only PIKfyve expression inhibits the cell-vacuolating activity of Helicobacter pylori VacA toxin. Ikonomov, O.C., Sbrissa, D., Yoshimori, T., Cover, T.L., Shisheva, A. J. Biol. Chem. (2002)

27. Rab9 GTPase is required for replication of human immunodeficiency virus type 1, filoviruses, and measles virus. Murray, J.L., Javrakis, M., McDonald N.J., Yilla, M., Sheng, J., Bellini, W.J., et al. J. Virol. (2005)

28. The phosphoinositide kinase PIKfyve is vital in early embryonic development: Preimplantation lethality of PIKfyve-/- embryos but normality of PIKfyve+/- mice. Ikonomov OC, Sbrissa D, Delvecchio K, Xie Y, Jin J-P, Rappolee D, Sisheva A. J Biol Chem (2011)