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A mammalian ortholog of Saccharomyces cerevisiae Vac14 that associates with and up-regulates PIKfyve phosphoinositide 5-kinase activity.

ABSTRACT: Multivesicular body morphology and size are controlled in part by PtdIns(3,5)P(2), produced in mammalian cells by PIKfyve-directed phosphorylation of PtdIns(3)P. Here we identify human Vac14 (hVac14), an evolutionarily conserved protein, present in all eukaryotes but studied principally in yeast thus far, as a novel positive regulator of PIKfyve enzymatic activity. In mammalian cells and tissues, Vac14 is a low-abundance 82-kDa protein, but its endogenous levels could be up-regulated upon ectopic expression of hVac14. PIKfyve and hVac14 largely cofractionated, populated similar intracellular locales, and physically associated. A small-interfering RNA-directed gene-silencing approach to selectively eliminate endogenous hVac14 rendered HEK293 cells susceptible to morphological alterations similar to those observed upon expression of PIKfyve mutants deficient in PtdIns(3,5)P(2) production. Largely decreased in vitro PIKfyve kinase activity and unaltered PIKfyve protein levels were detected under these conditions. Conversely, ectopic expression of hVac14 increased the intrinsic PIKfyve lipid kinase activity. Concordantly, intracellular PtdIns(3)P-to-PtdIns(3,5)P(2) conversion was perturbed by hVac14 depletion and was elevated upon ectopic expression of hVac14. These data demonstrate a major role of the PIKfyve-associated hVac14 protein in activating PIKfyve and thereby regulating PtdIns(3,5)P(2) synthesis and endomembrane homeostasis in mammalian cells.

SUBMITTER: Sbrissa D

PROVIDER: S-EPMC529046 | biostudies-literature | 2004 Dec

REPOSITORIES: biostudies-literature

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A mammalian ortholog of Saccharomyces cerevisiae Vac14 that associates with and up-regulates PIKfyve phosphoinositide 5-kinase activity.

Sbrissa Diego D Ikonomov Ognian C OC Strakova Jana J Dondapati Rajeswari R Mlak Krzysztof K Deeb Robert R Silver Robert R Shisheva Assia A

Molecular and cellular biology 20041201 23

Multivesicular body morphology and size are controlled in part by PtdIns(3,5)P(2), produced in mammalian cells by PIKfyve-directed phosphorylation of PtdIns(3)P. Here we identify human Vac14 (hVac14), an evolutionarily conserved protein, present in all eukaryotes but studied principally in yeast thus far, as a novel positive regulator of PIKfyve enzymatic activity. In mammalian cells and tissues, Vac14 is a low-abundance 82-kDa protein, but its endogenous levels could be up-regulated upon ectopi ...[more]

PMID: 15542851

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Project description:Although PIKFYVE phosphoinositide kinase inhibitors can selectively eliminate PIKFYVE-dependent human cancer cells in vitro and in vivo, the basis for this selectivity has remained elusive. Here we show that the sensitivity of cells to the PIKFYVE inhibitor WX8 is not linked to PIKFYVE expression, macroautophagic/autophagic flux, the BRAFV600E mutation, or ambiguous inhibitor specificity. PIKFYVE dependence results from a deficiency in the PIP5K1C phosphoinositide kinase, an enzyme required for conversion of phosphatidylinositol-4-phosphate (PtdIns4P) into phosphatidylinositol-4,5-bisphosphate (PtdIns[4,5]P2/PIP2), a phosphoinositide associated with lysosome homeostasis, endosome trafficking, and autophagy. PtdIns(4,5)P2 is produced via two independent pathways. One requires PIP5K1C; the other requires PIKFYVE and PIP4K2C to convert PtdIns3P into PtdIns(4,5)P2. In PIKFYVE-dependent cells, low concentrations of WX8 specifically inhibit PIKFYVE in situ, thereby increasing the level of its substrate PtdIns3P while suppressing PtdIns(4,5)P2 synthesis and inhibiting lysosome function and cell proliferation. At higher concentrations, WX8 inhibits both PIKFYVE and PIP4K2C in situ, which amplifies these effects to further disrupt autophagy and induce cell death. WX8 did not alter PtdIns4P levels. Consequently, inhibition of PIP5K1C in WX8-resistant cells transformed them into sensitive cells, and overexpression of PIP5K1C in WX8-sensitive cells increased their resistance to WX8. This discovery suggests that PIKFYVE-dependent cancers could be identified clinically by low levels of PIP5K1C and treated with PIKFYVE inhibitors.Abbreviations: DMSO: dimethylsulfoxide; ELISA: enzyme-linked immunosorbent assay; LC3-I: microtubule associated protein light chain 3-I; LC3-II: microtubule associated protein light chain 3-II; MS: mass spectrometry; PtdIns: phosphatidylinositol; PtdIns3P: PtdIns-3-phosphate; PtdIns4P: PtdIns-4-phosphate; PtdIns5P: PtdIns-5-phosphate; PtdIns(3,5)P2: PtdIns-3,5-bisphosphate; PtdIns(4,5)P2/PIP2: PtdIns-4,5-bisphosphate; PtdIns(3,4,5)P3/PIP3: PtdIns-3,4,5-trisphosphate; PIKFYVE: phosphoinositide kinase, FYVE-type zinc finger containing; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; PI4KA: phosphatidylinositol 4-kinase alpha; PI4KB: phosphatidylinositol 4-kinase beta; PI4K2A: phosphatidylinositol 4-kinase type 2 alpha; PI4K2B: phosphatidylinositol 4-kinase type 2 beta; PIP4K2A: phosphatidylinositol-5-phosphate 4-kinase type 2 alpha; PIP4K2B: phosphatidylinositol-5-phosphate 4-kinase type 2 beta; PIP4K2C: phosphatidylinositol-5-phosphate 4-kinase type 2 gamma; PIP5K1A: phosphatidylinositol-4-phosphate 5-kinase type 1 alpha; PIP5K1B: phosphatidylinositol-4-phosphate 5-kinase type 1 beta; PIP5K1C: phosphatidylinositol-4-phosphate 5-kinase type 1 gamma; WX8: 1H-indole-3-carbaldehyde (4-anilino-6-[4-morpholinyl]-1,3,5-triazin-2-yl)hydrazone.

Project description:Pre-mRNA splicing involves the stepwise assembly of a pre-catalytic spliceosome, followed by its catalytic activation, splicing catalysis and disassembly. Formation of the pre-catalytic spliceosomal B complex involves the incorporation of the U4/U6.U5 tri-snRNP and of a group of non-snRNP B-specific proteins. While in Saccharomyces cerevisiae the Prp38 and Snu23 proteins are recruited as components of the tri-snRNP, metazoan orthologs of Prp38 and Snu23 associate independently of the tri-snRNP as members of the B-specific proteins. The human spliceosome contains about 80 proteins that lack obvious orthologs in yeast, including most of the B-specific proteins apart from Prp38 and Snu23. Conversely, the tri-snRNP protein Spp381 is one of only five S. cerevisiae splicing factors without a known human ortholog.Using InParanoid, a state-of-the-art method for ortholog inference between pairs of species, and systematic BLAST searches we identified the human B-specific protein MFAP1 as a putative ortholog of the S. cerevisiae tri-snRNP protein Spp381. Bioinformatics revealed that MFAP1 and Spp381 share characteristic structural features, including intrinsic disorder, an elongated shape, solvent exposure of most residues and a trend to adopt ?-helical structures. In vitro binding studies showed that human MFAP1 and yeast Spp381 bind their respective Prp38 proteins via equivalent interfaces and that they cross-interact with the Prp38 proteins of the respective other species. Furthermore, MFAP1 and Spp381 both form higher-order complexes that additionally include Snu23, suggesting that they are parts of equivalent spliceosomal sub-complexes. Finally, similar to yeast Spp381, human MFAP1 partially rescued a growth defect of the temperature-sensitive mutant yeast strain prp38-1.Human B-specific protein MFAP1 structurally and functionally resembles the yeast tri-snRNP-specific protein Spp381 and thus qualifies as its so far missing ortholog. Our study indicates that the yeast Snu23-Prp38-Spp381 triple complex was evolutionarily reprogrammed from a tri-snRNP-specific module in yeast to the B-specific Snu23-Prp38-MFAP1 module in metazoa, affording higher flexibility in spliceosome assembly and thus, presumably, in splicing regulation.

Dataset Information

A mammalian ortholog of Saccharomyces cerevisiae Vac14 that associates with and up-regulates PIKfyve phosphoinositide 5-kinase activity.

Publications

A mammalian ortholog of Saccharomyces cerevisiae Vac14 that associates with and up-regulates PIKfyve phosphoinositide 5-kinase activity.

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