Vac14 Protein Multimerization Is a Prerequisite Step for Fab1 Protein Complex Assembly and Function

Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P₂) helps control various endolysosome functions including organelle morphology, membrane recycling, and ion transport. Further highlighting its importance, PtdIns(3,5)P₂ misregulation leads to the development of neurodegenerative diseases like Charcot-Marie-Tooth disease. The Fab1/PIKfyve lipid kinase phosphorylates PtdIns(3)P into PtdIns(3,5)P₂ whereas the Fig4/Sac3 lipid phosphatase antagonizes this reaction. Interestingly, Fab1 and Fig4 form a common protein complex that coordinates synthesis and degradation of PtdIns(3,5)P₂ by a poorly understood process. Assembly of the Fab1 complex requires Vac14/ArPIKfyve, a multimeric scaffolding adaptor protein that coordinates synthesis and turnover of PtdIns(3,5)P₂. However, the properties and function of Vac14 multimerization remain mostly uncharacterized. Here we identify several conserved C-terminal motifs on Vac14 required for self-interaction and provide evidence that Vac14 likely forms a dimer. We also show that monomeric Vac14 mutants do not support interaction with Fab1 or Fig4, suggesting that Vac14 multimerization is likely the first molecular event in the assembly of the Fab1 complex. Finally, we show that cells expressing monomeric Vac14 mutants have enlarged vacuoles that do not fragment after hyperosmotic shock, which indicates that PtdIns(3,5)P₂ levels are greatly abated. Therefore, our observations support an essential role for the Vac14 homocomplex in controlling PtdIns(3,5)P₂ levels.     

ArPIKfyve homomeric and heteromeric interactions scaffold PIKfyve and Sac3 in a complex to promote PIKfyve activity and functionality

PtdIns(3,5)P(2) (with PtdIns indicating phosphatidylinositol) is vital in the differentiation and development of multicellular organisms because knockout of the PtdIns(3,5)P(2)-synthesizing enzyme PIKfyve (phosphoinositide kinase for position 5 containing a FYVE finger domain) or its associated regulator ArPIKfyve is lethal. In previous work with endogenous proteins, we identified that Sac3, a phosphatase that turns over PtdIns(3,5)P(2), associates with the PIKfyve-ArPIKfyve biosynthetic complex. However, whether the three proteins suffice for the organization/maintenance of this complex [referred to as the PAS (PIKfyve-ArPIKfyve-Sac3) complex], how they interact with one another, and what the functional relevance of this ternary association would be remained unresolved. Using co-immunoprecipitation analyses in transfected mammalian cells with increased or decreased levels of the three proteins, singly or in double versus triple combinations, herein we report that the triad is sufficient to form and maintain the PAS complex. ArPIKfyve is the principal organizer interacting with both Sac3 and PIKfyve, whereas Sac3 is permissive for maximal PIKfyve-ArPIKfyve association in the PAS complex. We further identified that ArPIKfyve scaffolds the PAS complex through homomeric interactions, mediated via its conserved C-terminal domain. Introduction of the C-terminal peptide fragment of the ArPIKfyve-ArPIKfyve contact sites effectively disassembled the PAS complex and reduced the in vitro PIKfyve lipid kinase activity. Exploring insulin-regulated GLUT4 translocation in 3T3L1 adipocytes as a functional readout, a process that is positively regulated by PIKfyve activity and ArPIKfyve levels, we determined that ectopic expression of the ArPIKfyve C-terminal peptide inhibits GLUT4 surface accumulation. Our data indicate that the PAS complex is organized to provide optimal PIKfyve functionality and is maintained via ArPIKfyve homomeric and heteromeric interactions.     

ArPIKfyve Regulates Sac3 Protein Abundance and Turnover: DISRUPTION OF THE MECHANISM BY Sac3I41T MUTATION CAUSING CHARCOT-MARIE-TOOTH 4J DISORDER*

The mammalian phosphatidylinositol (3,5)-bisphosphate (PtdIns(3,5)P₂) phosphatase Sac3 and ArPIKfyve, the associated regulator of the PtdIns3P-5 kinase PIKfyve, form a stable binary complex that associates with PIKfyve in a ternary complex to increase PtdIns(3,5)P₂ production. Whether the ArPIKfyve-Sac3 subcomplex functions outside the PIKfyve context is unknown. Here we show that stable or transient expression of ArPIKfyve^WT in mammalian cells elevates steady-state protein levels and the PtdIns(3,5)P₂-hydrolyzing activity of Sac3, whereas knockdown of ArPIKfyve has the opposite effect. These manipulations do not alter the Sac3 mRNA levels, suggesting that ArPIKfyve might control Sac3 protein degradation. Inhibition of protein synthesis in COS cells by cycloheximide reveals remarkably rapid turnover of expressed Sac3^WT (t_½ = 18.8 min), resulting from a proteasome-dependent clearance as evidenced by the extended Sac3^WT half-life upon inhibiting proteasome activity. Coexpression of ArPIKfyve^WT, but not the N- or C-terminal halves, prolongs the Sac3^WT half-life consistent with enhanced Sac3 protein stability through association with full-length ArPIKfyve. We further demonstrate that mutant Sac3, harboring the pathogenic Ile-to-Thr substitution at position 41 found in patients with CMT4J disorder, is similar to Sac3^WT with regard to PtdIns(3,5)P₂-hydrolyzing activity, association with ArPIKfyve, or rapid proteasome-dependent clearance. Remarkably, however, neither is the steady-state Sac3^I41T elevated nor is the Sac3^I41T half-life extended by coexpressed ArPIKfyve^WT, indicating that unlike with Sac3^WT, ArPIKfyve fails to prevent Sac3^I41T rapid loss. Together, our data indentify a novel regulatory mechanism whereby ArPIKfyve enhances Sac3 abundance by attenuating Sac3 proteasome-dependent degradation and suggest that a failure of this mechanism could be the primary molecular defect in the pathogenesis of CMT4J.     

The PIKfyve–ArPIKfyve–Sac3 triad in human breast cancer: Functional link between elevated Sac3 phosphatase and enhanced proliferation of triple negative cell lines

The phosphoinositide 5-kinase PIKfyve and 5-phosphatase Sac3 are scaffolded by ArPIKfyve in the PIKfyve–ArPIKfyve–Sac3 (PAS) regulatory complex to trigger a unique loop of PtdIns3P–PtdIns(3,5)P₂ synthesis and turnover. Whereas the metabolizing enzymes of the other 3-phosphoinositides have already been implicated in breast cancer, the role of the PAS proteins and the PtdIns3P–PtdIns(3,5)P₂ conversion is unknown. To begin elucidating their roles, in this study we monitored the endogenous levels of the PAS complex proteins in cell lines derived from hormone-receptor positive (MCF7 and T47D) or triple-negative breast cancers (TNBC) (BT20, BT549 and MDA-MB-231) as well as in MCF10A cells derived from non-tumorigenic mastectomy. We report profound upregulation of Sac3 and ArPIKfyve in the triple negative vs. hormone-sensitive breast cancer or non-tumorigenic cells, with BT cell lines showing the highest levels. siRNA-mediated knockdown of Sac3, but not that of PIKfyve, significantly inhibited proliferation of BT20 and BT549 cells. In these cells, knockdown of ArPIKfyve had only a minor effect, consistent with a primary role for Sac3 in TNBC cell proliferation. Intriguingly, steady-state levels of PtdIns(3,5)P₂ in BT20 and T47D cells were similar despite the 6-fold difference in Sac3 levels between these cell lines. However, steady-state levels of PtdIns3P and PtdIns5P, both regulated by the PAS complex, were significantly reduced in BT20 vs. T47D or MCF10A cell lines, consistent with elevated Sac3 affecting directly or indirectly the homeostasis of these lipids in TNBC. Together, our results uncover an unexpected role for Sac3 phosphatase in TNBC cell proliferation. Database analyses, discussed herein, reinforce the involvement of Sac3 in breast cancer pathogenesis.     

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

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.     

Vacuole size control: regulation of PtdIns(3,5)P2 levels by the vacuole-associated Vac14-Fig4 complex, a PtdIns(3,5)P2-specific phosphatase

In the budding yeast Saccharomyces cerevisiae, phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2) is synthesized by a single phosphatidylinositol 3-phosphate 5-kinase, Fab1. Cells deficient in PtdIns(3,5)P2 synthesis exhibit a grossly enlarged vacuole morphology, whereas increased levels of PtdIns(3,5)P2 provokes the formation of multiple small vacuoles, suggesting a specific role for PtdIns(3,5)P2 in vacuole size control. Genetic studies have indicated that Fab1 kinase is positively regulated by Vac7 and Vac14; deletion of either gene results in ablation of PtdIns(3,5)P2 synthesis and the formation of a grossly enlarged vacuole. More recently, a suppressor of vac7Delta mutants was identified and shown to encode a putative phosphoinositide phosphatase, Fig4. We demonstrate that Fig4 is a magnesium-activated PtdIns(3,5)P2-selective phosphoinositide phosphatase in vitro. Analysis of a Fig4-GFP fusion protein revealed that the Fig4 phosphatase is localized to the limiting membrane of the vacuole. Surprisingly, in the absence of Vac14, Fig4-GFP no longer localizes to the vacuole. However, Fig4-GFP remains localized to the grossly enlarged vacuoles of vac7 deletion mutants. Consistent with these observations, we found that Fig4 physically associates with Vac14 in a common membrane-associated complex. Our studies indicate that Vac14 both positively regulates Fab1 kinase activity and directs the localization/activation of the Fig4 PtdIns(3,5)P2 phosphatase.     

PIKfyve-ArPIKfyve-Sac3 Core Complex: CONTACT SITES AND THEIR CONSEQUENCE FOR Sac3 PHOSPHATASE ACTIVITY AND ENDOCYTIC MEMBRANE HOMEOSTASIS*

The phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P₂) metabolizing enzymes, the kinase PIKfyve and the phosphatase Sac3, constitute a single multiprotein complex organized by the PIKfyve regulator ArPIKfyve and its ability to homodimerize. We previously established that PIKfyve is activated within the triple PIKfyve-ArPIKfyve-Sac3 (PAS) core. These data assign an atypical function for the phosphatase in PtdIns(3,5)P₂ biosynthesis, thus raising the question of whether Sac3 retains its PtdIns(3,5)P₂ hydrolyzing activity within the PAS complex. Herein, we address the issue of Sac3 functionality by a combination of biochemical and morphological assays in triple-transfected COS cells using a battery of truncated or point mutants of the three proteins. We identified the Cpn60_TCP1 domain of PIKfyve as a major determinant for associating the ArPIKfyve-Sac3 subcomplex. Neither Sac3 nor PIKfyve enzymatic activities affected the PAS complex formation or stability. Using the well established formation of aberrant cell vacuoles as a sensitive functional measure of localized PtdIns(3,5)P₂ reduction, we observed a mitigated vacuolar phenotype by kinase-deficient PIKfyve^K1831E if its ArPIKfyve-Sac3 binding region was deleted, suggesting reduced Sac3 access to, and turnover of PtdIns(3,5)P₂. In contrast, PIKfyve^K1831E, which displays intact ArPIKfyve-Sac3 binding, triggered a more severe vacuolar phenotype if coexpressed with ArPIKfyve^WT-Sac3^WT but minimal defects when coexpressed with ArPIKfyve^WT and phosphatase-deficient Sac3^D488A. These data indicate that Sac3 assembled in the PAS regulatory core complex is an active PtdIns(3,5)P₂ phosphatase. Based on these and other data, presented herein, we propose a model of domain interactions within the PAS core and their role in regulating the enzymatic activities.     

Regulation of Fab1 phosphatidylinositol 3-phosphate 5-kinase pathway by Vac7 protein and Fig4, a polyphosphoinositide phosphatase family member

The Saccharomyces cerevisiae FAB1 gene encodes the sole phosphatidylinositol 3-phosphate [PtdIns(3)P] 5-kinase responsible for synthesis of the polyphosphoinositide PtdIns(3,5)P(2). VAC7 encodes a 128-kDa transmembrane protein that localizes to vacuolar membranes. Both vac7 and fab1 null mutants have dramatically enlarged vacuoles and cannot grow at elevated temperatures. Additionally, vac7Delta mutants have nearly undetectable levels of PtdIns(3,5)P(2), suggesting that Vac7 functions to regulate Fab1 kinase activity. To test this hypothesis, we isolated a fab1 mutant allele that bypasses the requirement for Vac7 in PtdIns(3,5)P(2) production. Expression of this fab1 allele in vac7Delta mutant cells suppresses the temperature sensitivity, vacuolar morphology, and PtdIns(3,5)P(2) defects normally exhibited by vac7Delta mutants. We also identified a mutant allele of FIG4, whose gene product contains a Sac1 polyphosphoinositide phosphatase domain, which suppresses vac7Delta mutant phenotypes. Deletion of FIG4 in vac7Delta mutant cells suppresses the temperature sensitivity and vacuolar morphology defects, and dramatically restores PtdIns(3,5)P(2) levels. These results suggest that generation of PtdIns(3,5)P(2) by the Fab1 lipid kinase is regulated by Vac7, whereas turnover of PtdIns(3,5)P(2) is mediated in part by the Sac1 polyphosphoinositide phosphatase family member Fig4.     

PIKfyve: Partners, significance, debates and paradoxes

Key components of membrane trafficking and signaling machinery in eukaryotic cells are proteins that bind or synthesize phosphoinositides. PIKfyve, a product of an evolutionarily conserved single-copy gene has both these features. It binds to membrane phosphatidylinositol (PtdIns)3P and synthesizes PtdIns(3,5)P2 and PtdIns5P. Molecular functions of PIKfyve are elusive but recent advances are consistent with a key role in the course of endosomal transport. PIKfyve dysfunction induces endosome enlargement and profound cytoplasmic vacuolation, likely as a result of impaired normal endosome processing and membrane exit out of endosomes. Multicellular organisms with genetically impaired function of PIKfyve or that of the PIKfyve protein partners regulating PtdIns(3,5)P2 homeostasis display severe disorders, including embryonic/perinatal death. This review describes recent advances on PIKfyve functionality in higher eukaryotes, with particular reference to biochemical and genetic insights in PIKfyve protein partners.     

ArPIKfyve-PIKfyve interaction and role in insulin-regulated GLUT4 translocation and glucose transport in 3T3-L1 adipocytes

Insulin activates glucose transport by promoting translocation of the insulin-sensitive fat/muscle-specific glucose transporter GLUT4 from an intracellular storage compartment to the cell surface. Here we report that an optimal insulin effect on glucose uptake in 3T3-L1 adipocytes is dependent upon expression of both PIKfyve, the sole enzyme for PtdIns 3,5-P(2) biosynthesis, and the PIKfyve activator, ArPIKfyve. Small-interfering RNAs that selectively ablated PIKfyve or ArPIKfyve in this cell type depleted the PtdIns 3,5-P(2) pool and reduced insulin-activated glucose uptake to a comparable degree. Combined loss of PIKfyve and ArPIKfyve caused further PtdIns 3,5-P(2) ablation that correlated with greater attenuation in insulin responsiveness. Loss of PIKfyve-ArPIKfyve reduced insulin-stimulated Akt phosphorylation and the cell surface accumulation of GLUT4 or IRAP, but not GLUT1-containing vesicles without affecting overall expression of these proteins. ArPIKfyve and PIKfyve were found to physically associate in 3T3-L1 adipocytes and this was insulin independent. In vitro labeling of membranes isolated from basal or insulin-stimulated 3T3-L1 adipocytes documented substantial insulin-dependent increases of PtdIns 3,5-P(2) production on intracellular membranes. Together, the data demonstrate for the first time a physical association between functionally related PIKfyve and ArPIKfyve in 3T3-L1 adipocytes and indicate that the novel ArPIKfyve-PIKfyve-PtdIns 3,5-P(2) pathway is physiologically linked to insulin-activated GLUT4 translocation and glucose transport.     

Phosphatidylinositol-3,5-bisphosphate: no longer the poor PIP2

Phosphoinositides play an important role in organelle identity by recruiting effector proteins to the host membrane organelle, thus decorating that organelle with molecular identity. Phosphatidylinositol-3,5-bisphos- phate [PtdIns(3,5)P(2) ] is a low-abundance phosphoinositide that predominates in endolysosomes in higher eukaryotes and in the yeast vacuole. Compared to other phosphoinositides such as PtdIns(4,5)P(2) , our understanding of the regulation and function of PtdIns(3,5)P(2) remained rudimentary until more recently. Here, we review many of the recent developments in PtdIns(3,5)P(2) function and regulation. PtdIns(3,5)P(2) is now known to espouse functions, not only in the regulation of endolysosome morphology, trafficking and acidification, but also in autophagy, signaling mediation in response to stresses and hormonal cues and control of membrane and ion transport. In fact, PtdIns(3,5)P(2) misregulation is now linked with several human neuropathologies including Charcot-Marie-Tooth disease and amyotrophic lateral sclerosis. Given the functional versatility of PtdIns(3,5)P(2) , it is not surprising that regulation of PtdIns(3,5)P(2) metabolism is proving rather elaborate. PtdIns(3,5)P(2) synthesis and turnover are tightly coupled via a protein complex that includes the Fab1/PIKfyve lipid kinase and its antagonistic Fig4/Sac3 lipid phosphatase. Most interestingly, many PtdIns(3,5)P(2) regulators play simultaneous roles in its synthesis and turnover.     

Assembly of a Fab1 phosphoinositide kinase signaling complex requires the Fig4 phosphoinositide phosphatase

Phosphatidylinositol-3,5-bisphosphate [PtdIns(3,5)P₂] regulates several vacuolar functions, including acidification, morphology, and membrane traffic. The lipid kinase Fab1 converts phosphatidylinositol-3-phosphate [PtdIns(3)P] to PtdIns(3,5)P₂. PtdIns(3,5)P₂ levels are controlled by the adaptor-like protein Vac14 and the Fig4 PtdIns(3,5)P₂-specific 5-phosphatase. Interestingly, Vac14 and Fig4 serve a dual function: they are both implicated in the synthesis and turnover of PtdIns(3,5)P₂ by an unknown mechanism. We now show that Fab1, through its chaperonin-like domain, binds to Vac14 and Fig4 and forms a vacuole-associated signaling complex. The Fab1 complex is tethered to the vacuole via an interaction between the FYVE domain in Fab1 and PtdIns(3)P on the vacuole. Moreover, Vac14 and Fig4 bind to each other directly and are mutually dependent for interaction with the Fab1 kinase. Our observations identify a protein complex that incorporates the antagonizing Fab1 lipid kinase and Fig4 lipid phosphatase into a common functional unit. We propose a model explaining the dual roles of Vac14 and Fig4 in the synthesis and turnover of PtdIns(3,5)P₂.     

Acquisition of unprecedented phosphatidylinositol 3,5-bisphosphate rise in hyperosmotically stressed 3T3-L1 adipocytes, mediated by ArPIKfyve-PIKfyve pathway

Unlike yeast, where hyperosmotic stress induces a dramatic increase in phosphatidylinositol 3,5-bisphosphate (PtdIns 3,5-P(2)) synthesis, in mammalian cells, although activating a complex array of signaling events, hyperosmotic stress fails to up-regulate PtdIns 3,5-P(2), indicating the PtdIns 3,5-P(2) pathway is not involved in mammalian osmo-protective responses. Here we report an unexpected and marked PtdIns 3,5-P(2) increase in response to hyperosmotic stress in differentiated 3T3-L1 adipocytes. Because this effect was not observed in the precursor preadipocytes, a specific role during acquisition of the adipocyte phenotype and transition into insulin-responsive cells could be suggested. However, acute insulin action did not result in a measurable PtdIns 3,5-P(2) rise, indicating the PtdIns 3,5-P(2) pathway is a specific hyperosmotically activated signaling cascade selectively operating in differentiated 3T3-L1 adipocytes. Hyperosmolarity activates different components of several kinase cascades, including p38 mitogen-activated protein and tyrosine kinases, but these appear to be separate from the activated PtdIns 3,5-P(2) pathway. Because PtdIns 3,5-P(2) is primarily produced by PIKfyve-catalyzed synthesis and requires the upstream activator hVac14 (called herein ArPIKfyve) that physically associates with and activates PIKfyve, we examined the contribution of ArPIKfyve-PIKfyve for the hyperosmotic stress-induced rise in PtdIns 3,5-P(2). Small interfering RNA-directed gene silencing to selectively deplete ArPIKfyve or PIKfyve in 3T3-L1 adipocytes determined the ArPIKfyve-PIKfyve axis fully accountable for the hyperosmotically activated PtdIns 3,5-P(2). Together these results reveal a previously uncharacterized PtdIns 3,5-P(2) pathway activated selectively in hyperosmotically stressed 3T3-L1 adipocytes and suggest a plausible role for PtdIns 3,5-P(2) in the osmo-protective response mechanism in this cell type.     

PIKfyve Regulation of Endosome-Linked Pathways

The phosphoinositide 5-kinase (PIKfyve) is a critical enzyme for the synthesis of PtdIns(3,5) P ₂, that has been implicated in various trafficking events associated with the endocytic pathway. We have now directly compared the effects of siRNA-mediated knockdown of PIKfyve in HeLa cells with a specific pharmacological inhibitor of enzyme activity. Both approaches induce changes in the distribution of CI-M6PR and trans-Golgi network (TGN)-46 proteins, which cycles between endosomes and TGN, leading to their accumulation in dispersed punctae, whilst the TGN marker golgin-245 retains a perinuclear disposition. Trafficking of CD8-CI-M6PR (retromer-dependent) and CD8-Furin (retromer-independent) chimeras from the cell surface to the TGN is delayed following drug administration, as is the transport of the Shiga toxin B-subunit. siRNA knockdown of PIKfyve produced no defect in epidermal growth factor receptor (EGFR) degradation, unless combined with knockdown of its activator molecule Vac14, suggesting that a low threshold of PtdIns(3,5) P ₂ is necessary and sufficient for this pathway. Accordingly pharmacological inhibition of PIKfyve results in a profound block to the lysosomal degradation of activated epidermal growth factor (EGF) and Met receptors. Immunofluorescence revealed EGF receptors to be trapped in the interior of a swollen endosomal compartment. In cells starved of amino acids, PIKfyve inhibition leads to the accumulation of the lipidated form of GFP-LC3, a marker of autophagosomal structures, which can be visualized as fluorescent punctae. We suggest that PIKfyve inhibition may render the late endosome/lysosome compartment refractory to fusion with both autophagosomes and with EGFR-containing multivesicular bodies.     

Localized PtdIns 3,5-P2 synthesis to regulate early endosome dynamics and fusion

Perturbations in the intracellular PtdIns 3,5-P₂ pool or the downstream transmission of PtdIns 3,5-P₂ signals often result in a gradual development of gross morphological changes in the pleiomorphic multivesicular endosomes, culminating with the appearance of cytoplasmic vacuoles. To identify the onset of PtdIns 3,5-P₂ functional requirements along the endocytic system, in this study we characterized the morphological changes associated with early expression of the dominant-negative kinase-deficient form (K1831E) of the PtdIns 3,5-P₂-producing kinase PIKfyve, before the formation of cytoplasmic vacuoles in transfected COS cells. Enlarged PIKfyve^K1831E-positive vesicles co-localizing with dilated EEA1- and Rab5a^WT-positive perinuclear endosomes were observed (WT, wild type). This was dependent on the presence of active forms of Rab5 and the generation of PtdIns 3-P-enriched platforms on early endosomess. Because PIKfyve^WT did not substantially colocalize with EEA1- or Rab5-positive endosomes in COS cells, the dynamic PIKfyve-catalyzed PtdIns 3-to-PtdIns 3,5-P₂ switch was suggested to drive away PIKfyve^WT from early endosomes toward later compartments. Late endosomes/lysosomes marked by LAMP1 or Rab7 were dislocated from their typical perinuclear position upon PIKfyve^K1831E early expression. Cytosols derived from cells stably expressing PIKfyve^K1831E stimulated endosome fusion in vitro, whereas PIKfyve^WT-enriched cytosols had the opposite effect, consistent with PtdIns 3,5-P₂ production negatively regulating the endosome fusion. Together, our data indicate that PtdIns 3,5-P₂ defines specific endosome platforms at the onset of the degradation pathway to regulate the complex process of membrane remodeling and dynamics. carrier vesicle; multivesicular bodies; PIKfyve; Rab5/EEA1/PtdINS3-P platforms; Rab7; LAMP1     

Snx10 and PIKfyve are required for lysosome formation in osteoclasts

Bone resorption and organelle homeostasis in osteoclasts require specialized intracellular trafficking. Sorting nexin 10 (Snx10) is a member of the sorting nexin family of proteins that plays crucial roles in cargo sorting in the endosomal pathway by its binding to phosphoinositide(3)phosphate (PI3P) localized in early endosomes. We and others have shown previously that the gene encoding sorting Snx10 is required for osteoclast morphogenesis and function, as osteoclasts from humans and mice lacking functional Snx10 are dysfunctional. To better understand the role and mechanisms by which Snx10 regulates vesicular transport, the aim of the present work was to study PIKfyve, another PI3P-binding protein, which phosphorylates PI3P to PI(3,5)P2. PI(3,5)P2 is known to be required for endosome/lysosome maturation, and the inhibition of PIKfyve causes endosome enlargement. Overexpression of Snx10 also induces accumulation of early endosomes suggesting that both Snx10 and PIKfyve are required for normal endosome/lysosome transition. Apilimod is a small molecule with specific, nanomolar inhibitory activity on PIKfyve but only in the presence of key osteoclast factors CLCN7, OSTM1, and Snx10. This observation suggests that apilimod's inhibitory effects are mediated by endosome/lysosome disruption. Here we show that both Snx10 and PIKfyve colocalize to early endosomes in osteoclasts and coimmunoprecipitate in vesicle fractions. Treatment with 10 nM apilimod or genetic deletion of PIKfyve in cells resulted in the accumulation of early endosomes, and in the inhibition of osteoclast differentiation, lysosome formation, and secretion of TRAP from differentiated osteoclasts. Snx10 and PIKfyve also colocalized in gastric zymogenic cells, another cell type impacted by Snx10 mutations. Apilimod-specific inhibition of PIKfyve required Snx10 expression, as it did not inhibit lysosome biogenesis in Snx10-deficient osteoclasts. These findings suggest that Snx10 and PIKfyve are involved in the regulation of endosome/lysosome homeostasis via the synthesis of PI(3,5)P2 and may point to a new strategy to prevent bone loss.     

Sac3 Is an Insulin-regulated Phosphatidylinositol 3,5-Bisphosphate Phosphatase: GAIN IN INSULIN RESPONSIVENESS THROUGH Sac3 DOWN-REGULATION IN ADIPOCYTES*

Insulin-regulated stimulation of glucose entry and mobilization of fat/muscle-specific glucose transporter GLUT4 onto the cell surface require the phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P₂) pathway for optimal performance. The reduced insulin responsiveness observed under ablation of the PtdIns(3,5)P₂-synthesizing PIKfyve and its associated activator ArPIKfyve in 3T3L1 adipocytes suggests that dysfunction of the PtdIns(3,5)P₂-specific phosphatase Sac3 may yield the opposite effect. Paradoxically, as uncovered recently, in addition to turnover Sac3 also supports PtdIns(3,5)P₂ biosynthesis by allowing optimal PIKfyve-ArPIKfyve association. These opposing inputs raise the key question as to whether reduced Sac3 protein levels and/or hydrolyzing activity will produce gain in insulin responsiveness. Here we report that small interfering RNA-mediated knockdown of endogenous Sac3 by ∼60%, which resulted in a slight but significant elevation of PtdIns(3,5)P₂ in 3T3L1 adipocytes, increased GLUT4 translocation and glucose entry in response to insulin. In contrast, ectopic expression of Sac3^WT, but not phosphatase-deficient Sac3^D488A, reduced GLUT4 surface abundance in the presence of insulin. Endogenous Sac3 physically assembled with PIKfyve and ArPIKfyve in both membrane and soluble fractions of 3T3L1 adipocytes, but this remained insulin-insensitive. Importantly, acute insulin markedly reduced the in vitro C8-PtdIns(3,5)P₂ hydrolyzing activity of Sac3. The insulin-sensitive Sac3 pool likely controls a discrete PtdIns(3,5)P₂ subfraction as the high pressure liquid chromatography-measurable insulin-dependent elevation in total [³H]inositol-PtdIns(3,5)P₂ was minor. Together, our data identify Sac3 as an insulin-sensitive phosphatase whose down-regulation increases insulin responsiveness, thus implicating Sac3 as a novel drug target in insulin resistance.Insulin simulation of glucose uptake in fat and muscle, which is mediated by the facilitative fat/muscle-specific glucose transporter GLUT4, is essential for maintenance of whole-body glucose homeostasis (1–7). In basal states GLUT4 is localized in the cell interior, cycling slowly between the plasma membrane and one or more intracellular compartments. Insulin action profoundly activates movements of preformed postendosomal GLUT4 storage vesicles toward the cell surface and their subsequent plasma membrane fusion, thereby increasing the rate of glucose transport >10-fold. Defective signaling/execution of GLUT4 translocation is considered to be a common feature in insulin resistance and type 2 diabetes (8, 9). However, the molecular and cellular regulatory mechanisms whereby insulin activates GLUT4 membrane dynamics and glucose transport are still not fully understood. More than 60 protein and phospholipid intermediate players are currently implicated in orchestrating the overall process (1–7). A central role is attributed to the highest phosphorylated member of the phosphoinositide (PI)³ family, i.e. phosphatidylinositol (PtdIns) (3,4,5)P₃ (3). PtdIns(3,4,5)P₃ is generated at the cell surface by the action of wortmannin-sensitive class 1A PI3K that is activated via the insulin-stimulated IR/IR receptor substrate signaling pathway. Inositol polyphosphate 5-phosphatases SHIP or SKIP and 3-phosphatase PTEN rapidly convert PtdIns(3,4,5)P₃ to PtdIns(3,4)P₂ and PtdIns(4,5)P₂, respectively, thereby terminating insulin signal through class 1A PI3K (10–13). The class 1A PI3K-opposing function of these lipid phosphatases has provided an appealing prospect that inhibition of their hydrolyzing activities could produce significant efficacy in the treatment of type 2 diabetes and obesity (14–16).It has recently become apparent that signals by other PIs act in parallel with that of PtdIns(3,4,5)P₃ in integrating the IR-issued signal with GLUT4 surface translocation (3, 4). One such signaling molecule is PtdIns(3,5)P₂, whose functioning as a positive regulator in 3T3L1 adipocyte responsiveness to insulin has been supported by several lines of experimental evidence. Thus, expression of dominant-negative kinase-deficient mutants of PIKfyve, the sole enzyme for PtdIns(3,5)P₂ synthesis (17, 18), inhibits insulin-induced gain of surface GLUT4 without noticeable aberrations of cell morphology (19). Likewise, reduction in the intracellular PtdIns(3,5)P₂ pool through siRNA-mediated PIKfyve depletion reduces GLUT4 cell-surface accumulation and glucose transport activation in response to insulin (20). Concordantly, loss of ArPIKfyve, a PIKfyve activator that physically associates with PIKfyve to facilitate PtdIns(3,5)P₂ intracellular production (21, 22), also decreases insulin-stimulated glucose uptake in 3T3L1 adipocytes (20). Combined ablation of PIKfyve and ArPIKfyve produces a greater decrease in this effect, correlating with a greater reduction in the intracellular PtdIns(3,5)P₂ pool (20). Finally, pharmacological inhibition of PIKfyve activity powerfully reduces the net insulin effect on glucose uptake (23). These observations indicate positive signaling through the PtdIns(3,5)P₂ pathway and suggest that arrested PtdIns(3,5)P₂ turnover might potentiate insulin-regulated activation of glucose uptake.Sac3, a product of a single-copy gene in mammals, is a recently characterized phosphatase implicated in PtdIns(3,5)P₂ turnover (24). Our observations in several mammalian cell types have revealed that Sac3 plays an intricate role in the PtdIns(3,5)P₂ homeostatic mechanism. It is a constituent of the PtdIns(3,5)P₂ biosynthetic PIKfyve-ArPIKfyve complex and facilitates the association of these two (24, 25). Intriguingly, only if the PIKfyve-ArPIKfyve-Sac3 triad (known as the “PAS complex”) is intact will the PIKfyve enzymatic activity be activated (25). Thus, Sac3 not only catalyzes PtdIns(3,5)P₂ turnover but also promotes PtdIns(3,5)P₂ synthesis by functioning as an adaptor for the efficient association of PIKfyve with, and activation by, ArPIKfyve (25). Given these two seemingly opposing inputs, a critical question is whether reduction in Sac3 protein levels or phosphatase activity would facilitate or mitigate insulin action on glucose uptake and GLUT4 translocation. We demonstrate here that reduced levels of Sac3 potentiate, whereas ectopic expression of active Sac3 phosphatase reduces insulin responsiveness of GLUT4 translocation and glucose transport in 3T3L1 adipocytes. Whereas insulin action does not affect the PIKfyve kinase-Sac3 phosphatase association, it markedly inhibits the Sac3 hydrolyzing activity. We suggest that increased PtdIns(3,5)P₂ local availability through Sac3 phosphatase inhibition links insulin signaling to its effect on GLUT4 vesicle dynamics and glucose transport.     

PIKfyve Kinase and SKD1 AAA ATPase define distinct endocytic compartments. Only PIKfyve expression inhibits the cell-vacoulating activity of Helicobacter pylori VacA toxin

The mammalian phosphatidylinositol (PtdIns)- 5-P/PtdIns-3,5-P(2)-producing kinase PIKfyve and AAA ATPase SKD1, as their yeast counterparts, are implicated in the formation and function of multivesicular bodies/late endosomes. Point mutations inhibiting the enzyme activities convert PIKfyve and SKD1 into dominant-negative mutants (PIKfyve(K1831E) and SKD1(E235Q)), whose expression in cells of kidney origin induces a vacuolation phenotype. This phenotype closely resembles the changes in late endosomal-lysosomal morphology that occur following cell exposure to the vacuolating cytotoxin (VacA) from Helicobacter pylori. Here we have examined the possible functional relationship between PIKfyve and SKD1 as well as the role of these enzymes in the molecular mechanism of VacA-induced intracellular vacuolation. When co-expressed in COS cells, PIKfyve(WT) reduced SKD1(E235Q)dependent vacuole formation, whereas SKD1(WT) did not alter the vacuolation induced by PIKfyve(K1831E). In addition, SKD1(E235Q) disrupted the normal distribution of PIKfyve(WT). Expression of PIKfyve(WT) in COS and HEK293 cells inhibited vacuolation induced by subsequent intoxication with VacA, and microinjection of the PIKfyve lipid product PtdIns-3,5-P(2) produced a similar inhibitory effect. In contrast, in COS cells expressing SKD1(WT), VacA induced the formation of characteristic vacuoles with an efficiency similar to that in the control cells. These observations demonstrate that, although PIKfyve and SKD1 are functionally related, only PIKfyve regulates VacA action, and suggest that the inhibition of PIKfyve PtdIns-3,5-P(2)-producing activity is a key molecular event in VacA-induced cellular vacuolation.     

Vac14 controls PtdIns(3,5)P(2) synthesis and Fab1-dependent protein trafficking to the multivesicular body

BACKGROUND: The PtdIns3P 5-kinase Fab1 makes PtdIns(3,5)P(2), a phosphoinositide essential for retrograde trafficking between the vacuole/lysosome and the late endosome and also for trafficking of some proteins into the vacuole via multivesicular bodies (MVB). No regulators of Fab1 were identified until recently. RESULTS: Visual screening of the Eurofan II panel of S. cerevisiae deletion mutants identified YLR386w as a novel regulator of vacuolar function. Others recently identified this ORF as encoding the vacuolar inheritance gene VAC14. Like fab1 mutants, yeast lacking Vac14 have enlarged vacuoles that do not acidify correctly. FAB1 overexpression corrects these defects. vac14Delta cells make very little PtdIns(3,5)P(2), and hyperosmotic shock does not stimulate PtdIns(3,5)P(2) synthesis in the normal manner, implicating Vac14 in Fab1 regulation. We also show that, like fab1Delta mutants, vac14Delta cells fail to sort GFP-Phm5 to the MVB and thence to the vacuole: irreversible ubiquitination of GFP-Phm5 overcomes this defect. In the BY4742 genetic background, loss of Vac14 causes much more penetrant effects on phosphoinositide metabolism and vacuolar trafficking than does loss of Vac7, another regulator of Fab1. Vac14 contains motifs suggestive of a role in protein trafficking and interacts with several proteins involved in clathrin-mediated membrane sorting and phosphoinositide metabolism. CONCLUSIONS: Vac14 and Vac7 are both upstream activators of Fab1-catalysed PtdIns(3,5)P(2) synthesis, with Vac14 the dominant contributor to the hierarchy of control. Vac14 is essential for the regulated synthesis of PtdIns(3,5)P(2), for control of trafficking of some proteins to the vacuole lumen via the MVB, and for maintenance of vacuole size and acidity.     

Mammalian cell morphology and endocytic membrane homeostasis require enzymatically active phosphoinositide 5-kinase PIKfyve

The dual specificity mammalian enzyme PIKfyve phosphorylates in vitro position d-5 in phosphatidylinositol (PtdIns) and PtdIns 3-P, itself or exogenous protein substrates. Here we have addressed the crucial questions for the identity of the lipid products and the role of PIKfyve enzymatic activity in mammalian cells. CHO, HEK293, and COS cells expressing PIKfyve(WT) at high levels and >90% efficiencies increased selectively the intracellular PtdIns 3,5-P(2) production by 30--55%. In these cell types PtdIns 5-P was undetectable. A kinase-deficient point mutant, PIKfyve(K1831E), transiently transfected into these or other cells elicited a dramatic dominant phenotype. Subsequent to a dilation of the PIKfyve-containing vesicles, PIKfyve(K1831E)-expressing cells progressively accumulated multiple swollen lucent vacuoles of endosomal origin, first in the perinuclear cytoplasm and then toward the cell periphery. Despite their drastically altered morphology, the PIKfyve(K1831E)-expressing cells were viable and functionally active, evidenced by several criteria. This phenotype was completely reversed by introducing PIKfyve(WT) into the PIKfyve(K1831E)-transfected cells. Disruptions of the localization signal in the PIKfyve kinase-deficient mutant yielded a PIKfyve(K1831E Delta fyve) protein, incompetent to vacuolate cells, implying that an active PIKfyve enzyme at distinct late endocytic membranes is crucial for normal cell morphology. This was further substantiated by examining the vacuolation-induced potency of several pharmacological stimuli in cells expressing high PIKfyve(WT) levels. Together, the results indicate that PIKfyve enzymatic activity, possibly through the generation of PtdIns 3,5-P(2), and/or yet to be identified endogenous phosphoproteins, is critical for cell morphology and endomembrane homeostasis.