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.     

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     

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.     

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.     

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.     

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

Perturbations in phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2)-synthesizing enzymes result in enlarged endocytic organelles from yeast to humans, indicating evolutionarily conserved function of PtdIns(3,5)P2 in endosome-related events. This is reinforced by the structural and functional homology of yeast Vac14 and human Vac14 (ArPIKfyve), which activate yeast and mammalian PtdIns(3,5)P2-producing enzymes, Fab1 and PIKfyve, respectively. In yeast, PtdIns(3,5)P2-specific phosphatase, Fig4, in association with Vac14, turns over PtdIns(3,5)P2, but whether such a mechanism operates in mammalian cells and what the identity of mammalian Fig4 may be are unknown. Here we have identified and characterized Sac3, a Sac domain phosphatase, as the Fig4 mammalian counterpart. Endogenous Sac3, a widespread 97-kDa protein, formed a stable ternary complex with ArPIKfyve and PIKfyve. Concordantly, Sac3 cofractionated and colocalized with ArPIKfyve and PIKfyve. The intrinsic Sac3(WT) phosphatase activity preferably hydrolyzed PtdIns(3,5)P2 in vitro, although the other D5-phosphorylated polyphosphoinositides were also substrates. Ablation of endogenous Sac3 by short interfering RNAs elevated PtdIns(3,5)P2 in (32)P-labeled HEK293 cells. Ectopically expressed Sac3(WT) in COS cells colocalized with and dilated EEA1-positive endosomes, consistent with the PtdIns(3,5)P2 requirement in early endosome dynamics. In vitro reconstitution of carrier vesicle formation from donor early endosomes revealed a gain of function upon Sac3 loss, whereas PIKfyve or ArPIKfyve protein depletion produced a loss of function. These data demonstrate a coupling between the machinery for PtdIns(3,5)P2 synthesis and turnover achieved through a physical assembly of PIKfyve, ArPIKfyve, and Sac3. We suggest that the tight regulation in PtdIns(3,5)P2 homeostasis is mechanistically linked to early endosome dynamics in the course of cargo transport.     

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.     

The Phosphoinositide‐Gated Lysosomal Ca2+ Channel,TRPML1, Is Required for Phagosome Maturation

Macrophages internalize and sequester pathogens into a phagosome. Phagosomes then sequentially fuse with endosomes and lysosomes, converting into degradative phagolysosomes. Phagosome maturation is a complex process that requires regulators of the endosomal pathway including the phosphoinositide lipids. Phosphatidylinositol‐3‐phosphate and phosphatidylinositol‐3,5‐bisphosphate (PtdIns(3,5)P₂), which respectively control early endosomes and late endolysosomes, are both required for phagosome maturation. Inhibition of PIKfyve, which synthesizes PtdIns(3,5)P₂, blocked phagosome–lysosome fusion and abated the degradative capacity of phagosomes. However, it is not known how PIKfyve and PtdIns(3,5)P₂ participate in phagosome maturation. TRPML1 is a PtdIns(3,5)P₂‐gated lysosomal Ca²⁺ channel. Because Ca²⁺ triggers membrane fusion, we postulated that TRPML1 helps mediate phagosome–lysosome fusion. Using Fcγ receptor‐mediated phagocytosis as a model, we describe our research showing that silencing of TRPML1 hindered phagosome acquisition of lysosomal markers and reduced the bactericidal properties of phagosomes. Specifically, phagosomes isolated from TRPML1‐silenced cells were decorated with lysosomes that docked but did not fuse. We could rescue phagosome maturation in TRPML1‐silenced and PIKfyve‐inhibited cells by forcible Ca²⁺ release with ionomycin. We also provide evidence that cytosolic Ca²⁺ concentration increases upon phagocytosis in a manner dependent on TRPML1 and PIKfyve. Overall, we propose a model where PIKfyve and PtdIns(3,5)P₂ activate TRPML1 to induce phagosome–lysosome fusion.      

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.     

The Fab1/PIKfyve Phosphoinositide Phosphate Kinase Is Not Necessary to Maintain the pH of Lysosomes and of the Yeast Vacuole

Lysosomes and the yeast vacuole are degradative and acidic organelles. Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P₂), a master architect of endolysosome and vacuole identity, is thought to be necessary for vacuolar acidification in yeast. There is also evidence that PtdIns(3,5)P₂ may play a role in lysosomal acidification in higher eukaryotes. Nevertheless, these conclusions rely on qualitative assays of lysosome/vacuole pH. For example, quinacrine, an acidotropic fluorescent base, does not accumulate in the vacuoles of fab1Δ yeast. Fab1, along with its mammalian ortholog PIKfyve, is the lipid kinase responsible for synthesizing PtdIns(3,5)P₂. In this study, we employed several assays that quantitatively assessed the lysosomal and vacuolar pH in PtdIns(3,5)P₂-depleted cells. Using ratiometric imaging, we conclude that lysosomes retain a pH < 5 in PIKfyve-inhibited mammalian cells. In addition, quantitative fluorescence microscopy of vacuole-targeted pHluorin, a pH-sensitive GFP variant, indicates that fab1Δ vacuoles are as acidic as wild-type yeast. Importantly, we also employed fluorimetry of vacuoles loaded with cDCFDA, a pH-sensitive dye, to show that both wild-type and fab1Δ vacuoles have a pH < 5.0. In comparison, the vacuolar pH of the V-ATPase mutant vph1Δ or vph1Δ fab1Δ double mutant was 6.1. Although the steady-state vacuolar pH is not affected by PtdIns(3,5)P₂ depletion, it may have a role in stabilizing the vacuolar pH during salt shock. Overall, we propose a model in which PtdIns(3,5)P₂ does not govern the steady-state pH of vacuoles or lysosomes.     

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.     

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.     

Inhibition of PIKfyve by YM-201636 Dysregulates Autophagy and Leads to Apoptosis-Independent Neuronal Cell Death

The lipid phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P ₂), synthesised by PIKfyve, regulates a number of intracellular membrane trafficking pathways. Genetic alteration of the PIKfyve complex, leading to even a mild reduction in PtdIns(3,5)P ₂, results in marked neurodegeneration via an uncharacterised mechanism. In the present study we have shown that selectively inhibiting PIKfyve activity, using YM-201636, significantly reduces the survival of primary mouse hippocampal neurons in culture. YM-201636 treatment promoted vacuolation of endolysosomal membranes followed by apoptosis-independent cell death. Many vacuoles contained intravacuolar membranes and inclusions reminiscent of autolysosomes. Accordingly, YM-201636 treatment increased the level of the autophagosomal marker protein LC3-II, an effect that was potentiated by inhibition of lysosomal proteases, suggesting that alterations in autophagy could be a contributing factor to neuronal cell death.     

Production of phosphatidylinositol 5‐phosphate via PIKfyve and MTMR3 regulates cell migration

Although phosphatidylinositol 5‐phosphate (PtdIns5P) is present in many cell types and its biogenesis is increased by diverse stimuli, its precise cellular function remains elusive. Here we show that PtdIns5P levels increase when cells are stimulated to move and we find PtdIns5P to promote cell migration in tissue culture and in a Drosophila in vivo model. First, class III phosphatidylinositol 3‐kinase, which produces PtdIns3P, was shown to be involved in migration of fibroblasts. In a cell migration screen for proteins containing PtdIns3P‐binding motifs, we identified the phosphoinositide 5‐kinase PIKfyve and the phosphoinositide 3‐phosphatase MTMR3, which together constitute a phosphoinositide loop that produces PtdIns5P via PtdIns(3,5)P₂. The ability of PtdIns5P to stimulate cell migration was demonstrated directly with exogenous PtdIns5P and a PtdIns5P‐producing bacterial enzyme. Thus, the identified phosphoinositide loop defines a new role for PtdIns5P in cell migration.     

Phosphatidylinositol 3,5‐Bisphosphate‐Rich Membrane Domains in Endosomes and Lysosomes

Phosphatidylinositol 3,5‐bisphosphate (PtdIns(3,5)P₂) has critical functions in endosomes and lysosomes. We developed a method to define nanoscale distribution of PtdIns(3,5)P₂ using freeze‐fracture electron microscopy. GST‐ATG18‐4×FLAG was used to label PtdIns(3,5)P₂ and its binding to phosphatidylinositol 3‐phosphate (PtdIns(3)P) was blocked by an excess of the p40^phox PX domain. In yeast exposed to hyperosmotic stress, PtdIns(3,5)P₂ was concentrated in intramembrane particle (IMP)‐deficient domains in the vacuolar membrane, which made close contact with adjacent membranes. The IMP‐deficient domain was also enriched with PtdIns(3)P, but was deficient in Vph1p, a liquid‐disordered domain marker. In yeast lacking either PtdIns(3,5)P₂ or its effector, Atg18p, the IMP‐deficient, PtdIns(3)P‐rich membranes were folded tightly to make abnormal tubular structures, thus showing where the vacuolar fragmentation process is arrested when PtdIns(3,5)P₂ metabolism is defective. In HeLa cells, PtdIns(3,5)P₂ was significantly enriched in the vesicular domain of RAB5‐ and RAB7‐positive endosome/lysosomes of the tubulo‐vesicular morphology. This biased distribution of PtdIns(3,5)P₂ was also observed using fluorescence microscopy, which further showed enrichment of a retromer component, VPS35, in the tubular domain. This is the first report to show segregation of PtdIns(3,5)P₂‐rich and ‐deficient domains in endosome/lysosomes, which should be important for endosome/lysosome functionality.      

Changing phosphoinositides “on the fly”: how trafficking vesicles avoid an identity crisis

Joining an antagonistic phosphoinositide (PtdInsP) kinase and phosphatase into a single protein complex may regulate rapid and local PtdInsP changes. This may be important for processes such as membrane fission that require a specific PtdInsP and that are innately local and rapid. Such a complex could couple vesicle formation, with erasing of the identity of the donor organelle from the vesicle prior to its fusion with target organelles, thus preventing organelle identity intermixing. Coordinating signals are postulated to switch the relative activities of the kinase and phosphatase in a spatio‐temporal manner that matches membrane fission events. The discovery of two such complexes supports this hypothesis. One regulates the interconversion of phosphatidylinositol and PtdIns(3)P by joining the Vps34 PtdIns 3‐kinase and the myotubularin 3‐phosphatases. The other regulates the interconversion between PtdIns(3)P and PtdIns(3,5)P₂ through the Fab1/PIKfyve kinase and the Fig4/mFig4 phosphatase. These lipids are essential components of the endosomal identity code.     

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.     

Arabidopsis FAB1/PIKfyve Proteins Are Essential for Development of Viable Pollen

Phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P₂] is a phospholipid that has a role in controlling membrane trafficking events in yeast and animal cells. The function of this lipid in plants is unknown, although its synthesis has been shown to be up-regulated upon osmotic stress in plant cells. PtdIns(3,5)P₂ is synthesized by the PIKfyve/Fab1 family of proteins, with two orthologs, FAB1A and FAB1B, being present in Arabidopsis (Arabidopsis thaliana). In this study, we attempt to address the role of this lipid by analyzing the phenotypes of plants mutated in FAB1A and FAB1B. It was not possible to generate plants homozygous for mutations in both genes, although single mutants were isolated. Both homozygous single mutant plant lines exhibited a leaf curl phenotype that was more marked in FAB1B mutants. Genetic transmission analysis revealed that failure to generate double mutant lines was entirely due to inviability of pollen carrying mutant alleles of both FAB1A and FAB1B. This pollen displayed severe defects in vacuolar reorganization following the first mitotic division of development. The presence of abnormally large vacuoles in pollen at the tricellular stage resulted in the collapse of the majority of grains carrying both mutant alleles. This demonstrates a crucial role for PtdIns(3,5)P₂ in modulating the dynamics of vacuolar rearrangement essential for successful pollen development. Taken together, our results are consistent with PtdIns(3,5)P₂ production being central to cellular responses to changes in osmotic conditions.Phosphoinositides make up a minor fraction of total membrane lipids in all eukaryotic organisms. Their production is spatially restricted to the cytoplasmic leaflet of specific organellar membranes and temporally regulated by phosphatidylinositol (PtdIns) kinases and phosphatases. Three of the five hydoxyl groups of PtdIns can be phosphorylated, either singly or combinatorially, to produce seven different phosphoinositides. These different phosphoinositides can recruit and/or activate proteins with specific phosphoinositide-binding domains and have been implicated in the regulation of many important cellular functions, including membrane trafficking, cell growth, and cytoskeleton remodeling (Di Paolo and De Camilli, 2006).In animal cells, phosphorylation at the 3 position of PtdIns and its phosphorylated derivatives can be carried out by three different classes of PtdIns 3-kinase (classes I–III; Cantley, 2002). Plants and yeast only have class III PtdIns 3-kinases that are orthologs of the Saccharomyces cerevisiae protein Vps34p (Mueller-Roeber and Pical, 2002). Vps34p orthologs are thought to use PtdIns as their sole lipid substrate and produce PtdIns 3-phosphate (PtdIns3P). PtdIns3P is involved in endosomal/lysosomal protein sorting in eukaryotic cells in addition to cellular signaling events (Backer, 2008).In plants, PtdIns3P is essential for normal growth and development. Arabidopsis (Arabidopsis thaliana) plants carrying a VPS34 antisense construct have severe developmental defects (Welters et al., 1994). Furthermore, using pharmacological inhibitors of PtdIns3P production and analysis of transgenic plants defective in downstream signaling from PtdIns3P, it has been shown that this lipid has a role to play in many diverse physiological processes, such as root hair growth (Lee et al., 2008a). The phenotypes observed in studies of PtdIns3P function in plants are consistent with a role in endosomal and vacuolar trafficking in plants (Kim et al., 2001; Lee et al., 2008a), as in other eukaryotes. Recently, an attempt to generate vps34 homozygous mutant plant lines was unsuccessful due to failure of the mutant vps34 allele to transmit through the male germ line (Lee et al., 2008b).Importantly, PtdIns3P is the precursor to another phosphoinositide, PtdIns 3,5-bisphosphate [PtdIns(3,5)P₂], which also has vital roles in endosomal trafficking in eukaryotes (Dove et al., 2009). Thus, it is possible that some of the effects in plants attributed to PtdIns3P in previous studies may actually be due to an inability of cells to produce PtdIns(3,5)P₂. PtdIns(3,5)P₂ is produced by the PtdIns3P 5-kinases PIKfyve and Fab1p in animal and yeast cells, respectively. PIKfyve/Fab1p proteins have an N-terminal FYVE domain necessary for binding to PtdIns3P-containing membranes, a central Cpn60_TCP1 (for HSP chaperonin T complex 1) homology domain, and a C-terminal kinase domain. In Arabidopsis, there are a number of genes encoding putative Fab1p homologs, but only two of them, FAB1A (At4g33240) and FAB1B (At3g14270), encode proteins having FYVE domains at their N termini (Mueller-Roeber and Pical, 2002). It is likely that these proteins are PtdIns3P 5-kinases in Arabidopsis. Despite the importance of PtdIns(3,5)P₂ in yeast and animals, very little is known about its function in plants. However, it has been shown that hyperosmotic stress can induce the rapid synthesis of PtdIns(3,5)P₂ in cell suspension cultures from a number of plant species (Meijer and Munnik, 2003) and in pollen tubes from tobacco (Nicotiana tabacum; Zonia and Munnik, 2004). This production is consistent with a requirement for PtdIns(3,5)P₂ in vacuolar membrane reorganization, as water moves from the vacuole to the cytosol upon cells being placed under hyperosmotic stress. In animal cells, defective PtdIns(3,5)P₂ production leads to cytoplasmic vacuolation of endosome-derived membranes (Ikonomov et al., 2001; Jefferies et al., 2008). It seems that there is a general requirement in all eukaryotes for PtdIns(3,5)P₂ production in endomembrane remodeling. This remodeling could be mediated by proteins that bind to PtdIns(3,5)P₂. A number of candidates have been identified, including yeast Svp1p (Dove et al., 2004), its mammalian homolog WIP149 (Jeffries et al., 2004), CHMP3 (Whitley et al., 2003), and Ent3p (Friant et al., 2003).In this study, we aimed to further investigate the role of PtdIns(3,5)P₂ in plant physiology and the function of PIKfyve/Fab1p orthologs in Arabidopsis by generating mutant plant lines homozygous for T-DNA insertions in both FAB1A and FAB1B. We failed to generate double homozygous fab1a/fab1b knockout plants but observed subtle phenotypes in both fab1a and fab1b single homozygous plants. The data show that pollen with a fab1a/fab1b genotype has an abnormal vacuolar phenotype and does not contribute to the next generation. Our data are consistent with the hypothesis that the male gametophytic defect observed in vps34 mutant pollen (Lee et al., 2008b) is due to an inability of this pollen to generate PtdIns(3,5)P₂ and is not a direct result of the lack of PtdIns3P.     

Overexpression of FAB1A-GFP recruits SNX2b on the endosome membrane in snx1–1 mutant in Arabidopsis

Phosphatidylinositol 3,5-bisphosphate [PI(3,5)P₂] is one of the phosphoinositides that controls endosomal trafficking events in eukaryotes. PtdIns(3,5)P₂ is produced from PI(3)P by phosphatidylinositol 3-phosphate 5-kinase FAB1/PIKfyve. Recently, we reported that FAB1 predominantly localizes on the SNX1-residing late endosomes and a loss-of FAB1 function causes the release of late endosomal effector proteins, ARA7/RABF2b and SORTING NEXIN 1 from the endosome membrane, indicating that FAB1 or its product PtdIns(3,5)P₂ mediates the maturation process of the late endosomes. Intriguingly, the ectopic expression of FAB1A could complement the sucrose-dependent seedling growth phenotype of snx1–1 mutant. Here, we demonstrated that the depletion of SNX1 causes the release of SNX2b-mRFP from the endosomal membrane. However, overexpression of FAB1A-GFP reassembles SNX2b-mRFP on the endosomal membrane despite the absence of SNX1. From these results, we proposed that SNX2b homodimer or SNX2a/SNX2b heterodimer might function as functional Sorting Nexin complex instead of SNX1 to attach the endosomal membrane by binding of overproduced PI(3,5)P₂ in Arabidopsis.     

Structure–activity relationship study,target identification,and pharmacological characterization of a small molecular IL-12/23 inhibitor,APY0201

Interleukin-12 (IL-12) and IL-23 are proinflammatory cytokines and therapeutic targets for inflammatory and autoimmune diseases, including inflammatory bowel diseases, psoriasis, rheumatoid arthritis, and multiple sclerosis. We describe the discovery of APY0201, a unique small molecular IL-12/23 production inhibitor, from activated macrophages and monocytes, and demonstrate ameliorated inflammation in an experimental model of colitis. Through a chemical proteomics approach using a highly sensitive direct nanoflow LC–MS/MS system and bait compounds equipped with the FLAG epitope associated regulator of PIKfyve (ArPIKfyve) was detected. Further study identified its associated protein phosphoinositide kinase, FYVE finger-containing (PIKfyve), as the target protein of APY0201, which was characterized as a potent, highly selective, ATP-competitive PIKfyve inhibitor that interrupts the conversion of phosphatidylinositol 3-phosphate (PtdIns3P) to PtdIns(3,5)P₂. These results elucidate the function of PIKfyve kinase in the IL-12/23 production pathway and in IL-12/23-driven inflammatory disease pathologies to provide a compelling rationale for targeting PIKfyve kinase in inflammatory and autoimmune diseases.