Functional dissociation between PIKfyve-synthesized PtdIns5P and PtdIns(3,5)P2 by means of the PIKfyve inhibitor YM201636

PIKfyve is an essential mammalian lipid kinase with pleiotropic cellular functions whose genetic knockout in mice leads to preimplantation lethality. Despite several reports for PIKfyve-catalyzed synthesis of phosphatidylinositol 5-phosphate (PtdIns5P) along with phosphatidylinositol-3,5-biphosphate [PtdIns(3,5)P(2)] in vitro and in vivo, the role of the PIKfyve pathway in intracellular PtdIns5P production remains underappreciated and the function of the PIKfyve-synthesized PtdIns5P pool poorly characterized. Hence, the recently discovered potent PIKfyve-selective inhibitor, the YM201636 compound, has been solely tested for inhibiting PtdIns(3,5)P(2) synthesis. Here, we have compared the in vitro and in vivo inhibitory potency of YM201636 toward PtdIns5P and PtdIns(3,5)P(2). Unexpectedly, we observed that at low doses (10-25 nM), YM201636 inhibited preferentially PtdIns5P rather than PtdIns(3,5)P(2) production in vitro, whereas at higher doses, the two products were similarly inhibited. In cellular contexts, YM201636 at 160 nM inhibited PtdIns5P synthesis twice more effectively compared with PtdIns(3,5)P(2) synthesis. In 3T3L1 adipocytes, human embryonic kidney 293 and Chinese hamster ovary (CHO-T) cells, levels of PtdIns5P dropped by 62-71% of the corresponding untreated controls, whereas those of PtdIns(3,5)P(2) fell by only 28-46%. The preferential inhibition of PtdIns5P versus PtdIns(3,5)P(2) at low doses of YM201636 was explored to probe contributions of the PIKfyve-catalyzed PtdIns5P pool to insulin-induced actin stress fiber disassembly in CHO-T cells, GLUT4 translocation in 3T3L1 adipocytes, and induction of aberrant cellular vacuolation in these or other cell types. The results provide the first experimental evidence that the principal pathway for PtdIns5P intracellular production is through PIKfyve and that insulin effect on actin stress fiber disassembly is mediated entirely by the PIKfyve-produced PtdIns5P pool.     

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     

Arraying glycomics: a novel bi-functional spacer for one-step microscale derivatization of free reducing glycans

Glycan array development is limited by the complexity of efficiently generating derivatives of free reducing glycans with primary amines or other functional groups. A novel bi-functional spacer with selective reactivity toward the free glycan and a second functionality, a primary amine, was synthesized. We demonstrated an efficient one-step derivatization of various glycans including naturally isolated N-glycans, O-glycans, milk oligosaccharides, and bacterial polysaccharides in microgram scale. No protecting group manipulations or activation of the anomeric center was required. To demonstrate its utility for glycan microarray fabrication, we compared glycans with different amine-spacers for incorporation onto an amine-reactive glass surface. Our study results revealed that glycans conjugated with this bi-functional linker were effectively printed and detected with various lectins and antibodies.     

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 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.     

Phosphatidylinositol 3-phosphate-interacting domains in PIKfyve. Binding specificity and role in PIKfyve. Endomenbrane localization.

PIKfyve is a phosphatidylinositol (PtdIns) 3-phosphate (P)-metabolizing enzyme, which, in addition to a C-terminally positioned catalytic domain, harbors several evolutionarily conserved domains, including a FYVE finger. The FYVE finger domains are thought to direct the protein localization to intracellular membrane PtdIns 3-P. Recent studies with several FYVE domain proteins challenge this general concept. Here we have examined the binding of PIKfyve's FYVE domain to PtdIns 3-P in vitro and in vivo and a plausible contribution of this binding mechanism for the intracellular localization of the full-length protein. We document now a specific and high affinity interaction of a recombinantly produced PIKfyve FYVE domain peptide fragment with PtdIns 3-P-containing liposomes that requires the presence of the conservative core of basic residues within the FYVE domain. PIKfyve localization to membranes of the late endocytic pathway was found to be absolutely dependent on the presence of an intact FYVE finger. Cell treatment with PI 3-kinase inhibitor wortmannin dissociated endosome-bound PIKfyve, indicating that the protein targeted the membrane PtdIns 3-P. An enzymatically inactive peptide fragment of the PIKfyve catalytic domain was found to also specifically bind to PtdIns 3-P-containing liposomes, with residue Lys-1999 being critical in the interaction. This binding, however, was of relatively low affinity and, in the cellular context, was found ineffective in directing the molecule to PtdIns 3-P-enriched endosomes. Collectively, these results demonstrate that interaction of the FYVE domain with PtdIns 3-P is absolutely necessary for PIKfyve targeting to the membranes of the late endocytic pathway and determine PIKfyve as a downstream effector of PtdIns 3-P.     

Mutation of the COG complex subunit gene COG7 causes a lethal congenital disorder

The congenital disorders of glycosylation (CDG) are characterized by defects in N-linked glycan biosynthesis that result from mutations in genes encoding proteins directly involved in the glycosylation pathway. Here we describe two siblings with a fatal form of CDG caused by a mutation in the gene encoding COG-7, a subunit of the conserved oligomeric Golgi (COG) complex. The mutation impairs integrity of the COG complex and alters Golgi trafficking, resulting in disruption of multiple glycosylation pathways. These cases represent a new type of CDG in which the molecular defect lies in a protein that affects the trafficking and function of the glycosylation machinery.     

Formation of Advanced Glycation Endproducts from Low Molecular Weight Model Compounds

The process of glycation was studied in 12 model systems containing carbohydrates (Glc, Fru) and peptides (Gly-Gly, Gly-Phe, Phe-Gly, Gly-Lys) or acetylated amino acids (Ac-Lys, Ac-Arg) in order to clarify the role of different structural elements of the reacting components. The course of reaction was followed by the changes of the UV spectra of the reaction systems. The results show that the reactivity of the NH₂ group correlates with its pK_a value. The presence of benzene ring in the amino component accelerates glycation. Strong correlation between the intensity of the fluorescence and the absorption at 325 nm was found for all reaction systems.     

Glycan microarrays for screening sialyltransferase specificities

Here we demonstrate that glycan microarrays can be used for high-throughput acceptor specificity screening of various recombinant sialyltransferases. Cytidine-5'-monophospho-N-acetylneuraminic acid (CMP-Neu5Ac) was biotinylated at position 9 of N-acetylneuraminic acid (Neu5Ac) by chemoenzymatic synthesis generating CMP-9Biot-Neu5Ac. The activated sugar nucleotide was used as donor substrate for various mammalian sialyltranferases which transferred biotinylated sialic acids simultaneously onto glycan acceptors immobilized onto a microarray glass slide. Biotinylated glycans detected with fluorescein-streptavidin conjugate to generate a specificity profile for each enzyme both confirming previously known specificities and reveal additional specificity information. Human alpha2,6sialyltransferase-I (hST6Gal-I) also sialylates chitobiose structures (GlcNAcbeta1-4GlcNAc)(n) including N-glycans, rat alpha2,3sialyltransferase (rST3Gal-III) tolerates fucosylated acceptors such as Lewis(a), human alpha2,3sialyltransferase-IV (hST3Gal-IV) broadly sialylates oligosaccharides of types 1-4 and porcine alpha2,3sialyltransferase-I (pST3Gal-I) sialylates ganglio-oligosaccharides and core 2 O-glycans in our array system. Several of these sialyltransferases perform a substitution reaction and exchange a sialylated acceptor with a biotinylated sialic acid but are restricted to the most specific acceptor substrates. Thus, this method allows for a rapid generation of enzyme specificity information and can be used towards synthesis of new carbohydrate compounds and expand the glycan array compound library.     

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.