Sac3 Is an Insulin-regulated Phosphatidylinositol 3,5-bisphosphate phosphatase. Gain in insulin responsiveness through Sac3 down-regulation in adipocytes

Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 08/2009; 284(36):23961-71. DOI: 10.1074/jbc.M109.025361
Source: PubMed


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)P(2)) pathway for optimal performance. The reduced insulin responsiveness observed under ablation of the PtdIns(3,5)P(2)-synthesizing PIKfyve and its associated activator ArPIKfyve in 3T3L1 adipocytes suggests that dysfunction of the PtdIns(3,5)P(2)-specific phosphatase Sac3 may yield the opposite effect. Paradoxically, as uncovered recently, in addition to turnover Sac3 also supports PtdIns(3,5)P(2) 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 approximately 60%, which resulted in a slight but significant elevation of PtdIns(3,5)P(2) 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)P(2) hydrolyzing activity of Sac3. The insulin-sensitive Sac3 pool likely controls a discrete PtdIns(3,5)P(2) subfraction as the high pressure liquid chromatography-measurable insulin-dependent elevation in total [(3)H]inositol-PtdIns(3,5)P(2) 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.

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    ABSTRACT: 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 PIKfyveK1831E if its ArPIKfyve-Sac3 binding region was deleted, suggesting reduced Sac3 access to, and turnover of PtdIns(3,5)P2. In contrast, PIKfyveK1831E, which displays intact ArPIKfyve-Sac3 binding, triggered a more severe vacuolar phenotype if coexpressed with ArPIKfyveWT-Sac3WT but minimal defects when coexpressed with ArPIKfyveWT and phosphatase-deficient Sac3D488A. 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.
    Preview · Article · Oct 2009 · Journal of Biological Chemistry
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    ABSTRACT: Phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P(2)] 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)P(2) 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)P(2) in modulating the dynamics of vacuolar rearrangement essential for successful pollen development. Taken together, our results are consistent with PtdIns(3,5)P(2) production being central to cellular responses to changes in osmotic conditions.
    Full-text · Article · Oct 2009 · Plant physiology
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    ABSTRACT: 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 ArPIKfyveWT 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 Sac3WT (t½ = 18.8 min), resulting from a proteasome-dependent clearance as evidenced by the extended Sac3WT half-life upon inhibiting proteasome activity. Coexpression of ArPIKfyveWT, but not the N- or C-terminal halves, prolongs the Sac3WT 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 Sac3WT with regard to PtdIns(3,5)P2-hydrolyzing activity, association with ArPIKfyve, or rapid proteasome-dependent clearance. Remarkably, however, neither is the steady-state Sac3I41T elevated nor is the Sac3I41T half-life extended by coexpressed ArPIKfyveWT, indicating that unlike with Sac3WT, ArPIKfyve fails to prevent Sac3I41T 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.
    Preview · Article · Aug 2010 · Journal of Biological Chemistry
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