title: ArPIKfyve regulates Sac3 protein abundance and turnover: disruption of the mechanism by Sac3I41T mutation causing Charcot-Marie-Tooth 4J disorder.
abstract: The mammalian phosphatidylinositol (3,5)-bisphosphate (PtdIns(3,5)P(2)) 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)P(2) 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)P(2)-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((1/2)) = 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)P(2)-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.
authors.affiliation: Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan 48201.
authors.forename: Ognian C
authors.initials: OC
authors.lastname: Ikonomov
authors.affiliation: Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan 48201.
authors.forename: Diego
authors.initials: D
authors.lastname: Sbrissa
authors.affiliation: Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan 48201.
authors.forename: Jason
authors.initials: J
authors.lastname: Fligger
authors.affiliation: Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan 48201.
authors.forename: Khortnal
authors.initials: K
authors.lastname: Delvecchio
authors.affiliation: Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan 48201. Electronic address: ashishev@med.wayne.edu.
authors.forename: Assia
authors.initials: A
authors.lastname: Shisheva
mesh_terms: D041721:3T3-L1 Cells; D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002607:Charcot-Marie-Tooth Disease / Q000235:genetics / Q000378:metabolism*; D002522:Chlorocebus aethiops; D005420:Flavoproteins / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000235:genetics; D055785:Gene Knockdown Techniques; D006207:Half-Life; D006801:Humans; D006868:Hydrolysis; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000235:genetics / Q000378:metabolism; D000072180:Phosphoinositide Phosphatases; D010744:Phosphoric Monoester Hydrolases; D046988:Proteasome Endopeptidase Complex / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding
chemical_list: D002352:Carrier Proteins; D005420:Flavoproteins; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D046912:Multiprotein Complexes; D019869:Phosphatidylinositol 3-Kinases; D018129:Phosphatidylinositol Phosphates; D000072180:Phosphoinositide Phosphatases; D010744:Phosphoric Monoester Hydrolases; D046988:Proteasome Endopeptidase Complex; C494631:VAC14 protein, human; C525133:Vac14 protein, mouse; C106336:phosphatidylinositol 3,5-diphosphate; C492139:PIKFYVE protein, human; C116930:Pikfyve protein, mouse; C522215:FIG4 protein, human; C522216:Fig4 protein, mouse