title: Class III PI 3-kinase is the main source of PtdIns3P substrate and membrane recruitment signal for PIKfyve constitutive function in podocyte endomembrane homeostasis.
abstract: The evolutionarily conserved PIKfyve, which synthesizes PtdIns5P from PtdIns, and PtdIns(3,5)P2 from PtdIns3P, requires PtdIns3P as both an enzyme substrate and a membrane recruitment signal. Whereas the PtdIns3P source is undetermined, class III PI3K (Vps34), the only evolutionarily conserved of the eight mammalian PI3Ks, is presumed as a main candidate. A hallmark of PIKfyve deficiency is formation of multiple translucent cytoplasmic vacuoles seen by light microscopy in cells cultured in complete media. Such an aberrant phenotype is often observed in cells from conditional Vps34 knockout (KO) mice. To clarify the mechanism of Vps34 KO-triggered vacuolation and the PtdIns3P source for PIKfyve functionality, here we have characterized a podocyte cell type derived from Vps34fl/fl mice, which, upon Cre-mediated gene KO, robustly formed cytoplasmic vacuoles resembling those in PikfyveKO MEFs. Vps34wt, expressed in Vps34KO podocytes restored the normal morphology, but only if the endogenous PIKfyve activity was intact. Conversely, expressed PIKfyvewt rescued completely the vacuolation only in PikfyveKO MEFs but not in Vps34KO podocytes. Analyses of phosphoinositide profiles by HPLC and localization patterns by a PtdIns3P biosensor revealed that Vps34 is the main supplier of localized PtdIns3P not only for PIKfyve activity but also for membrane recruitment. Concordantly, Vps34KO podocytes had severely reduced steady-state levels of both PtdIns(3,5)P2 and PtdIns5P, along with PtdIns3P. We further revealed a plausible physiologically-relevant Vps34-independent PtdIns3P supply for PIKfyve, operating through activated class I PI3Ks. Our data provide the first evidence that the vacuolation phenotype in Vps34KO podocytes is due to PIKfyve dysfunction and that Vps34 is a main PtdIns3P source for constitutive PIKfyve functionality.
authors.affiliation: Department of Physiology, Wayne State University School of Medicine, Detroit, MI 48201, USA.
authors.forename: Ognian C
authors.initials: OC
authors.lastname: Ikonomov
authors.affiliation: Department of Physiology, Wayne State University School of Medicine, Detroit, MI 48201, USA.
authors.forename: Diego
authors.initials: D
authors.lastname: Sbrissa
authors.affiliation: Division of Nephrology, University of Michigan, Ann Arbor, MI 48109, USA.
authors.forename: Madhusudan
authors.initials: M
authors.lastname: Venkatareddy
authors.affiliation: Department of Pharmacology, Wayne State University School of Medicine, Detroit, MI 48201, USA.
authors.forename: Ellen
authors.initials: E
authors.lastname: Tisdale
authors.affiliation: Division of Nephrology, University of Michigan, Ann Arbor, MI 48109, USA.
authors.forename: Puneet
authors.initials: P
authors.lastname: Garg
authors.affiliation: Department of Physiology, Wayne State University School of Medicine, Detroit, MI 48201, USA. Electronic address: ashishev@med.wayne.edu.
authors.forename: Assia
authors.initials: A
authors.lastname: Shisheva
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000378:metabolism* / Q000648:ultrastructure; D058546:Class III Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D003470:Culture Media; D017353:Gene Deletion; D006706:Homeostasis; D007425:Intracellular Membranes / Q000378:metabolism*; D018345:Mice, Knockout; D010641:Phenotype; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D050199:Podocytes / Q000378:metabolism* / Q000648:ultrastructure; D015398:Signal Transduction; D013379:Substrate Specificity; D014617:Vacuoles / Q000378:metabolism / Q000648:ultrastructure
chemical_list: D003470:Culture Media; D018129:Phosphatidylinositol Phosphates; C106336:phosphatidylinositol 3,5-diphosphate; C055525:phosphatidylinositol 3-phosphate; C113567:phosphatidylinositol 5-phosphate; D019869:Phosphatidylinositol 3-Kinases; D058546:Class III Phosphatidylinositol 3-Kinases; C116930:Pikfyve protein, mouse
keywords: Class I PI 3-kinase; Class III PI 3-kinase; Endomembrane homeostasis; PIKfyve; Phosphatidylinositol phosphates; Vps34 knockout podocytes