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 Vps34KO-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 present evidence for 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.

Distinct Requirements for Vacuolar Protein Sorting 34 Downstream Effector Phosphatidylinositol 3-Phosphate 5-Kinase in Podocytes Versus Proximal Tubular Cells

“…Our recent studies have shown that cultured Vps34-deficient podocytes cannot be rescued by re-expression of Vps34 when PIKfyve activity is chemically inhibited. 21 These data suggest that the abnormal vacuolation phenotype observed in Vps34-deleted cultured podocytes is caused by failed PtdIns(3,5)P2 synthesis. This study was undertaken to determine the role of PIKfyve in glomerular podocytes and proximal tubular cells in mouse models with the respective tissue-specific PIKfyve gene deletions.…”

“…35 Formation of translucent cytoplasmic vacuoles is a proven sensitive assay for perturbed PIKfyve function and hence, reduced PtdIns(3,5)P2 levels as observed in a number of dividing cells, including podocytes in culture. 21,25 Isolated glomeruli from the mTmG fl/fl , PIKfyve fl/fl,Cre+ mice were plated on culture dishes. At day 0, there was expression of both red and green fluorescence in the glomeruli ( Figure 3A).…”

“…[17][18][19][20] In our recent study, we showed that the Vps34 downstream effector phosphatidylinositol 3-phosphate 5-kinase (PIKfyve) is responsible for the vacuolation phenotype observed in Vps34-deleted podocytes in culture. 21 Replenishment of Vps34 in cultured Vps34-deficient cells did rescue the vacuolation phenotype but only in the presence of intact PIKfyve activity. 21 PIKfyve is an Fyve (Fab1p, YOTB, Vac1p, and early endosome antigen 1 [EEA1]) finger-containing phosphoinositide (PI) kinase that phosphorylates PtdIns and Vps34-generated PtdIns(3)P on the 5-hyrdroxyl position to make the low-abundance signaling lipids phosphatidylinositol-5-phosphate [PtdIns(5)P]and phosphatidylinositol-3,5-phosphate 2 [PtdIns(3,5)P2], respectively.…”

“…21 Replenishment of Vps34 in cultured Vps34-deficient cells did rescue the vacuolation phenotype but only in the presence of intact PIKfyve activity. 21 PIKfyve is an Fyve (Fab1p, YOTB, Vac1p, and early endosome antigen 1 [EEA1]) finger-containing phosphoinositide (PI) kinase that phosphorylates PtdIns and Vps34-generated PtdIns(3)P on the 5-hyrdroxyl position to make the low-abundance signaling lipids phosphatidylinositol-5-phosphate [PtdIns(5)P]and phosphatidylinositol-3,5-phosphate 2 [PtdIns(3,5)P2], respectively. [22][23][24] In contrast to PtdIns(3)P, which is found in early endosomal compartments, PtdIns (3,5)P2 is thought to predominate in late endosomes and/or lysosomes.…”

Vps34 regulates Rab7 and late endocytic trafficking through recruitment of the GTPase-activating protein Armus

“…PIKfyve is recruited to PI(3)P-positive endosomes through its FYVE domain, where it phosphorylates PI(3)P to generate PI(3,5)P 2 . This phospholipid has been demonstrated to regulate ILV formation and endosome size (Ikonomov et al, 2003(Ikonomov et al, , 2015. In addition, ILV formation is also regulated by the ESCRT complexes, and the ESCRT-0 factor (Hrs) is a PI(3)P and Rab5 effector (Bache et al, 2003;Falguieres et al, 2008;Lindmo and Stenmark, 2006).…”