Regulation of Mammalian Autophagy by Class II and III PI 3-Kinases through PI3P Synthesis

Abstract

Synthesis of phosphatidylinositol-3-phosphate (PI3P) by Vps34, a class III phosphatidylinositol 3-kinase (PI3K), is critical for the initial steps of autophagosome (AP) biogenesis. Although Vps34 is the sole source of PI3P in budding yeast, mammalian cells can produce PI3P through alternate pathways, including direct synthesis by the class II PI3Ks; however, the physiological relevance of these alternate pathways in the context of autophagy is unknown. Here we generated Vps34 knockout mouse embryonic fibroblasts (MEFs) and using a higher affinity 4x-FYVE finger PI3P-binding probe found a Vps34-independent pool of PI3P accounting for ~35% of the total amount of this lipid species by biochemical analysis. Importantly, WIPI-1, an autophagy-relevant PI3P probe, still formed some puncta upon starvation-induced autophagy in Vps34 knockout MEFs. Additional characterization of autophagy by electron microscopy as well as protein degradation assays showed that while Vps34 is important for starvation-induced autophagy there is a significant component of functional autophagy occurring in the absence of Vps34. Given these findings, class II PI3Ks (α and β isoforms) were examined as potential positive regulators of autophagy. Depletion of class II PI3Ks reduced recruitment of WIPI-1 and LC3 to AP nucleation sites and caused an accumulation of the autophagy substrate, p62, which was exacerbated upon the concomitant ablation of Vps34. Our studies indicate that while Vps34 is the main PI3P source during autophagy, class II PI3Ks also significantly contribute to PI3P generation and regulate AP biogenesis.

The human CD5L/AIM-CD36 axis: A novel autophagy inducer in macrophages that modulates inflammatory responses

“…PtdIns3P may also be produced via PIK3C3-independent mechanisms, including dephosphorylation of PtdIns(3,4,5)P 3 by PtdIns 5-phosphatases and 4-phosphatase 59 or through class II PI3K molecules that also contribute to PtdIns3P synthesis. 60 However, our data suggest that CD5L preferentially enhances PtdIns3P levels through PIK3C3, since the signal was abrogated in the presence of the PI3K/PIK3C3 inhibitor 3-MA.…”

“…They were kept in the fixative for 2 h at 4 C. They were then washed with the same buffer and postfixed with 1% osmium tetroxide in the same buffer containing 0.8% potassium ferricyanide at 4 C. Then the samples were dehydrated in acetone, infiltrated with Epon resin (Electron Microscopy Sciences, RT 14120) for 2 d, embedded in the same resin, and polymerized at 60 C for 48 h. Ultrathin sections were obtained using a Leica Ultracut UC6 ultramicrotome (Leica Microsystems, Vienna) and mounted on Formvar-coated copper grids. They were stained with 2% uranyl acetate in water and lead citrate.…”

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

“…The ratio of pS6 to total S6 of a representative experiment (of two independent experiments) is shown. (Devereaux et al, 2013), was corrected by wild-type, but not Vps34 kinase-dead (Fig. 1A).…”

“…These findings suggest two possible scenarios: (1) that PI(3)P is produced by alternative sources such as the class II PI3Ks (Devereaux et al, 2013;Falasca and Maffucci, 2012;Posor et al, 2013); and/or (2) that a compensatory mechanism independent of PI(3)P is triggered to cope with the loss of Vps34. Previous reports indicate that expression of the constitutively active mutant of Rab5 can bypass the loss of EEA1 membrane localization upon wortmannin treatment Simonsen et al, 1998), and that wortmannin activates Rab5 (Chen and Wang, 2001).…”

Differential regulatory functions of three classes of phosphatidylinositol and phosphoinositide 3-kinases in autophagy

“…111,169,170 The question of whether class III PtdIns3K is the exclusive kinase responsible for PtdIns3P generation during autophagy remained unsolved until a recent study by Devereaux et al revealed an alternative source of PtdIns3P in Pik3c3 knockout MEFs. 171 In Pik3c3 KO MEFs, it is surprising that PtdIns3P is still detectable using a higher affinity PtdIns3P probe and the PtdIns3P-binding protein WIPI1, and autophagosomes are still observed under starvation conditions; class II PI3K was confirmed as another contributor to the PtdIns3P pool during autophagy, and depletion of class II PI3K further decreases autophagic flux, which is inhibited by Pik3c3 deletion. 171 Thus, cells may ensure the adequate and timely production of autophagic PtdIns3P by involving more than one producer (Fig.…”