Class III PI3K Vps34 plays an essential role in autophagy and in heart and liver function

Abstract

A critical regulator of autophagy is the Class III PI3K Vps34 (also called PIK3C3). Although Vps34 is known to play an essential role in autophagy in yeast, its role in mammals remains elusive. To elucidate the physiological function of Vps34 and to determine its precise role in autophagy, we have generated Vps34 f/f mice, in which expression of Cre recombinase results in a deletion of exon 4 of Vps34 and a frame shift causing a deletion of 755 of the 887 amino acids of Vps34. Acute ablation of Vps34 in MEFs upon adenoviral Cre infection results in a diminishment of localized generation of phosphatidylinositol 3-phosphate and blockade of both endocytic and autophagic degradation. Starvation-induced autophagosome formation is blocked in both Vps34-null MEFs and liver. Liver-specific Albumin-Cre;Vps34 f/f mice developed hepatomegaly and hepatic steatosis, and impaired protein turnover. Ablation of Vps34 in the heart of muscle creatine kinase-Cre;Vps34 f/f mice led to cardiomegaly and decreased contractility. In addition, while amino acid-stimulated mTOR activation was suppressed in the absence of Vps34, the steady-state level of mTOR signaling was not affected in Vps34-null MEFs, liver, or cardiomyocytes. Taken together, our results indicate that Vps34 plays an essential role in regulating functional autophagy and is indispensable for normal liver and heart function.LC3 | SQSTM1/p62 | 3-MA | epidermal growth factor receptor | transferrin M acroautophagy (referred to as autophagy hereafter) is a dynamic membrane trafficking process that involves the delivery of intracellular content to lysosomes for degradation. A fully executed autophagy includes the formation of doublemembraned autophagosomes, the fusion of autophagosomes to late endosomes/lysosomes, and the digestion of the enclosed content by lysosomal hydrolases. Autophagy is constantly maintained at the basal level and is up-regulated in response to stress conditions, such as nutrient and energy limitation, hypoxia, and DNA damage. Autophagy is necessary for cellular and tissue homeostasis, by eliminating damaged organelles and misfolded proteins, and its dysregulation is implicated in developmental defects and numerous diseases (1-5).

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

“…116 In this model, PIK3C3 is part of the MTOR complex; amino acids stimulate an elevation in the intracellular Ca 2C level and Ca 2C -CALM/calmodulin binding, which increases PIK3C3 activity; consequently, PtdIns3Ps produced by PIK3C3 activate MTOR by replacing FKBP8/FKBP38 with RHEB. 116 This model is consistent with earlier studies showing that PIK3C3 is required for MTOR-RPS6KB1 signaling activity, 117,118 and is supported by the evidence that amino acid restimulation of MTOR activity is impaired in Pik3c3 KO MEFs 111 and MTOR activity is remarkably ablated in Pik3c3 KO embryos. 110 However, several issues are still under dispute concerning the amino acid-Ca 2C -CALM-PIK3C3-MTOR signaling model.…”

“…167,168 However, studies from several Pik3c3 knockout animal models suggest that PtdIns3P generation and autophagic function rely predominantly on PIK3C3 activity. 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.…”

“…Our group 121 and others 122 have demonstrated that the generation of amino acids by autophagy is essential for MTORC1 activity; thus, the impairment of autophagy by Pik3c3 knockdown or inhibition may influence this feedback circuit. Additionally, since a proportion of PIK3C3 complexes are involved in the endosomal system and are plausibly important for proper lysosomal function, 75,76,111,123 Pik3c3 ablation may affect the activation of MTORC1 by amino acids at the lysosome, which is dependent on the function of several lysosomal proteins 55-57 and normal lysosomal degradation. 122,124 In contrast, yeast Vps34 is the sole PtdIns3K and it functions only in vacuolar trafficking/autophagy but not in the TORC1 regulatory network; 125 whether and why mammalian PIK3C3 has evolved to harbor bifurcate functions in MTOR modulation and autophagy is a mystifying question.…”

“…90 Pik3c3 knockout mice are embryonic lethal, 110 whereas tissue specific ablation of Pik3c3 blocks autophagic degradation and protein turnover, and severely debilitates liver and heart function. 111 As the core catalytic subunit in the class III PtdIns3K complex, PIK3C3 per se is a point of regulatory convergence by diverse autophagy-modulating mechanisms. A study by Kim et al 112 demonstrated the presence of 2 different regulatory mechanisms controlling the role of PIK3C3 in autophagy via: (i) formation of different protein complexes and (ii) direct phosphorylation of PIK3C3.…”

Repetitive stimulation of autophagy-lysosome machinery by intermittent fasting preconditions the myocardium to ischemia-reperfusion injury

“…These data suggest that the consequences of LAMP2 deficiency (in Lamp2 heterozygous null mice) on global autophagic flux are only unmasked in the presence of a stressor such as fasting, which rapidly stimulates formation of autophagosomes, which are thereafter not processed efficiently, due to impaired autophagosome-lysosome fusion. Therefore, Lamp2 heterozygous null mice are well suited as a model system to evaluate the effects of impaired myocardial lysosome function and consequently impaired autophagy specifically under stress; as they do not display the disadvantages observed in other model systems employed to study the effects of impaired autophagy-lysosomal machinery, namely presence of cardiomyopathy in the unstressed state (as in mice with cardiac myocyte specific or germline ablation of proteins essential for autophagosome formation, ATG5, ATG7, and PIK3C3; 14,15,33,34 and in models of lysosomal dysfunction [reviewed in ref. 35]); or the paradoxical effects on autophagy observed with haploinsufficiency of BECN1.…”