The beta identity of class I PtdIns3K

Abstract

Autophagy is critically controlled by phosphatidylinositol 3-kinases (PtdIns3Ks). The common understanding for mammalian autophagy is that class I PtdIns3Ks inhibit autophagy by activating the Akt-TOR kinase cascade, whereas the class III PtdIns3K (Vps34) promotes autophagy by generating the phospholipid PtdIns(3)P. However, direct genetic evidence for a role of class I PtdIns3Ks in autophagy has been lacking. Using mice with a conditional deletion of the class I PtdIns3K catalytic subunit isoform p110α or p110β, we revealed an unexpected function of p110β as a positive regulator of autophagy.

PtdIns3Ks belong to the family of lipid kinases that phosphorylate the 3′-hydroxyl group of phosphatidylinositol and phosphoinositides. PtdIns3Ks are divided into three classes based on their sequence homology and substrate specificities. In vivo, class I PtdIns3Ks are thought to phosphorylate PtdIns(4,5)P₂ to produce PtdIns(3,4,5)P₃, which activates Akt-TOR kinase pathways. The class I PtdIns3Ks are further subdivided into class IA and class IB, based on the structures of their catalytic and regulatory subunits. Class IA PtdIns3Ks are composed of a p110α, p110β or p110δ catalytic subunit bound to a p85 regulatory subunit. Class III PtdIns3K is composed of only one member, Vps34, which phosphorylates PtdIns to generate PtdIns(3)P, a molecule key to membrane trafficking events.

The involvement of PtdIns3Ks in autophagy was first demonstrated in studies using PtdIns3K inhibitors and in vitro delivery of phospholipids. Wortmannin, a broad-spectrum PtdIns3K inhibitor that inhibits all classes of PtdIns3Ks, strongly inhibits autophagy. In vitro delivery of the class III PtdIns3K product, PtdIns(3) P, stimulates autophagy, whereas delivery of the class I PtdIns3K product, PtdIns(3,4,5)P₃, inhibits autophagy. These and other studies led to the notion that class I PtdIns3Ks inhibit autophagy by activating the Akt-TOR pathway, whereas class III PtdIns3K promotes autophagy by making PtdIns(3)P. In line with this concept, PTEN deficiency causes hyperactivation of Akt-TOR activities, and correspondingly suppresses autophagy. Although the use of PtdIns3K inhibitors paved the way for autophagy studies, the information obtained using the inhibitors is far from conclusive, and often controversial, due to their lack of specificity. For example, PI-103, a commonly used class I PtdIns3K inhibitor, also inhibits TOR; TGX-221, an inhibitor relatively specific for p110β, also inhibits Vps34 at higher concentrations; and 3-MA, a routinely used inhibitor of Vps34, inhibits class I PtdIns3Ks as well.

We studied the role of the class IA PtdIns3K catalytic subunits p110α and p110β in autophagy using mice with conditional deletion of either of these two ubiquitously expressed isoforms. We examined MEFs generated from these animals, as well as the liver and heart. While p110α deficiency leads to similar or slightly elevated autophagy, p110β deficiency results in markedly impaired autophagy both at the basal level and in response to a variety of autophagy-inducing conditions. This unexpected positive role of p110β in autophagy was further confirmed by both overexpression and knockdown approaches in human cell lines.

Our further study found that, mechanistically, p110β does not act through the Akt-TOR pathway to promote autophagy, as pharmacological inhibitors of Akt or TOR fail to induce autophagy in p110β-deficient cells. Rather, p110β modulates the intracellular level of PtdIns(3)P that is required for autophagosome formation. The amount of total cellular PtdIns(3)P in p110β^−/− MEFs is approximately 30% lower than in control cells. Conversely, overexpression of p110β enhances autophagy and stimulates PtdIns(3)P production in a number of cell lines. Consistent with the effect of p110β on the PtdIns(3)P level, we found the early steps of autophagosome formation are affected by p110β.

The question then became how p110β promotes PtdIns(3)P production. One theory is that p110β produces PtdIns(3) P by phosphorylating PtdIns. The enzyme does catalyze this reaction in vitro, but whether it does so in vivo is still unclear. A second theory is that the PtdIns(3,4,5) P₃ produced by p110β is converted to PtdIns(3)P by lipid phosphatases that have been reported to associate with p110β. To determine if the kinase activity of p110β is required for its ability to increase PtdIns(3)P levels and autophagy, we tested the effects of kinase-deficient p110β mutants. Strikingly, the kinasedeficient p110β mutants retain the ability to promote PtdIns(3)P production and autophagy. While these results do not completely rule out the involvement of its kinase activity, they clearly indicate that p110β can function as a scaffold to regulate PtdIns(3)P levels and autophagy. Indeed, both wild-type and kinase-deficient p110β are able to enhance Vps34 activity. In line with this, we found that p110β associates and colocalizes with the autophagy-promoting Vps34-Vps15-Beclin 1-Atg14L complex.

While the molecular details of how p110β regulates Vps34 activity remain to be further explored, our discovery of a positive role of p110β in regulating autophagy and Vps34 expands our understanding of the functions of class I PtdIns3Ks in higher organisms. The p110α and p110β isoforms share similar features, yet are distinct. p110α is catalytically more active than p110β, and activating mutations of p110α, but not p110β, are frequently observed in human cancers. By contrast, p110β may regulate processes such as autophagy by a scaffolding mechanism. Our work identifies Vps34 as an effector of p110β's scaffold activity, and implies that the ancient class III PtdIns3K is under the control of the newly evolved class IA PtdIns3K. Our study suggests that class I PtdIns3Ks not only mediate cell activation, but also regulate cellular homeostasis. The dual role of p110β in transducing receptor signals and promoting autophagy suggests that PtdIns3Ks may act as a sensor for hormone availability, and may prime cells for distinct activities. Unlike in unicellular organisms such as yeast, which do not contain class I PtdIns3Ks and whose autophagy is controlled by the class III PtdIns3K Vps34, the evolution of class I PtdIns3Ks enables autophagy in multicellular organisms to be initiated in a more rapid and precise fashion in response to hormone and growth factor availability.

Punctum to: Dou Z, Chattopadhyay M, Pan JA, Guerriero JL, Jiang YP, Ballou LM, et al. The class IA phosphatidylinositol 3-kinase p110-{beta} subunit is a positive regulator of autophagy. J Cell Biol. 2010;191:827–843. doi: 10.1083/jcb.201006056.