The class III PI3K (phosphoinositide 3-kinase), Vps34 (vacuolar protein sorting 34), was first identified as a regulator of vacuolar hydrolase sorting in yeast. Unlike other PI3Ks, the Vps34 lipid kinase specifically utilizes phosphatidylinositol as a substrate, producing the single lipid product PtdIns3P. While Vps34 has been studied for some time in the context of endocytosis and vesicular trafficking, it has more recently been implicated as an important regulator of autophagy, trimeric G-protein signalling, and the mTOR (mammalian target of rapamycin) nutrient-sensing pathway. The present paper will focus on studies that describe the regulation of hVps34 (human Vps34) intracellular targeting and enzymatic activity in yeast and mammalian cells.
- mammalian target of rapamycin (mTOR)
- phosphoinositide 3-kinase (PI3K)
- vacuolar protein sorting 34 (Vps34)
Structure of hVps34 [human Vps34 (vacuolar protein sorting 34)] and hVps15
Vps34 was cloned from Saccharomyces cerevisiae by Herman and Emr , and was first described as a gene essential for the sorting of CPY (carboxypeptidase Y) to the yeast vacuole . The cloning of the mammalian class IA PI3K, p110α  led to the direct demonstration that Vps34 had lipid kinase activity towards phosphatidylinositol . Vps34 homologues have been identified in unicellular organisms (S. cerevisiae, Candida albicans and Dictyostelium discoideum), Caenorhabditis elegans, Drosophila, as well as vertebrates [4–8]. All of the Vps34 enzymes exhibit considerable homology to other PI3Ks (phosphoinositide 3-kinases), particularly at the level of domain organization. As compared with the PI3Kγ structure solved by Williams and co-workers , Vps34 has a structurally uncharacterized 52–54-amino-acid N-terminal region, followed by C2, helical and kinase domains. The extreme C-terminal 11 residues of Vps34 are required for lipid kinase activity, independent from any effects on binding to Vps15 (see below) .
Vps15, also cloned by the Emr laboratory , contains an N-terminal myristoylation consensus sequence, and the yeast and mammalian enzymes are myristoylated [12,13]. Vps15 consists of a protein kinase domain followed by a central region containing HEAT (Huntingtin, elongation factor 3, the PR65/A subunit of protein phosphatase 2A and the lipid kinase Tor) repeats, and a series of C-terminal WD-40 domains. In yeast, Vps34 activity requires a functional copy of Vps15 . Vps15 and Vps34 form a membrane-associated complex, and membrane targeting of Vps34 requires Vps15 . More recent analysis of the Vps34–Vps15 interaction identified residues 837–864 in the C-terminus of Vps34 as sufficient to bind to the kinase and HEAT domains of Vps15 .
Regulation of hVps34 in membrane trafficking
The discovery that Vps34 was a PI3K  led to a number of studies demonstrating a role for PI3K, and the mammalian homologue (hVps34) in particular, in mammalian endocytic trafficking (reviewed in [16,17]). Given that Vps34 activity is abolished in ΔVps15 yeast or yeast expressing kinase-dead Vps15, Vps34 is presumed to be activated by Vps15 phosphorylation . However, it is not clear whether this is a regulated event. In addition to potentially regulating hVps34 by phosphorylation, mammalian hVps15 (formerly called p150 ) targets hVps34 to Rab5-positive endosomes. We have shown that hVps15 binds to activated Rab5 through its HEAT and WD-40 domains [18,19]. Moreover, overexpression of constitutively active Rab5 leads to the recruitment of both hVps15 and hVps34 to enlarged early endosomes . hVps15 also binds to the late endosomal GTPase Rab7, and both hVps34 and hVps15 co-localize to Rab7-positive late endosomes . The Rab5/7-mediated regulation/recruitment of hVps34 in endosomes is likely to play an important role in the recruitment of FYVE (Fab1p, YOTB, Vac1p and EEA1)/PX domain (Phox homology domain)-containing effectors [16,17].
hVps34/hVps15 binds Ca2+/calmodulin in vitro, and treatment of macrophages with the calmodulin inhibitor W7 decreases the accumulation of PtdIns3P (phosphatidylinositol 3-phosphate) in phagosomes, as measured by an EGFP (enhanced green fluorescent protein)-FYVE domain probe . This effect is cell-type-specific, as W7 has no effect on EGFP-FYVE localization in COS-7 cells [22,23]. In macrophages, the decrease in phagosomal PtdIns3P appears to represent an effect on recruitment rather than activity of hVps34, since W7 has no effect on the activity of hVps34 measured in immunoprecipitates from J774 macrophages (M.P. Byfield and J.M. Backer, unpublished work).
In Saccharomyces cerevisiae, Vps34 is present in two distinct heterotetrameric complexes containing Vps34, Vps15, Atg6/Vps30, and either Atg14 or Vps38 . The two complexes have independent functions, as deletion of Atg14 blocks only autophagy, whereas deletion of Vps38 blocks only vacuolar sorting. Atg14 and Vps38 do not have obvious mammalian homologues. The mammalian homologue of Atg6/Vps30 is beclin-1, a coiled-coil protein originally identified as a Bcl-2-interacting tumour suppressor . beclin-1 binds hVps34 in mammalian cells , and beclin-1−/+ mice show decreased autophagy and enhanced tumorigenesis [27,28]. However, with regard to hVps34-dependent trafficking, minimal effects were seen in stable beclin-1-knockdown cells . Thus the requirement for beclin in both trafficking and autophagy-related hVps34 functions may not hold true in mammals.
Regulation of hVps34 by heterotrimeric G-proteins
Dohlman and co-workers  have recently shown that Vps34 and Vps15 are required for pheromone signalling to MAPK (mitogen-activated protein kinase) in S. cerevisiae. Activation of MAPK by an activated mutant of Gpa1 was blocked by null alleles of Vps34 or Vps15, but not Vps30 or Vps38. Thus signalling by Vps34/Vps34 can occur independently of its functions in autophagy or vesicular trafficking. Interestingly, Vps34 bound preferentially to activated Gpa1, and cellular PtdIns3P levels were increased in strains expressing activated Gpa1. In contrast, Vps15 bound preferentially to the unactivated form of Gpa1. Given that the Vps15 WD-40 domains are predicted to form a β-propeller similar to those found in Gβ-proteins [19,30–33], and based on sequence homology between Gγ subunits and the known Vps15-binding protein Atg14, it was proposed that Gpa1/Vps15/Atg14 form a novel type of trimeric G-protein involved in pheromone activation of MAPK.
Regulation of hVps34 by cellular nutritional status
The regulation of hVps34 by nutrients has been of considerable recent interest. hVps34 is required for one of the major cellular responses to nutrient stress, the induction of autophagy in starved cells [24,34]. Furthermore, it has recently been shown that hVps34 is required for insulin- and nutrient-stimulated activation of the mTOR (mammalian target of rapamycin) substrates S6K1 (S6 kinase 1) and 4EBP1, suggesting that it may be part of the nutrient-regulated inputs to mTOR kinase [35,36].
Two recent papers suggest that the beclin-associated, autophagy-related fraction of cellular hVps34 can be regulated by nutrients via effects on accessory proteins. Levine and co-workers  showed that binding of beclin to Bcl-2 increases under nutrient-rich conditions, and that overexpression of Bcl-1 reduces the amount of beclin-associated hVps34. It was subsequently shown by Jung and co-workers  that the nutrient-independent binding of beclin-1 to a tumour suppressor candidate protein, UVRAG (UV radiation resistance-associated gene), leads to increased beclin-associated hVps34 activity. A model was proposed in which beclin-associated hVps34 is controlled by a tonic-positive regulator (UVRAG) and a nutrient-dependent negative regulator (Bcl-2). Displacement of Bcl-2 during nutrient starvation would lead to increased beclin-1-associated hVps34, consistent with a positive role for hVps34 in the induction of autophagy. Although the model would predict that beclin-1-associated hVps34 activity should increase during amino acid starvation, this issue has in fact been controversial, as direct measurements of beclin-associated hVps34 activity, using somewhat different methodologies, have shown both increased and decreased activity [35,39].
At odds with its role in autophagy, but consistent with its role as a positive regulator of mTOR signalling, total hVps34 activity is decreased by nutrient withdrawal. Decreased hVps34 enzyme activity, and decreased cellular PtdIns3P levels, has been observed in amino-acid-starved cells [35,36]. In addition, glucose starvation inhibits hVps34 activity independently of amino acid starvation . The mechanism by which amino acids regulate hVps34 is unknown at present. With regard to glucose, the AMP-regulated kinase AMPK (AMP-activated protein kinase) is activated by glucose starvation as well as other metabolic stresses [40–43], and its activation inhibits mTOR [44–47]. Byfield et al.  noted that activation of AMPK, by treatment of cells with AICAR (5-amino-4-imidazolecarboxamide riboside) or oligomycin, also inhibited hVps34. While the regulation of mTOR by AMPK is known to involve the insulin-regulated Akt–TSC1/2 (tuberous sclerosis complex 1/2) pathway, these results suggest that AMPK-mediated inhibition of hVps34 may also play a role in regulation of mTOR.
The requirement for hVps34 during nutrient-stimulated mTOR activity would seem to be contradictory with the requirement for hVps34 during starvation-induced autophagy, particularly since mTOR is an inhibitor of autophagy [24,34,48,49]. It is possible that the regulation of autophagy may be more complex in mammalian cells than in yeast. For example, mammalian cells contain class II PI3Ks, which could produce PtdIns3P to support autophagy despite inhibition of hVps34 [50,51]. In addition, endosomal formation of PtdIns3P from PtdIns(3,4)P2 by Type Ia inositol polyphosphate 4-phosphatase has recently been reported . Alternatively, starvation-induced inhibition of hVps34 might serve as a brake on autophagy. Recent studies in Drosophila have shown that dS6K, like hVps34, is a positive effector of autophagy . Nonetheless, dS6K is inhibited by amino acid starvation. The authors suggest that inhibition of S6K by amino acid starvation might limit autophagy during periods of prolonged starvation, thereby minimizing potential cellular damage. The inhibition of hVps34 by nutrient deprivation may play a similar role in mammalian cells.
This work was supported by NIH (National Institutes of Health) grant DK070679 (J.M.B.).
3rd Focused Meeting on PI3K Signalling and Disease: Biochemical Society Focused Meeting held at Bath Assembly Rooms, U.K., 6–8 November 2006. Organized and Edited by B. Hemmings (Friedrich Miescher Institute for Biomedical Research, Switzerland), B. Vanhaesebroeck (Ludwig Institute for Cancer Research, U.K.), S. Ward (Bath, U.K.) and M. Welham (Bath, U.K.).
Abbreviations: AMPK, AMP-activated protein kinase; EGFP, enhanced green fluorescent protein; FYVE, Fab1p, YOTB, Vac1p and EEA1; HEAT Huntingtin, elongation factor 3, the PR65/A subunit of protein phosphatase 2A and the lipid kinase Tor; MAPK, mitogen-activated protein kinase; mTOR, mammalian target of rapamycin; PI3K, phosphoinositide 3-kinase; S6K, S6 kinase; UVRAG, UV radiation resistance-associated gene; Vps34, vacuolar protein sorting 34
- © 2007 The Biochemical Society