Class II/III PI3Ks (phosphoinositide 3-kinases) produce the PtdIns(3)P lipid that is involved in intracellular vesicular trafficking. In contrast with class I PI3Ks, the potential signalling roles of class II/III PI3Ks are poorly understood. In a recent article in the Biochemical Journal, Bago and co-workers report that Vps34 (vacuolar protein sorting 34), the only class III PI3K, controls the activity of SGK3 (serum- and glucocorticoid-regulated protein kinase 3). Like other AGC kinases, the SGKs (SGK1, SGK2 and SGK3) are activated by dual phosphorylation. Unlike its cousins SGK1 and SGK2, SGK3 contains a PtdIns(3)P-binding domain, providing an additional element of regulation. The study by Bago et al. characterizes and makes extensive use of a Novartis Vps34 inhibitor (VPS34-IN1) that inhibits this PI3K isoform with nanomolar potency, without affecting other lipid kinases or more than 300 protein kinases. The authors show that this compound very rapidly reduced PtdIns(3)P levels at the endosome with concomitant loss of SGK3 phosphorylation. Co-inhibition of class I PI3Ks led to a further reduction in SGK3 activity, indicating that class I PI3Ks may also regulate SGK3 activity through an additional, currently unknown, mechanism. It remains to be assessed whether the novel PI3K–protein kinase connection established by this study is subject to acute cellular stimulation or is part of a constitutive housekeeping function. VPS34-IN1 will provide a useful tool to decipher the kinase-dependent functions of Vps34, with acute changes in SGK3 phosphorylation and subcellular localization being new biomarkers of Vps34 activity.

Phosphoinositides comprise only a small fraction of membrane lipids, yet are critical regulators of many cellular processes. These lipids are dynamically interconverted by specific kinases and phosphatases. PI3Ks (phosphoinositide 3-kinases) phosphorylate the 3′ hydroxy group of PtdIns, PtdIns(4)P and PtdIns(4,5)P2, generating 3-phophoinositide lipids that play critical roles in a range of biological responses, including cell growth, proliferation, metabolism and intracellular vesicular trafficking. Mammals have eight PI3K isoforms that are divided into three classes [1]. The best characterized ones are the class I PI3K isoforms (p110α, p110β, p110γ and p110δ). In response to stimuli, including growth factors, adhesion, antigen and others, these PI3K isoforms acutely phosphorylate PtdIns(4,5)P2 at the plasma membrane to generate the second messenger PtdIns(3,4,5)P3. This lipid and its derivative PtdIns(3,4)P2 co-ordinate the localization and function of multiple effector proteins through a subset of PH (pleckstrin homology) domains which selectively bind these lipids. One of the most studied downstream effectors of class I PI3Ks is Akt/PKB (protein kinase B). The class I PI3K pathway is currently one of the most targeted kinase pathways in drug development, with clinical trials in all phases of development [2].

In contrast, the roles of the class II PI3Ks (PI3K-C2α, PI3K-C2β and PI3K-C2γ) and Vps34 (vacuolar protein sorting 34), the single class III PI3K isoform, are much less well understood. These PI3Ks produce PtdIns(3)P, a lipid involved in endocytosis, membrane trafficking, autophagy and phagocytosis. These functions derive from the recruitment of effector proteins via specific PtdIns(3)P-binding domains, such as the FYVE (for Fab1, YOTB, Vac1 and EEA1) zinc-finger domain and PX (Phox homology) domain. Indeed, Vps34 controls PtdIns(3)P-mediated intracellular trafficking events [1,3]. Roles for PtdIns(3)P in acute cell signalling remain unknown, although there are indications that this lipid, in one way or another, can affect signalling [4].

Vps34 is the ancestral isoform of PI3K and is part of multiple distinct protein complexes that determine its biological functions [3]. Vps34 thus has protein scaffolding functions in addition to its catalytic activity. To date, the biological roles of Vps34 have mainly been assessed using siRNA or gene-deletion/knockout models, which cannot discriminate between kinase-dependent and -independent functions of Vps34. The generation of a small-molecule inhibitor of Vps34 to selectively assess the role of its catalytic function is therefore an important milestone. Indeed, previously used inhibitors of Vps34 (such as 3-methyladenine) are very non-selective and may even have confused the field as to the real roles played by Vps34. In 2010, the crystal structure of Drosophila Vps34 was reported, revealing unique features in the Vps34 ATP-binding site that allow the generation of inhibitors with selectivity over other PI3K isoforms [5]. It was therefore a matter of “finding a fitting shoe for (this) Cinderella” [6]. Several patents on Vps34 inhibitors have recently been published, revealing an interest from Pharma in this target [WO2012085815A1 (Novartis), WO2012085244A1 (Sanofi) and WO2012021615A1 (Millennium/Takeda)].

After analysing several compounds reported in the patent literature, Bago et al. [7] identified and characterized a bipyrimidinamine compound from the Novartis patent (WO2012085815A1) that has high selectivity and potency (IC50 <25 nM) for Vps34. Significantly, this study, published in a recent issue of the Biochemical Journal, shows that this particular compound (which they call VPS34-IN1) does not inhibit the activity of 25 other lipid kinases, including class I/II PI3Ks and 340 protein kinases [7]. This is particularly important as there was no publicly available information on the activity of the Vps34 inhibitors reported in the patent literature against class II PI3Ks or other relevant phosphoinositide kinases [such as PI4Ks (phosphoinositide 4-kinases) and PI5Ks (phosphoinositide 5-kinases)]. When administered to cultured cells, VPS34-IN1 decreases PtdIns(3)P levels in the endosomes within 1 min, without affecting PtdIns(3,4,5)P3 levels.

The authors then assessed a potential role for Vps34 in regulating the activity of SGK3 (serum- and glucocorticoid-regulated kinase 3) whose PX domain is known to bind PtdIns(3)P [8]. SGK3 is a member of the AGC kinase subfamily that also includes Akt/PKB, and regulates similar processes to those of Akt/PKB including cell growth and proliferation [9]. Like other AGC kinases, in order to gain full activation, SGK3 requires dual phosphorylation, namely on its activation loop in the kinase domain [in the so-called T-loop; by PDK1 (phosphoinositide-dependent kinase 1)] and on a hydrophobic motif in the C-terminus [presumably by mTORC2 (mammalian target of rapamycin complex 2)] [9]. Given that Vps34 generates PtdIns(3)P in the endosomal compartment where SGK3 has previously been found to be localized and activated [8], Bago et al. [7] tested a possible connection between these two kinases. Employing mutational analysis and lipid overlay assays, Bago et al. [7] confirmed that the PX domain of SGK3 preferentially binds PtdIns(3)P over other PtdIns species and is responsible for the endosomal localization of SGK3.

Treatment of cells with VPS34-IN1 was found to induce a very rapid (<1 min) ~50–60% reduction in SGK3 activity with concomitant reduction in both activation loop and hydrophobic motif phosphorylation, without affecting Akt/PKB phosphorylation. Interestingly, VPS34-IN1 did not inhibit the activity of SGK2 that lacks a PX domain. The fact that Vps34 inhibition suppresses phosphoinositide binding, endosomal localization and activity of SGK3 further establishes that endosomal localization is a prerequisite for the full activity of this kinase. The endosomal localization of SGK3 might be essential for co-localization with the kinases responsible for its activating phosphorylations.

Interestingly, Bago et al. [7] found that treatment of cells with class I PI3K inhibitors (GDC-0941 and BKM120, both of which do not inhibit Vps34) also suppressed SGK3 activity by ~40%, but with slightly slower kinetics (2–5 min). Interestingly, a combination of VPS34-IN1 and GDC-0941 reduced SGK3 activity by ~80–90%. On the basis of these findings, the authors suggest that SGK3 activity is regulated by two pools of PtdIns(3)P. The first would be synthesized by Vps34 at the endosome and has a very quick turnover (~30 s) when Vps34 is inhibited, whereas the second pool of PtdIns(3)P would be generated via sequential dephosphorylation of PtdIns(3,4,5)P3 by PtdIns 5-phosphatases {SHIP1/2 [SH2 (Src homology 2)-domain-containing inositol phosphatase 1/2]} and PtdIns 4-phosphatase [INPP4B (inositol polyphosphate-4-phosphatase, type II)] (Figure 1a). However, live imaging studies using GFP–2×FYVEHRS and GFP–SGK3 reporters failed to detect a putative class I PI3K-derived PtdIns(3)P pool at the plasma membrane, although it is possible that PtdIns(3)P levels at the plasma membrane are too low/diffuse for detection by these probes. Down-regulation of SHIP1/2 and INPP4B expression using siRNAs could provide stronger evidence for a PtdIns(3,4,5)P3-derived pool of PtdIns(3)P. Alternatively, class I PI3K-mediated SGK3 activation might be PtdIns(3)P-independent and, for example, rely on a PtdIns(3,4,5)P3/PtdIns(3,4)P2-dependent mTORC2 activation at the plasma membrane, phosphorylating SGK3 in that location (Figure 1a).

Dual regulation of the SGK3 AGC kinase by class I and class III PI3Ks

Figure 1
Dual regulation of the SGK3 AGC kinase by class I and class III PI3Ks

(a) SGK3 activity is regulated by PtdIns(3)P produced by Vps34, targeting SGK3 to the endosome where it is phosphorylated on its T-loop and hydrophobic motif. SGK3 is additionally regulated by class I PI3Ks, possibly at the plasma membrane. It is not clear whether this second level of SGK3 regulation is dependent on PtdIns(3)P produced via sequential dephosphorylation of PtdIns(3,4,5)P3 by the SHIP/INPP4 lipid phosphatases. (b) Analogy between the canonical class I PI3K pathway regulating the AGC kinase Akt/PKB via PtdIns(3,4,5)P3 production (left-hand panel) and the class III PI3K Vps34 controlling the AGC kinase SGK3 via PtdIns(3)P binding at the endosome (right-hand panel). It is not clear at present whether Vps34-mediated regulation of SGK3 is responsive to extracellular stimuli as is the case for the class I PI3Ks. PH, pleckstrin homology; PI, phosphoinositide.

Figure 1
Dual regulation of the SGK3 AGC kinase by class I and class III PI3Ks

(a) SGK3 activity is regulated by PtdIns(3)P produced by Vps34, targeting SGK3 to the endosome where it is phosphorylated on its T-loop and hydrophobic motif. SGK3 is additionally regulated by class I PI3Ks, possibly at the plasma membrane. It is not clear whether this second level of SGK3 regulation is dependent on PtdIns(3)P produced via sequential dephosphorylation of PtdIns(3,4,5)P3 by the SHIP/INPP4 lipid phosphatases. (b) Analogy between the canonical class I PI3K pathway regulating the AGC kinase Akt/PKB via PtdIns(3,4,5)P3 production (left-hand panel) and the class III PI3K Vps34 controlling the AGC kinase SGK3 via PtdIns(3)P binding at the endosome (right-hand panel). It is not clear at present whether Vps34-mediated regulation of SGK3 is responsive to extracellular stimuli as is the case for the class I PI3Ks. PH, pleckstrin homology; PI, phosphoinositide.

The Bago et al. [7] study suggests that Vps34 could regulate signalling pathways in a similar way to class I PI3Ks by generating PtdIns(3)P to target SGK3, in analogy with class I PI3Ks generating a PtdIns(3,4,5)P3-dependent signalling platform to activate Akt/PKB. Interaction with their respective 3-phosphoinositides targets these AGC kinases to the intracellular lipid compartment where they are activated by phosphorylation, by PDK1 in the activation loop of the catalytic domain and mTORC2 in the C-terminus [10] (Figure 1b). The differences in cellular localization between Akt/PKB and SGK3 probably explain how their functional specificity is achieved. It will be important to uncover the selective downstream targets of SGK3, Akt/PKB and other SGK isoforms, and to assess whether Vps34 is implicated in this regulation. Very importantly, it will be critical to test whether Vps34-mediated regulation of SGK3 phosphorylation is modulated by extracellular stimuli, such as amino acids or growth factors. Indeed, at the moment, there is no evidence that Vps34/SGK3 is a stimulus-responsive signalling pathway in the classical sense of the word.

The Vsp34 inhibitors reported by Bago et al. [7] and of other studies [11,12] (see Box 1) will be powerful tools to dissect the roles of the catalytic activity of Vps34 in cells and in disease, such as neurodegeneration and cancer. In this context, it is important to mention that interfering with biology in which Vps34 has been implicated, such as autophagy, could be therapeutically detrimental or beneficial. Indeed, autophagy has been shown to have positive and negative roles in cancer maintenance and development respectively [13]. Importantly, SGK3 has recently been shown to be involved in cancer-related biology other than autophagy. First, SGK3 co-localizes with the EGFR [EGF (epidermal growth factor) receptor] in early endosomes, with an unknown functional link [10]. EGFR signalling is often dysregulated in cancer, with EGFR signalling from the endosome being sufficient to activate proliferation and survival. VPS34-IN1 could be used to test the functional relevance of the EGFR–SGK3 interaction by assessing the potential involvement of Vps34 in signalling downstream of EGF. Secondly, there have been a number of reports that SGK3 might be an important effector in cancer cells harbouring PIK3CA mutations and reduced Akt/PKB signalling [14]. VPS34-IN1 could be a key tool to in identifying substrates of SGK3 that are critical for these functions.

On a general note, it is of interest that non-class I PI3Ks are now also being explored for drug development. However, strong disease indications for these kinases are still lacking, and it will be the interplay of pharmacological approaches, as reported by Bago et al. [7], together with genetic studies in model organisms, that will hopefully answer this critical question in the near future.

Box 1

A recent study from Novartis [11] has reported another Vps34 inhibitor, called PIK-III, from the patent containing VPS34-IN1. By resolving the first crystal structure of human Vps34 in complex with PIK-III, this study provides a clear molecular basis for selectivity over class I PI3K isoforms. The Novartis group further use PIK-III to discover novel autophagy substrates including NCOA4 (nuclear receptor co-activator 4) which is required for ferritin degradation in lysosomes and iron regulation in vivo.

A second study from Sanofi [12] reports another ATP-competitive inhibitor of Vps34 which prevents vesicle trafficking from late endosome to the lysosome and the induction of autophagy by blocking the formation of autophagosomes.

We thank Leon Murphy (Novartis Institutes for BioMedical Research, Cambridge, MA, U.S.A.) for sharing unpublished data and Maria Whitehead for critically reading this Commentary before submission.

Abbreviations

     
  • EGF

    epidermal growth factor

  •  
  • EGFR

    EGF receptor

  •  
  • INPP4B

    inositol polyphosphate-4-phosphatase, type II

  •  
  • mTORC2

    mammalian target of rapamycin complex 2

  •  
  • PDK1

    phosphoinositide-dependent kinase 1

  •  
  • PI3K

    phosphoinositide 3-kinase

  •  
  • PKB

    protein kinase B

  •  
  • PX

    Phox homology

  •  
  • SGK

    serum- and glucocorticoid-regulated kinase

  •  
  • SHIP

    SH2 (Src homology 2)-domain-containing inositol phosphatase

  •  
  • Vps34

    vacuolar protein sorting 34

FUNDING

Work in the laboratory of B.V. is supported by the Biotechnology and Biological Sciences Research Council [grant number BB/I007806/1], Cancer Research UK [grant number C23338/A15965) and the National Institute for Health Research (NIHR) University College London Hospitals Biomedical Research Centre.

References

References
1
Vanhaesebroeck
 
B.
Guillermet-Guibert
 
J.
Graupera
 
M.
Bilanges
 
B.
 
The emerging mechanisms of isoform-specific PI3K signalling
Nat. Rev. Mol. Cell Biol.
2010
, vol. 
11
 (pg. 
329
-
341
)
[PubMed]
2
Rodon
 
J.
Dienstmann
 
R.
Serra
 
V.
Tabernero
 
J.
 
Development of PI3K inhibitors: lessons learned from early clinical trials
Nat. Rev. Clin. Oncol.
2013
, vol. 
10
 (pg. 
143
-
153
)
[PubMed]
3
Backer
 
J. M.
 
The regulation and function of Class III PI3Ks: novel roles for Vps34
Biochem. J.
2008
, vol. 
410
 (pg. 
1
-
17
)
[PubMed]
4
Balla
 
T.
 
Phosphoinositides: tiny lipids with giant impact on cell regulation
Physiol. Rev.
2013
, vol. 
93
 (pg. 
1019
-
1137
)
[PubMed]
5
Miller
 
S.
Tavshanjian
 
B.
Oleksy
 
A.
Perisic
 
O.
Houseman
 
B. T.
Shokat
 
K. M.
Williams
 
R. L.
 
Shaping development of autophagy inhibitors with the structure of the lipid kinase Vps34
Science
2010
, vol. 
327
 (pg. 
1638
-
1642
)
[PubMed]
6
Miller
 
S.
Oleksy
 
A.
Perisic
 
O.
Williams
 
R. L.
 
Finding a fitting shoe for Cinderella: searching for an autophagy inhibitor
Autophagy
2010
, vol. 
6
 (pg. 
805
-
807
)
[PubMed]
7
Bago
 
R.
Malik
 
N.
Munson
 
M. J.
Prescott
 
A. R.
Davies
 
P.
Sommer
 
E.
Shpiro
 
N.
Ward
 
R.
Cross
 
D.
Ganley
 
I. G.
Alessi
 
D. R.
 
Characterization of VPS34-IN1, a selective inhibitor of Vps34, reveals that the phosphatidylinositol 3-phosphate-binding SGK3 protein kinase is a downstream target of class III phosphoinositide 3-kinase
Biochem. J.
2014
, vol. 
463
 (pg. 
413
-
427
)
[PubMed]
8
Tessier
 
M.
Woodgett
 
J. R.
 
Role of the Phox homology domain and phosphorylation in activation of serum and glucocorticoid-regulated kinase-3
J. Biol. Chem.
2006
, vol. 
281
 (pg. 
23978
-
23989
)
[PubMed]
9
Pearce
 
L. R.
Komander
 
D.
Alessi
 
D. R.
 
The nuts and bolts of AGC protein kinases
Nat. Rev. Mol. Cell Biol.
2010
, vol. 
11
 (pg. 
9
-
22
)
[PubMed]
10
Virbasius
 
J. V.
Song
 
X.
Pomerleau
 
D. P.
Zhan
 
Y.
Zhou
 
G. W.
Czech
 
M. P.
 
Activation of the Akt-related cytokine-independent survival kinase requires interaction of its phox domain with endosomal phosphatidylinositol 3-phosphate
Proc. Natl. Acad. Sci. U.S.A.
2001
, vol. 
98
 (pg. 
12908
-
12913
)
[PubMed]
11
Dowdle
 
W. E.
Nyfeler
 
B.
Nagel
 
J.
Elling
 
R. A.
Liu
 
S.
Triantafellow
 
E.
Menon
 
S.
Wang
 
Z.
Honda
 
A.
Pardee
 
G.
, et al 
Selective VPS34 inhibitor blocks autophagy and uncovers a role for NCOA4 in ferritin degradation and iron homeostasis in vivo
Nat. Cell Biol.
2014
 
doi:10.1038/ncb3053
[PubMed]
12
Ronan
 
B.
Flamand
 
O.
Vescovi
 
L.
Dureuil
 
C.
Durand
 
L.
Fassy
 
F.
Bachelot
 
M. F.
Lamberton
 
A.
Mathieu
 
M.
Bertrand
 
T.
, et al 
A highly potent and selective Vps34 inhibitor alters vesicle trafficking and autophagy
Nat. Chem. Biol.
2014
 
in the press
13
White
 
E.
DiPaola
 
R. S.
 
The double-edged sword of autophagy modulation in cancer
Clin. Cancer Res.
2009
, vol. 
15
 (pg. 
5308
-
5316
)
[PubMed]
14
Bruhn
 
M. A.
Pearson
 
R. B.
Hannan
 
R. D.
Sheppard
 
K. E.
 
AKT-independent PI3-K signaling in cancer: emerging role for SGK3
Cancer Manag. Res.
2013
, vol. 
5
 (pg. 
281
-
292
)
[PubMed]

Author notes

2

Bart Vanhaesebroeck is a consultant to Retroscreen (London, U.K.) and Karus Therapeutics (Oxford, U.K.).