Oncogene (2008) 27, 5464–5476 & 2008 Macmillan Publishers Limited All rights reserved 0950-9232/08 $32.00 www.nature.com/onc REVIEW Understanding PTEN regulation: PIP2, polarity and protein stability NR Leslie, IH Batty, H Maccario, L Davidson and CP Downes Division of Molecular Physiology, College of Life Sciences, University of Dundee, James Black Centre, Dundee, Scotland, UK The PTEN tumour suppressor is a lipid and protein (Figure 1) and may also dephosphorylate specific phosphatase that inhibits phosphoinositide 3-kinase protein substrates. In many cellular assays and in vivo, (PI3K)-dependent signalling by dephosphorylating phos- PTEN acts to suppress cell growth, proliferation and phatidylinositol 3,4,5-trisphosphate (PtdInsP3). Here, we survival and, in a more cell specific manner, plays a role discuss the concept of PTEN as an ‘interfacial enzyme’, in the establishment of polarity and inhibits the which exists in a high activity state when bound migration of several mammalian cell types (Liliental transiently at membrane surfaces containing its substrate et al., 2000; Suzuki et al., 2003, 2008; Lacalle et al., 2004; and other acidic lipids, such as PtdIns(4,5)P2 and Leslie and Downes, 2004). Many of the effects of PTEN phosphatidylserine (PtdSer). This mechanism ensures that on cell growth, proliferation and survival are believed to PTEN functions in a spatially restricted manner, and may be mediated through its inhibition of the PtdInsP3- explain its involvement in forming the gradients of dependent protein kinase Akt (Manning and Cantley, PtdInsP3, which are necessary for generating and/or 2007). However, the effects of PTEN on cell polarity sustaining cell polarity during motility, in developing and migration seem to be mediated more through other neurons and in epithelial tissues. Coordinating PTEN PtdInsP3-dependent pathways, in particular, the small activity with alternative mechanisms of PtdInsP3 metabolism, GTPases of the Rac, Cdc42 and Arf families, and may by the tightly regulated SHIP 5-phoshatases, synthesizing involve lipid phosphatase-independent actions of PTEN the independent second messenger PtdIns(3,4)P2, may (Liliental et al., 2000; Raftopoulou et al., 2004; Leslie also be important for cellular polarization in some cell et al., 2007; Dey et al., 2008). The differential effects of types. Superimposed on this interfacial mechanism are these pathways also highlight the distinction between the additional post-translational regulatory processes, which effects of PI3K/PTEN that depend simply on cellular generally act to reduce PTEN activity. Oxidation of the PtdInsP3 levels (signal amplitude) and those that depend active site cysteine residue by reactive oxygen species and critically on the subcellular targeting of activity con- phosphorylation of serine/threonine residues at sites in the tributing to PtdInsP3 gradients (signal localization) C-terminus of the protein inhibit PTEN. These phosphor- (Leslie et al., 2001; von Stein et al., 2005; Pinal et al., ylation sites also appear to play a role in regulating both 2006). stability and localization of PTEN, as does ubiquitination Regulation of PTEN activity and/or expression serves of PTEN. Because genetic studies in mice show that the not only to modulate the temporal and spatial distribu- level of expression of PTEN in an organism profoundly tion of PtdInsP3, but also its availability for an influences tumour susceptibility, factors that regulate alternative route of metabolism to PtdIns(3,4)P2 by PTEN, localization, activity and turnover should be 5-phosphatases. The ability to switch the output of important in understanding its biological functions as a PI3K signalling between PtdInsP3 and PtdIns(3,4)P2 is tumour suppressor. also likely to have important implications in cell biology Oncogene (2008) 27, 5464–5476; doi:10.1038/onc.2008.243 and pathological conditions such as cancer. Keywords: phosphatase; phosphoinositide; cell polarity; Akt; SHIP Targeting PTEN activity to acidic membranes rich in PtdIns(4,5)P2 Introduction PTEN is an interfacially activated enzyme To access its lipid substrate, PtdInsP3, the cytosolic pool The tumour suppressor PTEN is a dual-specificity of PTEN must associate at least transiently with phosphatase, which antagonizes phosphoinositide membrane surfaces. Studies comparing rates of meta- 3-kinase (PI3K)-dependent signalling by hydroly- bolism of PtdInsP3 in synthetic lipid vesicles versus inositol 1,3,4,5-tetrakisphosphate (the soluble head- sing phosphatidylinositol 3,4,5-trisphosphate (PtdInsP3) group of PtdInsP3) showed that this interfacial interac- tion was not passive, but could account for up to a Correspondence: Dr NR Leslie, Division of Molecular Physiology, College of Life Sciences, University of Dundee, James Black Centre, 10 000-fold difference in Kcat values for these two Dow Street, Dundee, Scotland DD1 5EH, UK. substrates (McConnachie et al., 2003). This led to our E-mail: [email protected] or [email protected] proposal of a model in which PTEN can exist in two PTEN regulation NR Leslie et al 5465 PI(4,5)P2 PI(3,4,5)P3 PI(3,4)P2 P P PTEN 5-phosphatases PI3K Akt, Btk Akt, TAPP Rac GEFs Lamellipodin / MIG10 Arf GEFs Growth Proliferation Survival Motility / Polarity Figure 1 A model of phosphoinositide 3-kinase (PI3K)-dependent signalling is shown, detailing the synthesis of PtdIns(3,4,5)P3 from PtdIns(4,5)P2 via phosphorylation at the third position of the inositol ring by PI3K. PTEN catalyses the conversion of PtdInsP3 back to PtdIns(4,5)P2, whereas 5-phosphatases, such as the SHIP proteins, convert PtdInsP3 to PtdIns(3,4)P2. These two PI3K-dependent signals, PtdInsP3 and PtdIns(3,4)P2, share several downstream binding targets, including Akt. However, each of them also have specific targets, such as the Arf GEFs GRP1 and ARNO, which bind selectively to PtdInsP3, and the TAPP and lamellipodin proteins, which bind selectively to PtdIns(3,4)P2. Still further proteins are known to bind to these lipid targets, but their relative specificities have not been analysed thoroughly. GEF, guanine-nucleotide exchange factor; TAPP, tandem pleckstrin-homology domain-containing protein. active states, one favouring metabolism of soluble binding (Lee et al., 1999; Das et al., 2003; Vazquez et al., substrates and the other, the interfacially activated 2006; Redfern et al., 2008) and analysis of activity state, necessary for efficient metabolism of PtdInsP3 against substrates in acidic membranes or vesicles (Downes et al., 2007b) (Figure 2). The interfacially (Campbell et al., 2003; McConnachie et al., 2003; Iijima active state was also found to favour PtdInsP3 as the et al., 2004; Walker et al., 2004). substrate, explaining why, despite being able to meta- Initial experiments addressing the effects of bolize PtdIns(3,4)P2, PtdIns(3,5)P2 and PtdIns3P under PtdIns(4,5)P2 and other acidic lipids within target some in vitro conditions, PTEN appears to be highly membranes or vesicles tended to focus on the role of specific for PtdInsP3 in the cellular environment. Also these basic lipids in recruiting PTEN onto these supporting this specificity, other enzymes are required to surfaces, increasing access to the substrate lipid (Das metabolize cellular PtdIns3P and PtdIns(3,5)P2 (myotu- et al., 2003; Walker et al., 2004). However, a role for bularins) or PtdIns(3,4)P2 (inositol polyphosphate acidic lipids in the direct regulation of PTEN activity 4-phosphatases) efficiently. was also proposed (Campbell et al., 2003; Iijima et al., 2004). This latter proposal has now been strongly supported by data from the groups of Alonso Ross Regulation of PTEN by PtdIns(4,5)P2 and other acidic and Arne Gericke, indicating that acidic lipids, and lipids PtdIns(4,5)P2 in particular, actually cause a conforma- The scale of the interfacial activation when PTEN tional change within the enzyme and allosteric activa- associates with a membrane or vesicular substrate tion (Redfern et al., 2008). Infrared spectroscopy depends largely on the composition of the membrane experiments not only indicate that the N-terminus of and particularly its surface characteristics. These include PTEN binds specifically to PtdIns(4,5)P2 inducing an the overall surface charge, largely determined both by its increase in the PTEN a-helical content, but also that phosphatidylserine (PtdSer) content and, specifically, on simultaneous binding to PtdSer can also cause con- the presence of PtdIns(4,5)P2, both of which appear to formational alterations in PTEN (Redfern et al., 2008). mediate electrostatic interactions with the basic mem- This suggests that both recruitment and activity effects brane-binding surfaces of PTEN. Data supporting this enhance PTEN activity toward PtdInsP3 substrate that come from both direct analysis of PTEN’s lipid surface is present in membranes enriched with PtdIns(4,5)P2 and Oncogene PTEN regulation NR Leslie et al 5466 PI(4,5)P PI(3,4,5)P challenged this assumption, by developing well-vali- 2 3 dated ‘charge probes’ that display a localization on the plasma membrane that is dependent on electrostatic interactions between basic residues within the probe and anionic lipids, including PtdIns(4,5)P2 and PtdSer (Yeung et al., 2006). These and other experiments have shown that not only do these probes move off the high activity plasma membrane in response to depletion of membrane vs PIP3 PtdIns(4,5)P2 and PtdSer (Heo et al., 2006; Yeung et al., 2006), but, in macrophages, they also move away from the membrane during phagocytosis, selectively in the area of the phagocytic cup, and in contrast to fluorescent probes that localize to the plasma membrane through hydrophobic interactions (Yeung et al., 2006). An interesting aspect of the work of Heo et al. is that PtdInsP3 also appeared to be playing a part in controlling the electrostatic membrane recruitment of low activity high activity vs InsP and proteins containing polybasic regions, although, because protein substrates PtdInsP3 is present at such low concentrations in membranes, this seems to suggest either a surprising Figure 2 A hypothetical model of the interfacial activation of specificity in the interaction or an indirect mechanism PTEN at a membrane surface rich in PtdIns(4,5)P2 is shown.