Understanding PTEN Regulation: PIP2, Polarity and Protein Stability

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 interaction 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. PTEN is proposed to undergo a conformational change on (Yeung et al., 2006). Nevertheless, these data suggest an interaction with PtdIns(4,5)P2 and other membrane lipids, allowing unexpected possible autoregulatory feedback mechan- it to take up a conformation with high activity against its PtdInsP3 ism of PTEN regulation (Yeung et al., 2006). substrate. In solution, the relatively elevated activity of phosphory- Taken together, these studies suggest that the simple lated PTEN against inositol phosphates suggests that the C-terminal tail of PTEN may inhibit membrane interaction, while surface charge characteristic that appears to hold both allowing access to soluble substrates, but also that a less active these basic charge probes and PTEN onto the plasma conformation may exist. membrane can be modified by signalling, probably through phospholipase (PLC). This implicates both PIP2 and PtdSer in the recruitment of PTEN onto other acidic lipids, such as PtdSer. A potential membranes and also in independent allosteric activation functional consequence of this is suggested by experi- of the phosphatase. These studies also agree very well ments that identified pools of PtdInsP3 in the nuclear with recent results showing that in the social amoeba matrix and on the endoplasmic reticulum and other Dictyostelium discoideum PLC appears to act upstream endomembrane compartments in PTEN-null cells. Ex- of PTEN, regulating its membrane recruitment and pression of PTEN in these experiments reduced the activity (Keizer-Gunnink et al., 2007; Korthol et al., PtdInsP3 pool at the plasma membrane, but had no 2007). It will be interesting to see whether the regulation effect on the nuclear matrix or endoplasmic reticulum of PTEN by PLC will prove to be a conserved and pools, suggesting that cytosolic PTEN is unable to widely applicable regulatory mechanism. access substrate in the less-acidic endoplasmic reticular compartment (Lindsay et al., 2006). Further implication that PTEN activity is enhanced toward PtdInsP3 in regions of the plasma membrane that are relatively rich Metabolism of PtdInsP3 by the SHIP 5-phosphatases in PtdIns(4,5)P , especially in polarized cells, is dis- 2 In addition to the 3-phosphatase action of PTEN, cussed below. cellular PtdInsP3 concentrations are also regulated by the sequential activity of 5- and 4-phosphatases produ- Do changes in membrane charge represent a mechanism cing PtdIns(3,4)P2 and PtdIns3P, respectively. As these to rapidly regulate PTEN? molecules can act as distinct signals, the alternative, initial routes of PtdInsP removal by PTEN or 5- PTEN recruitment onto the plasma membrane is 3 phosphatases are not redundant but likely serve separate believed to rely on electrostatic interactions with anionic roles that confer flexibility to PI3K signalling. Thus, the lipids within this membrane, and probably additional capacity of PTEN to limit PtdInsP concentrations may protein–protein interactions (Leslie and Downes, 2004; 3 Vazquez et al., 2006). The acidic charged character of restrict metabolism by 5-phosphatases and thereby modulate both the magnitude and nature of the this membrane is believed to be contributed by several signalling output from PI3K (Figure 1). anionic lipids, including PtdSer, PtdIns(4,5)P2, PtdIns and phosphatidic acid (Yeung and Grinstein, 2007; Yeung et al., 2008). As such it was assumed by many Regulation of the SHIP 5-phosphatases researchers that this characteristic of the membrane Consistent with the view that the consequences of would remain relatively constant within a given cell. PtdInsP3 3- and 5-phosphatase activities are functionally However, recent data from Sergio Grinstein’s lab have distinct, none of the members of the family of inositol

Oncogene PTEN regulation NR Leslie et al 5467 polyphosphate/phosphoinositide 5-phosphatases is such that this achieves full capacity only following known to function as a tumour suppressor despite the stimulation. Furthermore, the ability to regulate evidence that, in contrast to the unique role of PTEN, 5-phosphatase activity independent of PI3K allows the several are involved in PtdInsP3 metabolism. Although balance of signalling output of the latter enzyme to be others, including the proline rich inositol polyphosphate switched between PtdInsP3 and PtdIns(3,4)P2. 5-phosphatase (Mochizuki and Takenawa, 1999) and the skeletal muscle inositol polyphosphate 5-phosphatase (Ijuin et al., 2000), may also be important, the best What are the functions of PtdIns(3,4)P2? characterized of these enzymes are the Src homology 2 Specific signalling functions have yet to be assigned to (SH2) domain-containing inositol polyphosphate PtdIns(3,4)P2, but are clearly implied by the several 5-phosphatases 1 and 2 (SHIP and SHIP2) (Krystal proteins, including the TAPP (tandem pleckstrin- et al., 1999; Rohrschneider et al., 2000; Backers et al., homology domain-containing protein) (Dowler et al., 2003; Dyson et al., 2005; Harris et al., 2008). 2000) and lamellipodin (Krause et al., 2004), which bind SHIP is restricted primarily to cells of haematopoietic to this lipid with high selectivity, and others, such origin, whereas SHIP2 is much more widely distributed, as DAPP1 (dual adaptor of phosphotyrosine and a feature that probably accounts for the distinct 3-phosphoinositides 1) (Dowler et al., 1999) and protein biological consequences resulting from the separate loss kinase B (Thomas et al., 2002), which preferentially bind of each enzyme (Helgason et al., 1998; Sleeman et al., both PtdIns(3,4)P2 and PtdInsP3. The degradation of 2005), with the deletion of SHIP2 having particular PtdIns(3,4)P2 is mediated by the types I and II inositol implications for insulin signalling (Sleeman et al., 2005; polyphosphate 4-phosphatases (Norris et al., 1995, Lazar and Saltiel, 2006). Nevertheless, SHIP and SHIP2 1997; Majerus et al., 1999), and the functional sig- share a similar, although not identical, multi-domain nificance of this activity is again implied both by the structure suggesting that these enzymes may be subject ability of the type I enzyme to regulate cell growth to complex regulation and/or may fulfil functions downstream of the GATA transcription factor (Vyas additional to, but likely integrated with, their PtdInsP3 et al., 2000) and by the neuronal loss characteristic of 5-phosphatase activity. Key features of these enzymes, Weeble mice that lack this protein (Nystuen et al., 2001) which confer the capacity for recruitment to multi- and express elevated cellular concentrations of protein complexes, include an N-terminal SH2 domain PtdIns(3,4)P2 (Shin et al., 2005). Both the type I and and, C-terminal to the common 5-phosphatase domain, II enzymes are expressed as a and b splice variants a proline rich region, which incorporates a double or which lack and contain a hydrophobic C-terminal tail, single NPXY motif in SHIP and SHIP2, respectively respectively. Like PTEN, the 4-phosphatases have the (Rohrschneider et al., 2000; Backers et al., 2003; Dyson essential CXXXXXR catalytic phosphatase motif char- et al., 2005). Consequently, each of these PtdInsP3 acteristic of the protein tyrosine phosphatase family of 5-phosphatases binds numerous protein partners enzymes and, thus, may be similarly susceptible to through either SH3 domain-directed or reversible inhibition by reactive oxygen species (ROS) (Downes tyrosine phosphorylation-mediated interactions. et al., 2007a). Thus, it is tempting to speculate that ROS However, as neither protein binding nor tyrosine produced endogenously downstream of PI3K-coupled phosphorylation has been shown to increase the specific receptors might inhibit both PTEN and 4-phosphatases, 5-phosphatase activity of SHIP or SHIP2 (Giuriato thereby promoting the accumulation of PtdIns(3,4)P2 in et al., 1997, 2002; Taylor et al., 2000; Blero et al., 2001), particular. Indeed, exogenously applied peroxide and the predominant effect of these modifications on other protein tyrosine phosphatase inhibitors, such as PtdInsP3 hydrolysis has been considered to reflect their vanadate analogues, dramatically and selectively in- associated re-localization from the cytosol to the vicinity crease cellular PtdIns(3,4)P2 above the normally very of their lipid substrate (Rohrschneider et al., 2000; low resting concentrations, consistent with an important Backers et al., 2003). Contrary to this view, two recent metabolic role for the potentially redox-sensitive studies suggest that the specific activity of both 4-phosphatase enzymes (Van der Kaay et al., 1999; 5-phosphatases is also subject to acute and dramatic Batty et al., 2007). The lipid product of 4-phosphatase regulation. The first shows that the activity of SHIP is activity is PtdIns3P whose role in vesicle trafficking is increased substantially by the allosteric binding of well established (Simonsen et al., 2001), and although it PtdIns(3,4)P2 to a putative C2-domain C-terminal to is not certain that the pools of this lipid produced by the 5-phosphatase domain, an effect that can be PtdInsP3 dephosphorylation are functionally equivalent reproduced by exogenously applied small molecules to those generated by class II or class III PI3Ks, the (Ong et al., 2007). The second demonstrates that, in formation of PtdIns3P represents a further distinguish- contrast to previous reports, the specific activity of ing feature of metabolism by this route. SHIP2 is markedly increased by the direct, PI3K- Thus, although PtdInsP3 is metabolized by PTEN and independent tyrosine phosphorylation of this protein by 5-phosphatases, these two pathways likely fulfil (Batty et al., 2007). Although important details of these distinct, although integrated, roles in PI3K signalling. mechanisms and their mutual exclusivity remain to be Although PTEN is believed to be constitutively active in established, both studies indicate that, in apparent resting and stimulated cells, 5-phosphatases, such as contrast to PTEN, PtdInsP3 5-phosphatase activity SHIP and SHIP2, are likely to be acutely regulated so can be acutely increased over a wide dynamic range that their low resting activity is dramatically increased

Oncogene PTEN regulation NR Leslie et al 5468 on stimulation by both their re-localization to sites of cell extract in vitro is also sensitive to selective CK2 PtdInsP3 synthesis, mediated by their capacity for inhibitors, when detected using phosphospecific anti- targeted protein interaction, and by marked increases bodies against P-S380, P-S385 or the combined 380/2/3 in their specific activity. Ultimately, therefore, the sites (Nick Leslie and James Murray, unpublished data). relative capacities of the two routes responding to Both analysis of the effects of phospho-mimicking differing prevailing regulatory influences can be ex- mutations and estimates of stoichiometry based on data pected to allow variability not only in the magnitude but comparing cell lysates with in vitro phosphorylated also in the spectrum of 3-phosphoinositide signals protein imply that, at least in several cell lines, the generated by PI3K, which, in conjunction with addi- stoichiometry of phosphorylation at these sites is quite tional temporal and spatial constraints on these, high, possibly greater than 50% (Vazquez et al., 2000; enhances the flexibility of PI3K-dependent signalling. Maccario et al., 2007). However, the detection of significant stimulation of phosphorylation at these sites, especially seen in neuronal tissues, would argue that the maintained basal phosphorylation levels must be much Regulation by phosphorylation lower in some cell types (Kim et al., 2004; Arevalo and Rodriguez-Tebar, 2006; Ning et al., 2006). It should be Serine/threonine phosphorylation of PTEN noted that although there have been relatively few The C-terminal, apparently unstructured tail of PTEN additional reports of the stoichiometry of phosphoryla- contains several sites of serine and threonine phosphory- tion of these sites being rapidly modulated, for example, lation (Figure 3). These form two separable groups during signalling, most of this work has been performed comprising a close cluster of four residues, Ser380, in primary cells and in vivo (Choi et al., 2004; Okamura Thr382, Thr383 and Ser385, and a pair of residues et al., 2006; Rafiq et al., 2006; Hoshino et al., 2007; Song slightly more N-terminal, Thr366 and Ser370. The 380– et al., 2007). 385 cluster sites all appear to be phosphorylated by Important early studies of these PTEN phosphoryla- CK2, based on evidence that these sites are excellent tion sites led to the proposal of a model in which substrates for CK2 in vitro, their phosphorylation is phosphorylated PTEN adopts a ‘closed’ conformation, sensitive to relatively selective CK2 inhibitors in cells which is more stable, less able to engage in PDZ and the kinase and PTEN appear to interact directly domain-dependent interactions and generally less active in pull-down experiments (Torres and Pulido, 2001; in cellular assays (Vazquez et al., 2001). Subsequent data Miller et al., 2002; Arevalo and Rodriguez-Tebar, 2006). have supported this model, adding details regarding In addition, the phosphorylation of these sites by a the effects of phosphorylation to suppress membrane

PI(4,5)P2 binding site

MTAIIKEIVSRRNKRR

C71 C124

C C

15186 352 403

Phosphatase domain C2 Domain

GSK3 CK2 CK2 P P P P P P VEEPSNPEASSSTSVTPDVSDNEPDHYRYSDTTDSDPENEPFDEDQHTQITKV T366/S370 380-385 PDZ binding motif Phosphorylation sites Figure 3 The 403-amino-acid PTEN protein is shown, divided into the N-terminal PTP family phosphatase domain (amino acids 7–185), the central C2 domain (186–351) and the reportedly unstructured tail (352–403). The N-terminal PtdIns(4,5)P2-binding site, comprising several basic residues clustered at amino acids 6–14is expanded with these basic residues shown in red. The C-terminal tail is also expanded, to show the PDZ-binding site boxed in blue and with phosphorylation sites shown in bold alongside their proposed kinases. The active site cysteine residue (C124) is shown, as is cysteine 71 (C71), both of which are targets for reactive oxygen species and can form a disulphide bond on oxidation with H2O2.

Oncogene PTEN regulation NR Leslie et al 5469 association (Das et al., 2003; Vazquez et al., 2006; phosphorylation have not been identified with great Odriozola et al., 2007), nuclear localization (Gil et al., confidence, mass spectrometry data indicate a tyrosine- 2006), PDZ binding (Tolkacheva et al., 2001; Valiente phosphorylated peptide within the phosphatase domain et al., 2005), ubiquitination and degradation (Tolkache- very close to its junction with the C2 domains (Guo va et al., 2001; Torres and Pulido, 2001; Wu et al., 2003), et al., 2008). Although the functional consequences of and phosphatase activity (Miller et al., 2002; Ning et al., tyrosine phosphorylation are currently unclear in 2006; Odriozola et al., 2007). Although this model has molecular terms, recent reports have associated PTEN stood the test of much further work, much of this tyrosine phosphorylation with regulation by RhoA and supportive data derive from the use of mutant proteins, ROCK (Li et al., 2005; Sanchez et al., 2005; Papakon- in which phosphorylation sites have been replaced by stanti et al., 2007). unphosphorylatable alanine residues. One such com- monly used mutant (PTEN 380A, 382A, 383A) has now been shown to have much greater catalytic activity in vitro than an unphosphorylated bacterially expressed Regulation of PTEN’s stability wild-type protein (Ning et al., 2006; Odriozola et al., 2007), strongly suggesting that in cellular experiments, The regulation of PTEN’s stability has been extensively this mutation will cause an activation far greater than studied, but the detailed mechanisms implicated and the that caused by simply blocking phosphorylation. pathways controlling stability are still poorly under- Although this does not call into question the current stood. As mentioned above, the phosphorylation state models for PTEN regulation, it does suggest that better of its C-terminal tail and interaction with specific experimental approaches are required to provide clearer partners via this C-terminal domain are the two main insight into this issue. mechanisms implicated in regulating PTEN’s stability. Also, in the unstructured C-terminal tail, Ser370 and As discussed, the protein kinase CK2 phosphorylates Thr366 appear to be phosphorylated by CK2 and a cluster of serine and threonine residues located near GSK3, respectively; this conclusion being similarly the C-terminus of PTEN. Dephosphorylation or alanine based on data from in vitro phosphorylation and mutation of these sites provokes a decrease in PTEN’s inhibitor analysis (Al-Khouri et al., 2005; Maccario stability in comparison with wild-type PTEN (Vazquez et al., 2007). Having been identified more recently, the et al., 2000; Torres and Pulido, 2001). Moreover, function of phosphorylation on these sites is rather recently our data showed that PTEN is phosphorylated unclear, although there is evidence for the suppression on threonine 366 and serine 370 by GSK3 and CK2, of activity (Al-Khouri et al., 2005), the cell-type-specific respectively, and that blocking phosphorylation on reduction in PTEN protein stability (Maccario et al., threonine 366 by either alanine mutation or GSK3 2007) and the mediation of a novel phosphatase- inhibition in glioblastoma cell lines, in this case, led to a independent tumour suppressor function (Okumura stabilization of the PTEN protein (Maccario et al., et al., 2005). This last study also identified a novel 2007). So, PTEN’s stability can be controlled by the interaction between PTEN and the FHA domain- phosphorylation state of multiple sites along its containing protein MSP58, mediated by a phos- C-terminal tail, apparently acting in opposition. phospecific interaction between the FHA domain and On the other hand, although the phosphorylation phosphothreonine 366 (Okumura et al., 2005). Despite state of PTEN has a big impact on its stability, PTEN’s the strong data for CK2 and GSK3 as the dominant interaction with specific partners also regulates its kinases responsible for PTEN C-terminal phosphoryla- stability. Indeed, PTEN associates with a variety tion in several cell types, other kinases are able to of scaffolding and/or regulatory molecules via its phosphorylate PTEN efficiently in vitro and may C-terminal tail (Leslie and Downes, 2004; Okahara contribute to regulating specific PTEN functions. et al., 2004; Takahashi et al., 2006). Notably, the PTEN PDZ-binding domain binds to several proteins, including MAGI-2 and MAST205. These interactions appear Tyrosine phosphorylation of PTEN to enhance PTEN’s stability, as interference with either Now there is strong evidence that PTEN can be these binding partners or PTEN’s ability to bind them phosphorylated on tyrosine residues. Initial studies greatly reduces PTEN’s stability (Wu et al., 2000; indicating PTEN tyrosine phosphorylation (Koul Subauste et al., 2005; Valiente et al., 2005). et al., 2002; Lu et al., 2003) have been backed up by The mechanisms associating C-terminal phosphoryla- data from a global analysis of tyrosine phosphorylation tion and PDZ-mediated protein–protein interactions to in lung tumours and cell lines, which indicated that stability are far from clear. A protein named PICT-1 tyrosine phosphorylation of PTEN was present in most (protein interacting with the C-tail-1) has been shown to examples of a definable rare subset of these tumours, but interact with the C terminus of PTEN, promoting both not detected in the great majority (Rikova et al., 2007; phosphorylation and stability of PTEN (Okahara et al., Guo et al., 2008). This suggests that PTEN tyrosine 2004). It has also been shown that during apoptosis, the phosphorylation may occur only in certain cell types PTEN protein is cleaved by caspase 3 at several target and circumstances, or that only an extremely small sites and that this cleavage is also suppressed by CK2- proportion of the cellular PTEN normally becomes mediated PTEN phosphorylation (Torres et al., 2003). tyrosine phosphorylated. Although the sites of tyrosine However, most data currently support a dominant role

Oncogene PTEN regulation NR Leslie et al 5470 for ubiquitin regulated proteasomal degradation in molecular mechanisms and oxidative changes that occur controlling PTEN’s stability. PTEN has two canonical in PTEN during redox signalling is yet to emerge, PEST motifs, a signature in many short-lived proteins further possibilities for PTEN redox regulation were degraded by the ubiquitin–proteasome pathway. More- raised by studies demonstrating PTEN inactivation over, treatment of many cell types with proteasome through nitrosylation and in response to cellular inhibitors enhances the levels of PTEN protein (Tolk- arachidonic acid treatment (Yu et al., 2005; Covey acheva et al., 2001; Torres and Pulido, 2001), and et al., 2007). recently an in vitro ubiquitination assay using cell Currently, it is clear that redox signalling plays a extracts or the ligase E3 Nedd4.1, along with cellular critical role in mediating the cellular responses to many ubiquitination experiments, has shown that PTEN is stimuli, including many growth factors, cytokines and both mono- and polyubiquitinated (Trotman et al., hormones (Rhee et al., 2005), and may contribute to 2007; Wang et al., 2007). Significantly, this work also several regulated steps in the PI3K signalling pathway, indicated that PTEN monoubiquitination controlled the including PTEN (Leslie, 2006). However, the experi- nuclear localization of PTEN, alongside the effects of mental challenges of studying redox signalling have polyubiquitination on PTEN’s stability. In addition, resulted in only a few studies in which these pathways Wang et al. (2007), using overexpression or RNAi have been elucidated in detail in more physiological approaches in 293T cells, implicated Nedd4.1 as the settings. Perhaps the best example proposed for the dominant ubiquitin ligase E3 for PTEN in their physiological significance of PTEN oxidation is cur- experiments. Interaction between PTEN and Nedd4.1 rently in muscle. In cardiac muscle, ischaemia and has been observed in several cell lines, and a novel reperfusion led to both the degradation and oxidation of mutual regulation between PTEN and Nedd4.1 is PTEN associated with the activation of Akt (Cai and suggested by the studies of Ahn et al. (2008). Given Semenza, 2005). Further studies implicating PTEN the emerging complexity and significance of ubiquitin in oxidation in both cardiac and skeletal muscle suggest regulating protein stability and other signalling systems, that the thioredoxin-interacting protein Txnip is re- it seems likely that much remains to be discovered quired for the maintenance of thioredoxin NADPH regarding how PTEN ubiquitination is controlled and levels, and efficient PTEN reduction, as muscle lacking regarding the roles of ubiquitin and possibly related Txnip displays elevated Akt activation and a redox- molecules in PTEN regulation. dependent loss of PTEN antigenicity (Hui et al., 2008).

PTEN oxidation activates PI3K-dependent signalling PTEN localization and activity in polarized cells

PTEN is a member of the diverse protein tyrosine Cellular localization of PTEN phosphatase superfamily, which share a catalytic me- The first descriptions of PTEN localization within cells, chanism requiring a reduced active site cysteine nucleo- either using fluorescently tagged protein or immuno- phile, the reactivity of which makes it sensitive to fluorescence, were obtained from a variety of cultured oxidation. In PTEN, it has been shown that oxidation cell lines, in which PTEN appears diffusely cytosolic, with H2O2 in vitro, or treatment of cells with H2O2,can with a somewhat variable nuclear component (Li and lead to the formation of a disulphide bond between the Sun, 1997; Leslie and Downes, 2004). It is clear that active site Cys124and another very closely located PTEN is enriched in the nucleus of some cell types cysteine, Cys71 (Lee et al., 2002) (Figure 3). Subsequent (Gimm et al., 2000; Perren et al., 2000). However, our work showed that the regulation of downstream PI3K- understanding of the mechanisms regulating this nuclear dependent Akt/PKB activation by H2O2 oxidative stress localization (Liu et al., 2005; Gil et al., 2006; Trotman is by PTEN oxidation, and that this oxidative inhibition et al., 2007) and, in particular, its functional conse- mechanism appears to contribute to the activation of quences is currently at a relatively early stage and has Akt/PKB in artificially LPS/PMA-stimulated macro- been described by others (Baker, 2007), and so will not phages, generating high levels of endogenous ROS be discussed further here. (Leslie et al., 2003). Oxidation of PTEN has now been Single-molecule studies using total internal reflection shown in cells stimulated with a range of peptide growth fluorescence microscopy in Dictyostelium and HEK293 factors, in which the stimulated generation of endogen- cells have shown that PTEN associates stably, but ous ROS is at greatly lower levels than that found in transiently, with the plasma membrane, with PTEN- macrophages, also accompanied by an oxidant-depen- YFP having a residence time ‘half-life’ on the plasma dent activation of downstream Akt signalling (Kwon membrane of a cultured HEK293 cell of around 150 ms et al., 2004; Seo et al., 2005). In these studies, PTEN (Vazquez et al., 2006). This work suggests that PTEN is oxidation was identified in insulin-stimulated neuroblas- probably abundant on most cell membranes relative to toma cells or HEK293 cells, in EGF-stimulated HeLa the cytosol, and possibly concentrated on some specific cells and in PDGF-stimulated NIH3T3 fibroblasts, membrane regions and domains, but this localization is using band-shift assays in addition to biotinylation often outside the detection limits of standard fluores- and alkylation protection methods (Kwon et al., 2004; cence microscopy. Nevertheless, recent analyses in vivo, Seo et al., 2005). Although a clear picture of the in primary cells or using cultured cells retaining

Oncogene PTEN regulation NR Leslie et al 5471 significant cellular architecture and polarity, have begun polarization of PTEN was shown originally in Droso- to suggest that PTEN is enriched at specialized sites, in phila (von Stein et al., 2005; Pinal et al., 2006), and later particular, at the apical domains of polarized epithelial in the chick embryo epiblast epithelium and in polarized cells (Li et al., 2003; von Stein et al., 2005; Chadborn MDCK kidney and T84colon cells in 3D culture (Leslie et al., 2006; Pinal et al., 2006; Leslie et al., 2007; Martin- et al., 2007; Martin-Belmonte et al., 2007). In Droso- Belmonte et al., 2007) (Figure 4). This epithelial phila, PDZ-dependent interaction with Bazooka/dPAR3 in the zonula adherens was implicated in the apical PTEN localization, and in these experiments, apical localization was limited to the Drosophila PTEN2 isoform, which has a C-terminal PDZ-binding sequence. Chemotaxis Biochemical evidence from mammalian epithelial cells also positions PTEN in apical adherens junction complexes, targeted through its PDZ-binding motif and the Par3 and MAGI proteins (Kotelevets et al., 2005; Subauste et al., 2005; Wu et al., 2007). It is perhaps worth mentioning that although some of Epithelial these localization studies have used fluorescently tagged polarisation PTEN proteins, the involvement of both termini in targeting the protein (to anionic lipids through the N-terminus and PDZ domains through the C-terminus) means that, where possible, localization of the endogenous protein is clearly preferable. In this regard, only Neuronal a few antibodies have been shown to be suitable for polarisation immunofluorescent analysis, with most consistent results probably being obtained using a well-validated monoclonal antibody named 6H2.1 (Perren et al., 1999; Pallares et al., 2005). In addition, N-terminal tagging of PTEN can be problematic, and can lead to a loss of cellular function (Lacalle et al., 2004). T cell activation

PTEN and the regulation of cellular polarity It has emerged over the past 5 or 6 years, that PTEN and PtdInsP3 play conserved roles in the determination of cell polarity in very diverse cell types (Figure 4). Data, Cytokinesis obtained ﬁrst from Dictyostelium cells (Parent et al., 1998; Meili et al., 1999; Funamoto et al., 2002; Iijima

= PIP3 and Devreotes, 2002) and later from neutrophils undergoing chemotaxis (Servant et al., 2000; Li et al., 2003), = PTEN and PI(4,5)P 2 show enrichment of PtdInsP3 at the leading edge of Figure 4 Several diverse cell types display similar patterns of these cells and localization of PTEN to the back, PTEN and phosphoinositide polarization. Enrichment of PTEN, although the localization of PTEN in neutrophils is still where identified, is shown in blue, and PtdInsP3 in specific controversial (Xu et al., 2003; Lacalle et al., 2004; Li membranes is shown in red. PtdInsP3 has been described at the et al., 2005). This work has now been extended to show leading edge of neutrophils, fibroblasts and Dictyostelium cells during chemotaxis; the basolateral membranes of breast and that Dictyostelium display a related but bipolar polar- kidney epithelial cell lines; the axonal growth cones of polarizing ization during cytokinesis, with PTEN localizing to the neurons; and in extending neurites, at the immunological synapse furrow and PtdInsP3 being enriched at the poles during T-cell activation, with a weaker localization at the opposite (Janetopoulos and Devreotes, 2006). In polarizing pole of these lymphocytes; and finally at the two poles of neurons, PtdInsP appears to be enriched at the growth Dictyostelium cells undergoing cytokinesis. Conversely, PTEN 3 localizes to the back of Dictyostelium cells, and possibly cone, in particular, in the developing axon during neutrophils, during chemotaxis, to the apical membrane of several neuronal polarization (Shi et al., 2003; Aoki et al., epithelial cell types, and to the furrow of Dictyostelium cells 2005), with PTEN being excluded from this region until undergoing cytokinesis. Very similar distributions have been growth cone collapse (Chadborn et al., 2006). Function- described for PtdIns(4,5)P2, being localized along with PTEN on the apical membranes of some epithelial cells and at the furrow of ally, Dictyostelium cells lacking PTEN have defects in dividing Dictyostelium cells. We are not aware of data localizing polarization and chemotaxis, and mutation of PTEN/ PtdIns(4,5)P2 during chemotaxis. It should be noted that although daf-18 in Caenorhabditis elegans or knockdown of a PtdInsP3 gradient and cell polarity are maintained for many PTEN expression in rat hippocampal neurons causes hours during T-cell activation, it appears that PI3K/PTEN aberrant neuronal polarization (Funamoto et al., 2002; signalling is not required for efficient cell polarization and activation in this circumstance (Costello et al., 2002; Harriague Iijima and Devreotes, 2002; Jiang et al., 2005; Adler and Bismuth, 2002). Further details and references are in the main et al., 2006). In contrast, several mammalian cell types, text. PIP3, phosphatidylinositol 3,4,5-trisphosphate. including B cells and fibroblasts, migrate faster when

Oncogene PTEN regulation NR Leslie et al 5472 lacking PTEN, although cellular polarization has not Several other factors would also support the existence of been studied in great detail in these cases (Liliental et al., distinct membrane domains containing relatively high 2000; Suzuki et al., 2003). and low ratios of PtdIns(4,5)P2 to PtdInsP3, including Most recently, the investigation of PTEN’s role in the apparent roles for PtdInsP3 in the activation of regulating cell polarity in MDCK kidney epithelial cells PLCg1 and the inhibition of RhoA (Bae et al., 1998; has provided the most detailed evidence to date that Falasca et al., 1998; Krugmann et al., 2004, 2006). In PTEN is also important in epithelial cell polarization addition, the low Km of class I PI3K for PtdIns(4,5)P2 and plays a role in the regulation of phosphoinositide means that generation of cellular PtdInsP3 by PI3K is gradients defining apical and basolateral membrane not affected by fluctuations in PtdIns(4,5)P2 concentra- domains within these cells (Martin-Belmonte et al., tions except under extreme conditions of PtdIns(4,5)P2 2007). This study showed that both PTEN and depletion, and PLC-induced reductions in PtdInsP3 PtdIns(4,5)P2 are enriched on the apical membrane of accumulation appear to be mediated through inhibition polarized MDCK cells, whereas PtdInsP3 is enriched on of PI3K activation, rather than substrate limitation the basolateral membrane, relative to the apical (Batty and Downes, 1996; Batty et al., 2004). membrane. This study also presented data showing that Annexin 2 binds to cdc42, as well as to PtdIns(4,5)P2, and that PTEN, Annexin2 and cdc42 are all apically PtdIns(3,4)P2 and polarity? localized and required for correct polarization and Evidence for the integrated synthesis and metabolism of lumen formation. These results build on earlier data PtdInsP3 is emerging in several signalling systems (Iijima from a mammary epithelial cell line, in which polariza- et al., 2002; Leslie, 2006), and it seems likely that in the tion in 3D culture was dependent on PI3K signalling production of signal gradients, for example, during and resulted in basolateral accumulation of PtdInsP3 directed cell migration, the coordination of both PI3K (Liu et al., 2004). The recent study in MDCK cells also activity and PtdInsP3 metabolism is critical (Iglesias and proposed that PTEN may act to generate PtdIns(4,5)P2 Devreotes, 2008). However, in some cases, it appears from PtdInsP3 in the apical domain, but as PtdInsP3 is a that the functionally significant route of metabolism is minor lipid believed almost always to be generated from not via PTEN, but via the PtdInsP3 5-phosphatase PtdIns(4,5)P2, which generally exists in at least a 100- SHIP. In murine neutrophils, deletion of the Pten gene fold excess, it seems unlikely that this proposal is had little or no effect on chemotaxis or overt polariza- correct. Further evidence for a role in regulating tion in response to fMLP or C5a, whereas deletion of epithelial architecture comes from the effects of PTEN Ship1 caused a major failure in polarization and cell on cell migration events in the early chick embryo. Here, motility (Nishio et al., 2007). Additional data support- PTEN inhibits the epithelial to mesenchymal transition ing a significant role for PtdIns(3,4)P2 and 5-phospha- that epiblast cells undergo before they can migrate tase action in regulating cellular polarity comes from toward the embryo periphery, and this effect requires studies of neuronal polarization in Caenorhabditis the C-terminal PDZ-binding motif and the protein elegans, in which aberrant polarization was caused by phosphatase activity of PTEN (Leslie et al., 2007). mutation of PI3K/age-1 or PTEN/daf-18 or the selective PTEN function is very frequently lost during the PtdIns(3,4)P2-binding target lamellipodin/MIG10 development of epithelial-derived tumours, and many (Adler et al., 2006). This suggests that in some polarized such tumours are believed to undergo a form of cell responses, the regulated role of PTEN may epithelial to mesenchymal transition before they metas- simply be to allow the re-direction of PtdInsP3 tasize. Together with the accumulating evidence that metabolism via 5-phosphatase action to PtdIns(3,4)P2, loss of cellular polarity and tissue architecture can be presumably through suppression of PTEN 3-phospha- driving forces in tumour progression rather than its tase activity. by-product (Wodarz and Nathke, 2007), this indicates that these studies of PTEN in epithelial biology may be very relevant to the tumour suppressor functions of PTEN. Conclusions Although PI3K signalling is not absolutely required for cell polarity, and other parallel pathways regulating Data now show that PTEN tumour suppressor activity directed cell migration and polarization exist, data from can act in a dose-dependent, haploinsufficient manner in diverse experimental models suggest that PI3K signal- well-studied mouse models of prostate cancer and ling may play a general role in many forms of cellular probably in other tumour types. This strengthens the polarization (Arimura and Kaibuchi, 2007; Franca-Koh need to understand the cellular mechanisms by which et al., 2007; Insall and Andrew, 2007), linking in with PTEN expression and activity are controlled. Here, we other polarity systems, such as the Par3/Par6/aPKC clearly still have much to learn, especially with novel complex. In particular, PI3K signalling appears to be data suggesting the existence of complex regulatory one mechanism that would act as part of a positive mechanism involving mono- and polyubiquitination, feedback system, interacting in particular with Rho additional phosphorylation sites, mechanisms of redox family GTPases and possibly actin, to establish a semi- regulation and even PTEN acetylation (Okumura et al., stable polarized state (Iijima et al., 2002; Weiner, 2002; 2006; Covey et al., 2007; Trotman et al., 2007; Guo Charest and Firtel, 2006; Van Keymeulen et al., 2006). et al., 2008).

Oncogene PTEN regulation NR Leslie et al 5473 Acknowledgements International Cancer Research and a consortium of pharma- ceutical companies comprising Astra Zeneca, Boehringer Research in the Inositol Lipid Signalling laboratory is Ingelheim, GlaxoSmithKline, Merck and Co., Merck KGaA funded by the Medical Research Council, the Association for and Pﬁzer.

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