Advances in Enzyme Regulation 49 (2009) 190–196

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advenzreg

The significance of PTEN’s protein phosphatase activity

Nick R. Leslie*, Helene Maccario, Laura Spinelli, Lindsay DavidsonDivision of Molecular Physiology, College of Life Sciences, University of Dundee, Dundee, DD1 5EH, UK

Introduction

PTEN (EC 3.1.3.67) was originally identified as a candidate tumour suppressor in 1997 (Li et al., 1997;Steck et al., 1997). Much of the excitement surrounding its discovery derived from its similarity to theprotein tyrosine phosphatase family and the expectation that its function would be directly to suppressoncogenic tyrosine kinase signalling. However, although initial studies showed that PTEN did indeedhave protein phosphatase activity (Furnari et al., 1997; Li and Sun, 1997; Myers et al., 1997), a strongphosphatase activity against the lipid PtdInsP3 was soon revealed (Maehama and Dixon, 1998). Thisdiscovery and a wealth of subsequent work over ten years have established the significance of PTEN asa PtdInsP3 phosphatase and regulator of the PI3K/Akt signalling pathway (Leslie and Downes, 2002;Salmena et al., 2008; Sulis and Parsons, 2003). However, while it is clear that PTEN has many diversebiological effects, several of these effects are not obviously linked to its lipid phosphatase activity, anda number of convincing studies have presented data supporting the additional significance of PTEN’sprotein phosphatase activity (Gildea et al., 2004; Gu et al., 1999; Ji et al., 2006; Leslie et al., 2007;Mahimainathan and Choudhury, 2004; Raftopoulou et al., 2004; Rong et al., 2005; Tamura et al., 1998;Vogelmann et al., 2005).

PTEN is one of the hundred or so members of the Protein Tyrosine Phosphatase (PTP) superfamily inthe human genome. This family contains classical tyrosine-specific phosphatases (EC 3.1.3.48), dualspecificity phosphatases that are active against both phosphorylated tyrosine and serine/threoninesubstrates and also more divergent members that dephosphorylate non-protein substrates includingphosphoinositide lipids, mRNA and complex carbohydrates (Alonso et al., 2004; Andersen et al., 2004).The protein phosphatase activity of PTEN will act weakly upon phosphoserine and phosphothreoninecontaining substrates, although data indicate that it favours phosphotyrosine and that in each caseacidic substrates are strongly favoured (Li and Sun, 1997; Myers et al., 1997). However, even against thebest substrate identified, a phosphorylated polymer of tyrosine and glutamic acid, the protein phos-phatase activity of PTEN was shown to be rather weak (Myers et al., 1997). Perhaps it is simplest toconclude from these biochemical experiments, considering the possibility that excellent as yetunstudied substrates may exist, that PTEN is either a weak protein phosphatase, or one that has highactivity only towards certain highly specific substrates. A corollary to this would be that the protein

* Corresponding author. Tel.: þ44 1382 386263.E-mail address: n.r.leslie@dundee.ac.uk (N.R. Leslie).

0065-2571/$ – see front matter � 2009 Elsevier Ltd. All rights reserved.doi:10.1016/j.advenzreg.2008.12.002

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phosphatase activity of PTEN is simply an inconsequential by-product of a PTP family enzyme with anactive site pocket large enough to accommodate PtdInsP3. However, the lack of protein phosphataseactivity reported for other PTP family lipid phosphatases would seem to argue that the proteinphosphatase activity of PTEN is not an evolutionary coincidence. This seems especially true for TPIP,a very closely related PtdInsP3 phosphatase that appears to have evolved originally from an ancestralPTEN-like enzyme (Walker et al., 2001) and lacks protein phosphatase activity, at least against phos-phorylated polyGluTyr (Nick Leslie and Nevin Perera, unpublished work).

Over the years, a number of studies have implicated protein phosphatase activity as beingresponsible for some of the biological effects of PTEN and we discuss some of these in detail here.However, despite the proposal of several candidate protein substrates, none of these have beenconfirmed with much confidence, and the identification of specific sites of dephosphorylation andother mechanistic details are mostly absent. An important point at this stage is that many of the studiesproposing specific substrates have used general anti-phosphotyrosine antibodies to detect proteinphosphorylation. Several of the proposed proteins contain many phosphorylated residues (Mahimai-nathan and Choudhury, 2004; Tamura et al., 1998; Vogelmann et al., 2005). For example, b-catenincontains 9 reported sites of tyrosine phosphorylation and 11 phosphorylatable Ser/Thr residues, thea and b PDGF receptors contain 13 and 14 potential phosphotyrosines respectively and FAK, 11.Therefore, it seems unlikely that the apparently weak protein phosphatase PTEN is an importantphysiological phosphatase for all, or even many, of these sites. Thus, the identification of specific sites

Fig. 1. A hypothetical model for the separable roles of PTEN against both PtdInsP3 and unknown protein substrates in the regulationof cell migration. PtdInsP3 is synthesised by phosphoinositide 3-kinase (PI3K), once activated upon engagement of receptor tyrosinekinases (RTKs) and also some integrins. PTEN dephosphorylates PtdInsP3 at the 3 position to regenerate PtdIns(4,5)P2, but also canact through its protein phosphatase activity to oppose the integrin driven activation of FAK, Src family kinases and possibly Rac.Although cell migration is presented as one entity, the regulation by PTEN/PI3K of the many complex sub-processes required formigration is unclear.

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that may be dephosphorylated by PTEN, and a rigorous in vitro characterisation of their quality assubstrates seems necessary.

A key tool in the experimental analysis of PTEN’s protein phosphatase activity has been a pointmutant of PTEN originally identified in two Cowden disease families, PTEN G129E (Liaw et al., 1997).This enzyme, with a mutation in the active site pocket, retains similar protein phosphatase activity towild-type PTEN, at least using polyGluTyrP as a substrate, yet has greatly reduced activity, approxi-mately 10% of the wild-type protein’s activity, against both the lipid PtdInsP3 and its soluble headgroupIns(1,3,4,5)P4 (Furnari et al., 1998; Myers et al., 1998). This identification of a mutation with selectiveloss of lipid phosphatase activity in Cowden disease patients is still one of the strongest pieces ofevidence indicating that the lipid phosphatase activity of PTEN is the key mechanism of tumoursuppression, at least in Cowden disease (Furnari et al., 1998; Myers et al., 1998). PTEN G129E has alsobeen used in many cellular experiments, along with other approaches to define the significance ofPTEN’s lipid phosphatase activity.

The regulation of cell migration

The best evidence for the significance of PTEN’s protein phosphatase activity comes from studies ofcell migration. Before the identification of PTEN’s lipid phosphatase activity, PTEN over-expression wasshown to be able to inhibit the migration and spreading of NIH3T3 and primary fibroblasts and thespreading of U87MG and DBTRG glioblastoma cells (Tamura et al., 1998). Significantly, expression ofPTEN G129E was shown to block cell spreading and affect cell morphology to the same extent as thewild-type enzyme. Also, antisense knockdown of endogenous PTEN expression in NIH3T3 cellssignificantly enhanced cell migration in the scratch wound assay used and FAK was proposed asa potential substrate for PTEN (Tamura et al., 1998). Subsequently, analysis of immortalised murinefibroblasts and primary murine B cells lacking PTEN showed that these cell types migrated faster thanwild-type cells and, in the case of the fibroblasts, it was shown that this enhanced migration wasunaffected by the expression of PTEN G129E, implicating elevated PtdInsP3 levels and the activation ofthe small GTPases rac1 and cdc42 in this enhanced migration (Liliental et al., 2000; Suzuki et al., 2003).

Several further studies have confirmed the capacity of PTEN G129E expression to inhibit cellmigration and invasion as efficiently as wild-type PTEN, when expressed in glioblastoma and bladdercancer cell lines (Dey et al., 2008; Gildea et al., 2004; Park et al., 2002; Raftopoulou et al., 2004). Thesestudies all implicate the protein phosphatase activity of PTEN as mediating effects on cell migration andinvasion, and although one study suggested that full length phosphatase dead PTEN could also inhibitinvasion (Maier et al., 1999), this has not been found in other studies. While most of these studies usedcultured cell lines in monolayer or Boyden chamber migration assays, similar data have also beenprovided from in vivo experiments. Both wild-type and G129E PTEN expression similarly inhibited theinvasion of bladder cancer cells in organotypic culture assays and orthotopic in vivo assays (Gildeaet al., 2004) and the in vivo migration of chick embryo mesoderm cells out from the primitive streak(Leslie et al., 2007). It is worth noting that while almost all of the studies in question employed highlevels of over-expression or did not determine expression levels relative to normal PTEN levels, at leastsome have engineered PTEN mutant expression at levels close to those found endogenously (Gildeaet al., 2004) and performed some experiments in primary astrocytes from wild-type and heterozygousPTEN mice (Dey et al., 2008).

Several studies have tried to dissect the molecular mechanisms by which the protein phosphataseactivity of PTEN may affect cell migration. One of the most significant of these contributions was thedemonstration that, in microinjected glioblastoma cell monolayers, the C2 domain of PTEN alone wasable to block cell migration (Raftopoulou et al., 2004). This important result was accompanied by datasuggesting that to inhibit migration, full length PTEN requires protein phosphatase activity to auto-dephosphorylate its C-terminal phosphorylation sites, specifically T383, allowing a conformationalopening up of the PTEN protein, and revealing the C2 domain inhibitory activity (Raftopoulou et al.,2004). A similar conclusion was supported by in vivo studies in the early chick embryo, in which theprotein phosphatase activity of PTEN was also implicated in opening up the PTEN structure to allowa phosphatase independent mechanism of migration inhibition (Leslie et al., 2007). However, in thiscase, cell migration away from the primitive streak appeared to be prevented specifically through

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a block in the epithelial to mesenchymal transition (EMT) that these cells normally undergo to enablemigration. Also, the block in EMT could be mediated by expression of the PDZ-binding C-terminal tailalone, but not by the adjacent C2 domain (Leslie et al., 2007).

In attempts to define the signalling pathways affected by PTEN’s protein phosphatase activity,several studies have identified effects on FAK phosphorylation (Park et al., 2002; Tamura et al., 1999),but this is not observed in all cell types in all studies (Liliental et al., 2000; Maier et al., 1999) and PTENG129E has also been shown potently to inhibit the expression of matrix metalloproteases (MMPs) (Parket al., 2002). A recent step forward in understanding these potential mechanisms was the observationthat both lipid and protein phosphatase activities contribute to the suppression by PTEN of the integrinstimulated activation of both rac1 and the Src family kinase, Fyn (Dey et al., 2008). This paper alsoshowed that expression of either wild-type or G129E PTEN or the Src family kinase inhibitor PP1inhibited glioblastoma cell migration (Dey et al., 2008).

Together these studies support a model (Fig. 1) by which PTEN can act to inhibit migration of severalcell types, but especially glioblastoma cells, through tyrosine dephosphorylation of a protein substratethat plays a role in the activation of FAK and Src family kinases upon integrin engagement (Dey et al.,2008; Park et al., 2002; Tamura et al., 1998; Tamura et al., 1999). It seems possible that other effects ofPTEN expression that appear to be mediated by its protein phosphatase activity, such as the inhibitionof EMT in the chick embryo primitive streak (Leslie et al., 2007), may also be caused by this mechanism.Such a model would also tie in with data suggesting that the protein phosphatase activity of PTEN couldbe responsible for the regulation by PTEN of cyclin D1 expression (Weng et al., 2001), a well defineddownstream target of integrin signalling (Walker and Assoian, 2005).

Other mechanisms, other substrates?

The regulation of the expression of the procoagulant, tissue factor (TF) in glioblastoma cells, by bothPTEN and hypoxia also implicates the protein phosphatase activity of PTEN as being significant (Ronget al., 2005). In cells in which TF expression had been induced by hypoxia, induced expression of eitherwild-type or G129E PTEN was able to suppress TF expression, whereas phosphatase dead G129R PTENwas not. Significantly, in this work, PTEN G129E expression had no effect on the phosphorylation ofeither Akt or the p42/p44 MAPKs (Rong et al., 2005).

A recent study identified an interaction between PTEN and the 5-HT2C serotonin receptor and,using PTEN G129E expression, implicated the protein phosphatase activity of PTEN in dephosphory-lating the 5-HT2C receptor (Ji et al., 2006). This study then went on to perform experiments usinga peptide derived from a polybasic loop within this receptor, which blocked the interaction of PTENwith the receptor in vitro. Used in rats, this peptide blocked the effects of D9-tetrahydrocannabinol(THC) on neuronal firing rate and blocked the behavioural effects of THC and nicotine in a placepreference test (Ji et al., 2006). Although this study was able co-immunoprecipitate the endogenousPTEN and 5-HT2C receptor proteins, specific regulated phosphorylation sites on the receptor were notidentified, and an indirect mechanism remains a possible explanation for these apparent effects on5-HT2C phosphorylation.

Direct dephosphorylation of b-catenin by PTEN has also been proposed, as part of an investigationinto the mechanism by which TGFb stimulation reduced cell–cell adhesion in pancreatic carcinomacells. TGFb stimulation led to a dissociation of PTEN from b-catenin and increased tyrosine phos-phorylation of the latter protein (Vogelmann et al., 2005). Based on experiments in which tyrosinephosphorylated b-catenin was incubated with immunoprecipitated PTEN and a reduction observed inthe b-catenin signal detected using a phosphotyrosine antibody, a direct mechanism was proposed(Vogelmann et al., 2005). Although these initial findings appear exciting, a more detailed mechanisticunderstanding must be a priority.

Autodephosphorylation versus other substrates?

It has been proposed that PTEN may autodephosphorylate its C-terminal phosphorylation sites(Vazquez et al., 2000), with specific data being presented regarding Thr383 phosphorylation andalso referred to above (Raftopoulou et al., 2004). In these experiments, expressing PTEN protein

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carrying unphosphorylatable acidic replacement mutations in the other known C-terminalphosphorylation sites, radioactive 32P phosphate was found at greatly elevated levels in catalyt-ically inactive PTEN relative to the catalytically active enzyme (Raftopoulou et al., 2004). Thesedata fit into a model for PTEN regulation in which the C-terminal tail of PTEN, when phosphor-ylated, binds into the phosphatase and C2 domains, resulting in a closed conformation that is bothmore stable and less biologically active (Vazquez et al., 2001). Although this model has been wellsupported by other subsequent data (Leslie and Downes, 2004), precise molecular details arelacking, in part because the crystal structure of PTEN lacks this C-terminal tail (Lee et al., 1999). Inseveral studies, in agreement with the model of Raftopoulou, non-enzymatic effects of PTENexpression have been identified that can be mediated by the naked C-terminal tail and/or appearto rely upon this open conformation, in support of the significance of autodephosphorylation(Leslie et al., 2007; Odriozola et al., 2007; Raftopoulou et al., 2004). However, these studies haveinvariably relied upon over-expression studies and have not excluded additional effects mediatedby the protein phosphatase activity of PTEN on other substrates. Thus, it is very difficult in thesecases to distinguish between effects mediated by the protein phosphatase activity throughautodephosphorylation, or through action upon other substrates (and in most other studies thesedistinct possibilities are not addressed). However, in some experimental models these possibili-ties have been investigated and the results strongly implicate activity against other substrates(Ning et al., 2006). It should also be noted that further phosphorylation sites have been identifiedthat were not considered in some earlier studies (Al-Khouri et al., 2005; Maccario et al., 2007).

Summary

Many hundreds of research papers over the last ten years have established the significance ofPTEN’s lipid phosphatase activity in mediating many of its effects on specific cellular processes inmany different cell types, including cell growth, proliferation, survival, and migration (Backman et al.,2002; Iijima et al., 2002; Leslie and Downes, 2002; Salmena et al., 2008). In some cases, detailedsignalling mechanisms have been identified by which these PtdInsP3-dependent effects are manifest(Kolsch et al., 2008; Manning and Cantley, 2007; Tee and Blenis, 2005). Further, in some settings, invivo data from, for example genetic deletion of PTEN, relates closely with independent manipulationof the PI3K/Akt signalling pathway (Bayascas et al., 2005; Chen et al., 2006; Crackower et al., 2002;Ma et al., 2005). Together these studies indicate that the dominant effects of PTEN function aremediated through its regulation of PtdInsP3-dependent signalling, but that its protein phosphataseactivity also contributes in some settings. These conclusions are of great importance given the intenseefforts underway to develop PI3K (EC 2.7.1.153) inhibitors as cancer therapeutics. The experimentsreviewed here have firmly established that the protein phosphatase activity of PTEN plays a role inthe regulation of cellular processes including migration. On the other hand, it has not been estab-lished beyond doubt that PTEN acts on substrates other than itself; no such substrates have beenconfidently identified and effector mechanisms for PTEN’s protein phosphatase activity are currentlyunclear. The goal for future research must be firstly to understand the signalling mechanisms bywhich PTEN protein phosphatase activity acts: whether this is through identifying substrates, orworking out how autodephosphorylation mediates its effects. Secondly, and critically, the significanceof PTEN’s protein phosphatase activity must be established in vivo. This can be achieved throughrelating the phenotypes intervening with both PTEN and with protein phosphatase effector pathwayswhen they are identified, and through the generation of mouse models expressing substrate selectivePTEN mutants. We should then be able to answer the important question of whether PTEN’s proteinphosphatase activity contributes to tumour suppression.

Acknowledgements

NRL is a Research Councils UK academic fellow and conducts research supported by the Associationfor International Cancer Research. Research in the Inositol Lipid Signalling laboratory is funded by theUK Medical Research Council, and a consortium of pharmaceutical companies comprising Astra Zeneca,Boehringer Ingelheim, GlaxoSmithKline, Merck-Serono and Pfizer.

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