Acute regulation of the tumour suppressor ...

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Biochemical Society Transactions (2004) Volume 32, part 2

Acute regulation of the tumour suppressor phosphatase, PTEN, by anionic lipids and reactive oxygen species C.P. Downes1 , S. Walker, G. McConnachie, Y. Lindsay, I.H. Batty and N.R. Leslie Division of Cell Signalling, School of Life Sciences, MSI/WTB Complex, University of Dundee, Dundee DD1 5EH, U.K.

Abstract PTEN (phosphatase and tensin homologue deleted on chromosome 10) is a member of the protein tyrosine phosphatase family that is structurally adapted to facilitate the metabolism of 3-phosphoinositide lipid second messengers, especially PtdIns(3,4,5)P3 . Cellular PTEN activity is restrained by the retention of C-terminally phosphorylated enzyme in the cytosol. Dephosphorylation by as yet undefined phosphatases initiates an electrostatic switch which targets PTEN specifically to the plasma membrane, where it binds through multiple positively charged residues in both the C2 and N-terminal domains and is susceptible to feedback regulation through proteolytic degradation. PTEN also forms signalling complexes with PDZ domaincontaining adaptors, such as the MAGUK (membrane-associated guanylate kinase) proteins, interactions which appear to be necessary for metabolism of localized pools of PtdIns(3,4,5)P3 involved in regulating actin cytoskeleton dynamics. TPIP [TPTE (transmembrane phosphatase with tensin homology) and PTEN homologous inositol lipid phosphatase] is a novel gene product which exists in multiply spliced forms. TPIPα has PtdIns(3,4,5)P3 3-phosphatase activity and is localized to the endoplasmic reticulum, via two transmembrane spanning regions, where it may metabolize PtdIns(3,4,5)P3 that appears to be unaffected by expressed PTEN. PTEN can be acutely regulated by oxidative stress and by endogenously produced reactive oxygen species. This mechanism provides a novel means to stimulate phosphoinositide 3-kinase-dependent signalling pathways, which may be important in circumstances where PtdIns(3,4,5)P3 and oxidants are produced concurrently.

Introduction Type I PI 3-kinases (phosphoinositide 3-kinases) convert PtdIns(4,5)P2 into PtdIns(3,4,5)P3 , a lipid second messenger. The PI 3-kinase signalling pathway regulates diverse cellular responses, including cell survival, proliferation, growth and motility, as well as many of the metabolic and transcriptional responses to insulin [1]. These responses are initiated by the specific binding of PtdIns(3,4,5)P3 to proteins that possess an appropriate phosphoinositide binding domain, most commonly a PH (pleckstrin homology) domain [2]. One of the most completely understood examples is the PI 3kinase-dependent activation of PKB (protein kinase B/Akt) by PDK1 (phosphoinositide-dependent kinase 1). Both PKB and PDK1 possess PH domains that bind PtdIns(3,4,5) P3 and are essential for the efficient phosphorylation and hence activation of PKB. PKB and PDK1 each phosphorylate

Key words: intracellular targeting, PtdIns(3,4,5)P3 , PTEN (phosphatase and tensin homologue deleted on chromosome 10), reactive oxygen species, TPIP (TPTE and PTEN homologous inositol lipid phosphatase), TPTE (transmembrane phosphatase with tensin homology). Abbreviations used: ER, endoplasmic reticulum; MAGUK, membrane-associated guanylate kinase; PDK, phosphoinositide-dependent kinase; PH, pleckstrin homology; PI 3-kinase, phosphoinositide 3-kinase; PKB, protein kinase B; PTEN, phosphatase and tensin homologue deleted on chromosome 10; ROS, reactive oxygen species; TPIP, TPTE and PTEN homologous inositol lipid phosphatase; TPTE, transmembrane phosphatase with tensin homology. 1 To whom correspondence should be addressed (e-mail [email protected]).

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a number of targets that mediate many of the cellular responses noted above [3]. Not surprisingly, therefore, PtdIns(3,4,5)P3 is subject to tight regulatory control of both its synthesis and its subsequent metabolism. These processes are frequently deregulated in tumours, the significance of which was given great impetus by the discovery that the tumour suppressor PTEN (phosphatase and tensin homologue deleted on chromosome 10) is a PtdIns(3,4,5)P3 3-phosphatase. It now appears that, in many cells, PTEN mediates the principal route of PtdIns(3,4,5)P3 metabolism and acts to antagonize signalling through the PI 3-kinase pathway [4–7]. Despite intensive efforts, however, relatively little is known about the regulation of PTEN activity. It seems likely that PTEN is constitutively active in cells, and its abundance can be regulated transcriptionally [8,9] and through phosphorylationdependent modulation of protein stability [10–12]. More specifically, PTEN may be recruited to substrate-rich membranes via protein–protein or protein–lipid interactions which, in some cases, appear necessary for catalytically active PTEN to function effectively in cells. In the present paper we review (i) the kinetic behaviour of PTEN, emphasizing the importance of non-substrate lipid binding domains, (ii) the intracellular targeting of PTEN, and (iii) the expanding family of PTEN-related gene products, and we also discuss

PI-3 Kinase in Signalling and Disease

Figure 1 Domain structures and regulatory motifs of PTEN and TPIPα PTEN comprises an N-terminal catalytic domain and a C-terminal C2 domain that have extensive contacts, as revealed in the three-dimensional structure of the protein [13]. At the extreme N-terminus is a conserved anionic lipid binding motif. The active site contains a reactive cysteine residue that forms a covalent intermediate with substrate and is susceptible to oxidation by ROS. Redox regulation of PTEN is possible owing to the reversible nature of this oxidation process, which involves the formation of a disulphide bond with a neighbouring cysteine residue. The extreme C-terminus contains several serine/threonine residues that retain PTEN in the cytosol when these residues are phosphorylated. Dephosphorylation appears to be required to target PTEN to the plasma membrane, where it gains access to its substrate, the lipid second messenger PtdIns(3,4,5)P3 . Some functions of PTEN require the targeting of PTEN to protein complexes via the PDZ domain binding motif at the extreme C-terminus of the protein. TPIPα retains several critical features of the PTEN protein and thus has PtdIns(3,4,5)P3 3-phosphatase activity. In addition TPIPα has an N-terminal extension with two membrane-spanning domains that localize the enzyme to the ER [inset at bottom left; reproduced with permission from Walker, Downes and Leslie (2001) Biochem. J. 360, 277–283]. Further details and references are given in the body of the text. GFP, green fluorescent ptotein; PDI, protein disulphide isomerase.

data suggesting that PTEN can be acutely regulated by cellular oxidants.

Interfacial behaviour of PTEN PTEN is a member of the dual-specificity protein phosphatase family, and the discovery that its major physiological substrates are lipid second messengers came initially as something of a surprise. PTEN, however, has several structural features which favour its use of substrates that are integral components of cell membranes [13]. It has a highly basic active-site pocket, that is both wider and deeper than in related phosphatases and thus favours

inositol polyphosphate-containing substrates. As illustrated in Figure 1, PTEN has its catalytic domain at the N-terminus, with a distinct Ca2+ -independent C2 domain towards the C-terminus. The latter can bind to phospholipid vesicles in vitro. We used unilamellar vesicles containing substrate lipids in a background of phosphatidylcholine to study the kinetic behaviour of PTEN [14]. Under these conditions, PTEN greatly preferred PtdIns(3,4,5)P3 over other 3-phosphoinositides and water-soluble substrates such as Ins(1,3,4,5)P4 . These differences could be attributed to the interfacial binding properties of the enzyme, as activity was found to be dependent on the surface concentration of the substrate lipid as well as its bulk concentration. The interfacial C 2004

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K m (defined as the surface concentration giving half maximal activity at non-limiting bulk substrate concentration) was found to be particularly low for PtdIns(3,4,5)P3 , reflecting the small proportion of this lipid in cellular membranes. Moreover, the rate enhancement comparing a lipid substrate with its cognate headgroup could not be explained simply in terms of enzyme ‘scooting’ on a lipid surface, implying that the interfacial binding step favourably orientates the active site or allosterically stimulates enzyme activity. The C2 domain of PTEN contains a highly basic sequence which has been proposed to be required for efficient binding to membranes and vesicles. Mutation of this sequence (K263 MLKKDK269 ) by replacing all the lysines and the methionine residue with alanine (PTEN M-CBR3) reduced binding to phospholipid vesicles as well as the biological activity of PTEN [13]. By contrast, PTEN M-CBR3 displays enhanced activity with soluble substrates both in vitro and in vivo [15]. A kinetic analysis of the M-CBR3 mutant revealed that the values of interfacial K m and V max were unaffected, whereas K s (the interfacial binding constant) was significantly increased [14]. Thus structural, binding and kinetic data all support the conclusion that the C2 domain of PTEN is required for efficient metabolism of lipid substrates through non-catalytic interfacial binding. The kinetic analyses also revealed that PTEN activity could be stimulated severalfold by seeding phosphatidylcholine vesicles with small amounts of non-substrate anionic lipids. Of the lipids tested, PtdIns(4,5)P2 was much the most effective and potent, but even phosphatidylserine could induce a large stimulation of enzyme activity. Surprisingly, this effect was not attributable to the C2 domain, but to a polybasic region at the extreme N-terminus of the protein suggested previously to be a putative PtdIns(4,5)P2 binding motif (C.P. Downes, S. Walker, G. McConnachie, Y. Lindsay, I.H. Batty and N.R. Leslie, unpublished work). As this polybasic motif is mutated in some human tumours, it is the subject of continuing investigation in our laboratory.

Intracellular targeting of PTEN Most studies of the intracellular localization of ectopically expressed or endogenous PTEN suggest that the protein is largely cytosolic, compatible with the idea that the noncatalytic membrane binding is of relatively low affinity. We have examined the subcellular localization of PtdIns(3,4,5)P3 , the major substrate of PTEN, using the PH domain of GRP1 (general receptor for phosphoinositides 1) as a probe, by quantitative immunogold electron microscopy, and found that it is present in a number of intracellular membranes as well as in the plasma membrane. Surprisingly, overexpression of PTEN reduced the PtdIns(3,4,5)P3 content of the plasma membrane, but not of other membrane pools of this lipid (C.P. Downes, D. McCoull and J. Lucocq, unpublished work). This result suggests that functionally active PTEN is targeted preferentially to plasma membranes, perhaps as a result of the interactions with anionic lipids (see above), C 2004

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which are relatively abundant in the inner leaflet of the plasma membrane. Recent results have thrown new light on the mechanisms underpinning the membrane recruitment of PTEN. Several studies have indicated that the C-terminal tail of PTEN is constitutively phosphorylated on five residues, possibly by the protein kinase CK2 [10–12]. Mimicking the dephosphorylation of the C-terminal tail, by substituting alanine for Ser380 , Thr382 and Thr383 , greatly enhanced binding to anionic vesicles in vitro and gave a pronounced plasma membrane localization when C-terminally green fluorescent protein-tagged proteins were expressed in HEK293 cells [16]. The results suggest that phosphorylated PTEN is mainly cytosolic. Dephosphorylation was suggested to cause a conformational change, resulting in an electrostatic switch due to the exposure of clusters of positively charged amino acids in both the C2 and N-terminal domains, that targets PTEN selectively to plasma membranes. The membraneassociated form of PTEN appears to be especially sensitive to degradation by proteases, thus explaining the earlier observation that dephosphorylated PTEN is turned over rapidly [16]. The protein phosphatases responsible for initiating this electrostatic switch remain to be identified, but one exciting possibility is that they could be subject to auto-dephosphorylation by the protein phosphatase activity of PTEN itself. The specific targeting of functionally active PTEN to the plasma membrane appears to be the result of protein–lipid interactions involving several clusters of positively charged amino acids. Since PTEN needs to be dephosphorylated to be targeted to plasma membranes, another possibility is that the relevant protein serine/threonine phosphatases are located there and not in intracellular membranes. PTEN also has the potential to be targeted to signalling complexes via protein–protein interactions. The extreme C-terminus of PTEN contains a consensus site for binding to PDZ domain-containing proteins. PTEN can thus interact with a number of such proteins, including the MAGUK (membrane-associated guanylate kinase) proteins, DLG (mammalian discs large), MAGI-1 (MAGUK with inverted orientation), -2 and -3, the microtubule-associated serine/threonine protein kinase MAST-205, and MUPP1 (multiPDZ-domain protein 1) [17–19]. We studied the biological importance of C-terminally mediated protein–protein interactions of PTEN using a truncation mutant lacking the last five C-terminal amino acids. The results showed that the PDZ domain binding sequence was dispensable for efficient downregulation of cellular PtdIns(3,4,5)P3 levels and several components of PI 3-kinase signalling pathways, including activation of PKB and p70S6 kinase. However, events involving PI 3-kinase-dependent regulation of the actin cytoskeleton, such as platelet-derived growth factor-induced membrane ruffling in Swiss 3T3 cells and cell spreading in PTEN null U87MG cells, did require an intact extreme C-terminus. Overall, the results suggest that a small, localized pool of PtdIns(3,4,5)P3 regulates the remodelling of the actin cytoskeleton required for membrane ruffling and cell

PI-3 Kinase in Signalling and Disease

spreading, and that the metabolism of such a functionally specific pool of this lipid requires precise targeting of PTEN via an interaction of the C-terminus with a PDZ domaincontaining adaptor protein [20,21]. As a small, but significant, percentage of glioblastomas carry mutations in the extreme C-terminus of PTEN, we analysed the phosphorylation state of PKB in one such tumour sample. As predicted, PKB phosphorylation remained suppressed, implicating the deregulation of PI 3-kinase-dependent processes other than PKB in the development of some tumours [21].

PTEN-related genes In an extensive database search, we identified, cloned, expressed and characterized two human homologues of PTEN (see Figure 1). These differ with respect to their subcellular location and lipid phosphatase activities. The previously cloned, but not characterized, testis-specific gene product TPTE (transmembrane phosphatase with tensin homology) [22] is localized to the plasma membrane, but appears to lack detectable phosphoinositide 3-phosphatase activity. A protein that was suggested to be the murine orthologue of TPTE (termed mPTEN) was also found to be testis-specific, was active as a phosphoinositide 3-phosphatase and appeared to be localized to the Golgi [23]. TPIP (TPTE and PTEN homologous inositol lipid phosphatase), on the other hand, occurs in several differentially spliced forms, of which two, TPIPα and TPIPβ, appear to be functionally distinct. TPIPβ lacked any detectable phosphoinositide 3-phosphatase activity and was localized in the cytosol. Interestingly, TPIPα displayed very similar phosphoinositide 3-phosphatase activity and specificity compared with PTEN itself in vitro and has two putative transmembrane spanning domains N-terminal to the catalytic domain [24]. Ectopically expressed TPIPα was associated with intracellular membranes and co-localized with an ER (endoplasmic reticulum) marker. Orientation studies, using protease protection assays, place the catalytic domain of TPIPα on the cytosolic surface of the ER (C.P. Downes, S. Walker, G. McConnachie, Y. Lindsay, I.H. Batty and N.R. Leslie, unpublished work). As our PtdIns(3,4,5)P3 localization studies identified significant quantities of this lipid in the ER and, as noted above, overexpression of PTEN did not affect the ER pool, we speculate that TPIPα may specifically metabolize PtdIns(3,4,5)P3 in the ER. Experiments to address this and hence to understand the functions of PtdIns(3,4,5)P3 in endomembranes are currently under way. TPTE, TPIPβ and several other TPIP splice variants are predicted to be catalytically inactive. Similar diversity exists elsewhere within the protein tyrosine phosphatase superfamily. It will therefore be important to establish whether any of the inactive variants of TPIP have cellular functions, for example by exerting dominant negative effects through either substrate binding or disruption of protein– protein interactions, as in the myotubularin family of enzymes [25].

Redox control of PTEN activity

ROS (reactive oxygen species), such as hydrogen peroxide, are damaging by-products of an aerobic lifestyle that also play important regulatory roles in eukaryotic cells [26,27]. For example, many growth factors stimulate the production of oxidants which appear to contribute to mitogenic responses. Relatively little is known, however, about the molecular mechanisms underpinning redox-dependent signalling processes. Members of the protein tyrosine phosphatase family, which includes PTEN, are good candidates for mediating some of the signalling functions of ROS. They all possess a highly reactive cysteine residue within their active sites that participates directly in catalysis, via a covalent intermediate, and must be in the reduced state for this to occur. There is increasing evidence for physiological regulation of certain protein tyrosine phosphatases, not only in response to severe oxidative stress (induced for example by exposure of cells to high concentrations of exogenous oxidants), but also by endogenous ROS produced in response to growth factors [28–30]. More recently, inactivation of PTEN was demonstrated in the presence of hydrogen peroxide which caused the formation of a disulphide bond between the activesite cysteine (Cys124 ) and Cys71 . Importantly, this process was reversible, and hence might function physiologically to regulate PTEN activity [31]. We adapted an assay procedure, originally described by Meng et al. [30], that allows the proportions of PTEN in the oxidized and reduced states at the point of cell lysis to be determined. This uses iodoacetic acid as an alkylating agent which binds covalently only to the reduced form of the enzyme. Subsequent immunoprecipitation and assay in standard reducing conditions fails to detect any PTEN that was in the active, reduced state at cell lysis in such conditions, but does detect cellular PTEN that was in the oxidized state, owing to it being protected from alkylation. This assay allowed us to show that addition of exogenous hydrogen peroxide to Swiss 3T3 cells could completely inactivate cellular PTEN. By using a PTEN null cell line (U87MG) with and without expression of exogenous PTEN, it was possible to show that activation of the PI 3-kinase pathway by oxidative stress was entirely dependent on PTEN expression. This is important, because it establishes that PI 3-kinase signalling is not being initiated by an upstream event such as inhibition of a tyrosine phosphatase causing hyperphosphorylation and hence activation of a receptor tyrosine kinase [32]. The production of ROS by the phagocytic NADPH oxidase complex in myeloid cells such as neutrophils and macrophages has been studied extensively [33,34]. To establish if PTEN is subject to redox regulation in response to the physiological production of endogenous ROS, we used the macrophage cell line RAW264.7. Stimulation of these cells with either phorbol ester (PMA) or lipopolysaccharide inactivated a small proportion (approx. 15%) of cellular PTEN, an effect that was reversed by an antioxidant (N-acetylcysteine) or an inhibitor of the NADPH oxidase (diphenyleneiodonium chloride). Lipopolysaccharide, PMA C 2004

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and insulin were all able to stimulate PI 3-kinase signalling in macrophages, as indicated by the phosphorylation and activation of PKB. Interestingly these responses were completely (PMA), partially (lipopolysaccharide) or not at all (insulin) sensitive to treatment with N-acetylcysteine or diphenyleneiodonium chloride, indicating both the lack of overt toxicity of these reagents and the variable role of ROS in response to these different treatments [32]. Overall, the results suggest that PTEN is a likely important physiological target for the effects of oxidative stress and endogenously produced ROS. Inhibition of PTEN represents a novel means of initiating PI 3-kinase signalling, bypassing the normal receptor-dependent activation of PI 3-kinases themselves. This may have important implications for circumstances in which PtdIns(3,4,5)P3 and oxidants are generated concurrently.

Conclusions PTEN plays a vital role in regulating the activity of a key second messenger, and its status as a tumour suppressor that is mutated in multiple human tumours is linked to the role of its substrate, PtdIns(3,4,5)P3 , in growth control, cell motility and the inhibition of apoptosis. While the structural and mechanistic basis of PTEN activity and substrate specificity is now well understood, the challenge has moved towards understanding its regulation. Inhibition by cellular oxidants seems likely to be one such mechanism, with relevance to the normal production of ROS in response to growth and chemotactic factors as well as to their pathological generation in relation to diseases such as stroke and in some tumours. This review has concentrated on the phosphoinositide 3-phosphatase activity of PTEN, as this has been crucially linked to its tumour suppressor function. It remains possible that some of the functions of PTEN require its protein phosphatase activity, a possibility that has received relatively little recent attention.

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