Distinct inactivation of PI3K signalling by PTEN and 5-phosphatases

Advances in Enzyme Regulation 52 (2012) 205–213

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Distinct inactivation of PI3K signalling by PTEN and 5-phosphatases Nick R. Leslie*, Miles J. Dixon, Martijn Schenning, Alex Gray, Ian H. Batty Division of Cell Signalling and Immunology, College of Life Sciences, University of Dundee, Dundee DD1 5EH, UK

Introduction In multicellular organisms, cell growth, survival and proliferation are each tightly and often co-ordinately controlled by extracellular signals along with intrinsic programmes. The dysregulation of these processes is also a key step in the evolution of cancer. A key mechanism by which cells interpret extracellular signals that stimulate cell growth and proliferation is carried out by a family of enzymes called the Class I Phosphoinositide 3-Kinases or PI3Ks (EC 2.7.1.153). The PI3Ks are tightly regulated enzymes that become activated when cells are exposed to any one of a range of stimuli, including many different growth factors, hormones, cytokines and extracellular membrane components. The class I PI3Ks phosphorylate the 3-position of the inositol ring of phosphatidylinositol 4,5-bisphosphate, also known as PtdIns(4,5)P2, to generate phosphatidylinositol 3,4,5-trisphosphate, also known as PtdIns(3,4,5)P3 or PIP3. In turn, downstream signalling is propagated through the binding of the PIP3 lipid to a diverse group of proteins that includes the oncogenic AKT protein kinases and that together play important roles in controlling cell behaviour. Interest has particularly fallen upon PI3K signalling from research communities studying cancer and diabetes, due to the critical role of enhanced PI3K signalling in many tumours and the significance of this signalling pathway in determining a cell’s response to insulin. There are also two other groups of PI 3-kinases, the class II and III enzymes, that utilise different phosphoinositide substrates, fulfil different cellular functions and will not be further considered here (Engelman et al., 2006). Two routes of PIP3 metabolism: 5-phosphatases or 3-phosphatases? It is clear that the synthesis of PIP3 by PI3Ks is the key step by which these enzymes control cell behaviour. However, once synthesised, PIP3 is seen to be metabolised by removal of a phosphate group either from the 3 position or from the 5 position. The first reaction is catalysed by the tumour suppressor, PTEN (EC 3.1.3.67), the only confirmed cellular PIP3 3-phosphatase (Salmena et al., 2008). Removal of phosphate from the 3-position of PIP3 by PTEN generates PtdIns(4,5)P2, and since the latter lipid is present in cells in great excess of PIP3, this reaction simply removes the PIP3 signal. Removal of * Corresponding author. Tel.: þ44 1382 386263. E-mail address: [email protected] (N.R. Leslie). 0065-2571/$ – see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.advenzreg.2011.09.010

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phosphate from the 5-position of PIP3 is carried out by a family of PI 5-phosphatases, the best recognised being SHIP1 and the more widely expressed SHIP2 (Rohrschneider et al., 2000). A key point regarding the activity of these 5-phosphatases is that this activity does not simply remove PIP3, but that the reaction product, PtdIns(3,4)P2, represents a distinct signal, with its own independent signalling roles (Downes et al., 2007). In this way, activation of PI3K usually leads to rapid increases in levels of both PIP3 and PtdIns(3,4)P2 and both lipids are recognised and bound by an overlapping, but distinct groups of proteins. Therefore both PIP3 and PtdIns(3,4)P2 must be considered to be the downstream lipid mediators of PI3K signalling, but despite a great deal of intensive research, we still have a rather poor picture of how the two PI3K lipid products contribute to the biological effects of PI3K activation. In this review we will discuss recent developments that have highlighted the importance of PtdIns(3,4)P2 and provide additional insight into how this PI3K lipid code is interpreted (Fig. 1). Inactivation of PI3K signalling by lipid phosphatases Removal of PIP3 by the tumour suppressor PTEN PTEN was identified simultaneously by two research groups as a candidate tumour suppressor in 1997 (Li et al., 1997; Steck et al., 1997). Although it is a divergent member of the large Protein Tyrosine Phosphatase (PTP) family, it was rapidly shown that its principal substrates are phosphoinositide lipids and that its tumour suppressor activity is thus mediated by inhibition of PI3K-dependent signalling (Maehama and Dixon, 1998; Myers et al., 1998; Stambolic et al., 1998). Subsequent detailed kinetic analysis shows that in vitro, PTEN strongly favours PIP3 over PtdIns(3,4)P2 by a factor of around 200 fold (McConnachie et al., 2003). It seems likely that in vivo, PTEN metabolises exclusively PIP3, and that the formation of PtdIns(3,4)P2 largely from PIP3 is responsible for observations that PTEN activity usually results in lower levels of both lipids. Whereas the PI3K enzymes themselves have very low activity in unstimulated cells, and display tightly regulated activation (Hawkins et al., 2006), it appears that PTEN is constitutively active and acts to maintain PIP3 (and therefore also PtdIns(3,4)P2) at a low level (Leslie and Foti, 2011). There have been a number of proposals that following receptor activation PTEN activity may be transiently inhibited by mechanisms such as oxidation, phosphorylation and ubiquitination, favouring PIP3 accumulation (Leslie and Foti, 2011). However, the physiological relevance of these PTEN regulatory mechanisms is currently unclear. It is probably this constitutive activity maintaining low PIP3 levels that is key to PTEN’s status as one of the most frequently mutated tumour suppressors in human cancer (Salmena et al., 2008).

Conversion of PIP3 to PtdIns(3,4)P2 by the SHIP 5-phosphatases There is a family of 10 inositol 5-phosphatases encoded within the human genome (Ooms et al., 2009). Although the substrate specificity of this diverse family extends to water soluble inositol

Fig. 1. A well-supported model for PI3K signalling is represented. Regulated activation of Class I PI3K enzymes converts PtdIns(4,5)P2 to PtdIns(3,4,5)P3 (PIP3). Metabolism of PIP3 by PTEN or by 5-phosphatases which generate PtdIns(3,4)P2 (here PI(3,4)P2) is shown. PtdIns(3,4)P2 is then metabolised by INPP4 4-phosphatases to the relatively abundant PtdIns(3)P. In all names here, PtdIns is abbreviated to PI.

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phosphates, most are phosphoinositide lipid phosphatases and several are capable of regulating cellular PIP3 levels, including the SKIP and INPP5E phosphatases. INPP5E, for example, appears to play a highly localised role in the regulation of phosphoinositide signalling, probably via PIP3 metabolism, specifically in the primary cilium (Bielas et al., 2009; Jacoby et al., 2009). However, it is the SHIP enzymes, the largely hematopoietic SHIP1 and the very widely expressed SHIP2, that have been most clearly demonstrated to be cellular PIP3 5-phosphatases and regulators of PI3K dependent signalling. They are both relatively large enzymes of around 145 kD which, in addition to their magnesium dependent catalytic domain, have in common an N-terminal phospho-tyrosine binding SH2 domain, as well as several poly-proline motifs and sites of tyrosine phosphorylation. SHIP2 also contains a Cterminal SAM domain. It was through phosphorylation by tyrosine kinase mediated signalling that SHIP1 was simultaneously identified by several groups (Rohrschneider et al., 2000), and increased activation of SHIP2 by tyrosine phosphorylation has been subsequently demonstrated (Batty et al., 2007). In accord with this evidence for stimulus driven activation of SHIP activity, these enzymes have been shown to be recruited into activated receptor signalling complexes (Backers et al., 2003) and appear to play a role to modulate stimulated PI3K signalling towards PtdIns3,4P2 rather than provide ongoing suppression of PIP3 levels (Blero et al., 2005; Liu et al., 1999; Sleeman et al., 2005). As would be predicted, the effects of SHIP activity on levels of PIP3 and PtdIns(3,4)P2 appear strongly influenced by the simultaneous activities of PI3K, PTEN and enzymes that remove metabolise PtdIns(3,4)P2 (possibly dominated by the INPP4 phosphatases). The INPP4 enzymes remove PtdIns(3,4)P2 and inhibit AKT phosphorylation In the mid-1990s a series of papers from Norris and Majerus characterised the type I and II inositol polyphosphate 4-phosphatases, now more commonly known by their gene names, INPP4A and INPP4B respectively (Norris et al., 1997; Norris and Majerus, 1994). These were purified in vitro as PtdIns(3,4)P2 phosphatases, although it took some years for data supporting this activity to emerge from cellular experiments (Gewinner et al., 2009; Sasaki et al., 2010; Shin et al., 2005). The INPP4 enzymes remove phosphate from the 4-position of PtdIns(3,4)P2 to form PtdIns(3)P which, given the greater abundance of PtdIns(3)P and alternate routes of its synthesis, has a much greater effect on cellular levels of PtdIns(3,4)P2 than on PtdIns(3)P levels. Interest in INPP4B has increased greatly in the last few years, since the presentation of data suggesting that it may be an important tumour suppressor (Gewinner et al., 2009). It has now been shown that in breast and prostate cancer cells, knockdown of INPP4B increases the activating phosphorylation of the AKT oncogenic protein kinases and that manipulation of INPP4B expression can also affect cell proliferation, colony formation in culture and in vivo tumorigenesis in xenograft experiments (Fedele et al., 2010; Gewinner et al., 2009; Hodgson et al., 2011). Combined with data from breast and prostate tumour samples revealing frequent deletion of one copy of INPP4B gene and loss of INPP4B protein expression (Fedele et al., 2010; Gewinner et al., 2009; Hodgson et al., 2011; Taylor et al., 2010), it now appears that both INPP4B and PTEN suppress tumour formation through their inhibition of PI3K signalling (Agoulnik et al., 2011). These data are in some contrast to the type I 4-phosphatase, since deletion of this gene in mice, Inpp4a, causes lethal excitotoxic neuronal death and elevated levels of PtdIns(3,4)P2 in the striatum (Sasaki et al., 2010) and Inpp4a null MEFs display elevated AKT phosphorylation (Ivetac et al., 2009). It is currently unclear whether INPP4A plays a tumour suppressing role in any tissues, and if not, what the functional differences are, between these two widely expressed and similar-looking enzymes. Overlapping but distinct functions for PIP3 and PtdIns(3,4)P2 Analysis of the functions of the enzymes that synthesise and metabolise PIP3 and PtdIns(3,4)P2 supports the existence of separate functions for these two lipids; in particular the distinct phenotypes observed in mice lacking Inpp4a, Ship and Ship2 when compared to the may mice lines lacking Pten in particular tissues (Liu et al., 1999; Sasaki et al., 2010; Sleeman et al., 2005; Suzuki et al., 2008). However, the clearest demonstration of the distinct biological functions fulfilled by the two lipids comes from analysis of the lipid binding proteins that respond to changes in the levels of these lipids and in turn propagate signalling downstream.

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How are PIP3 and PtdIns(3,4)P2 functionally different? The PH domain code Regulated changes in the abundance or locations of cellular phosphoinositide lipids enforce their many effects on cellular behaviour through proteins that are able to selectively bind to each lipid and respond with altered function (Di Paolo and De Camilli, 2006). Studies through the 1990s established a model for the regulation of the AKT kinases as a paradigm for stimulus-driven lipid based signal transduction. In this model, the N-terminal pleckstrin homology (PH) domain of AKT is able to bind either PIP3 or PtdIns(3,4)P2 with relatively high affinity. Therefore, when activation of PI3K increases cellular levels of PIP3 and PtdIns(3,4)P2, the binding of AKT to these lipids appears sufficient to drive the movement or translocation of the kinase onto the inner leaflet of the plasma membrane, where it becomes co-localised with the protein kinase PDK1 (that also binds PIP3 and PI(3,4)P2) which phosphorylates and activates AKT (Alessi and Downes, 1998; Lemmon and Ferguson, 2000). Since this time, a wealth of data have emerged to support this model for PI3K-regulated protein translocation as a model for signal transduction, although it also appears that highly selective lower affinity phosphoinositide binding can also play a role in the regulation of proteins on membrane surfaces without driving translocation by avidity (Campa et al., 2009; Lemmon, 2008; Park et al., 2008; Welch et al., 2002). Estimates of the number of human proteins that are able to be regulated by PI3K through binding selectively to PIP3 and PtdIns(3,4)P2, range from around 20 up towards 100 (Park et al., 2008; Stephens and Hawkins, 2011). The great majority of these identified proteins bind to these lipids through a PH domain. The large PH domain family has 323 representatives in the human genome, occurring in 276 different gene products and being structurally related to PTB domains. Although many PH domains bind selectively to PIP3 and PI(3,4)P2, this is not universal, as PH domains have been identified that bind with high selectivity to non-PI3K product lipids such as PtdIns(4,5)P2 (that of PLCd1 (Harlan et al., 1994)) and PtdIns(4)P (FAPP1 (Godi et al., 2004)) and it has also been shown that many, probably most, PH domains have little or no measurable affinity for phosphoinositide lipids (Yu et al., 2004). In addition, examples of additional domains have been recently identified as selective PIP3 binders; a divergent C2 domain in DOCK180 (Premkumar et al., 2010), a novel domain (termed an aPI domain) in IQGAP1 (Dixon et al., 2011), a PX domain in PLD1 (Lee et al., 2005) and a domain termed a PPO domain in OGT (Yang et al., 2008). This work has helped fuel speculation regarding a larger PIP3-binding proteome (Fig. 2). The relative contributions of PIP3 and PtdIns(3,4)P2 to PI3K dependent signal transduction would appear to depend upon the distinct sets of proteins that are able to bind and be regulated by these lipids (Table 1). Several proteins have been proposed to bind with similar affinities to both PIP3 and to PI(3,4)P2, including the AKT kinases, SWAP70 and DAPP1. Whether or not PtdIns(3,4)P2 plays a physiological role in AKT regulation has been somewhat controversial for some years, since the finding that both lipids were similarly efficient at binding to the AKT PH domain (Frech et al., 1997; Kavran et al., 1998) and promoting its activation in vitro (Alessi et al., 1997). However, recent evidence, in particular the demonstration that the INPP4 PtdIns(3,4)P2 phosphatases affect AKT activity strongly support a role for both PIP3 and PtdIns(3,4)P2 in AKT regulation, although whether any distinction between the roles of the two lipids exists in unclear (Gewinner et al., 2009; Hodgson et al., 2011; Ma et al., 2008). A relatively large number of proteins have been identified that bind through pleckstrin homology domains to PIP3 with apparent specificity. These include PDK1 and the TEC/BTK group of kinases, several activating exchange factors for the RAC GTPases, including PRex1 and 2, Tiam-1, Vav and regulators of the ARF GTPases including several members of the Cytohesin and Centaurin families. A very small number of PH domain containing proteins have been proposed to bind with high selectively to PtdIns(3,4)P2. This is most clear for the TAPP1 and TAPP2 proteins, with the PH domain of TAPP1 having been used and validated in several studies as a selective experimental probe for PtdIns(3,4)P2 (Dowler et al., 2000; Manna et al., 2007; Watt et al., 2004). The biological significance of TAPP1 and 2 regulation by PtdIns(3,4)P2 binding has been shown through the generation of mice carrying mutations in both TAPP1 and TAPP2 genes and expressing mutant proteins unable to bind to phosphoinositides (Wullschleger et al., 2011). The enhanced insulin sensitivity observed in these mice supports a role for the TAPP proteins and PtdIns(3,4)P2 specifically in the feedback downregulation of PI3K signalling. Given the limited number of selective PtdIns(3,4)P2 binding proteins and the relatively

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Fig. 2. Phylogenetic tree of human PH domains. The amino acid sequence of 323 PH domain sequences found in 276 human proteins was obtained from SMART (Sep. 2010) and input for analysis on the basis of sequence divergence into Geneious Pro (version 5.4.6 www.geneious.com). The clustering of PH domains with well-established 3-phosphorylated lipid binding capability are indicated.

modest phenotype of the TAPP knockin mice, a tempting speculation regarding the function of this lipid is that it serves to maintain the activation of the AKT kinases without activating specifically PIP3 responsive proteins including many regulators of GTPase signalling. When attempting to assess the roles of these lipid based signalling pathways, it should be borne in mind that experiments to determine the phosphoinositide binding characteristics of proteins are technically challenging and there have been many examples of conflicting published data. Therefore, the proposed binding specificities of the many proteins for which only one or two experiments have been presented should be considered with some caution. Table 1 Reported selectivities of phosphoinositide binding proteinsa. PtdIns(3,4,5)P3

Both lipids

PtdIns(3,4)P2

Cytohesins (GRP1, ARNO) BTK/TEC/ITK PDK1 PREX1 and 2 ARAP1 and 3/Centaurin d TIAM1 SH3BP2 FGD6 VAV SOS IQGAP1 (aPI domain) PLD1 (PX domain) DOCK180 (C2 domain) OGT (PPO domain)

AKT1, 2 and 3 DAPP SWAP70 IRGM1 (amphipathic helix)

TAPP1 and 2 Lamellipodin

a

Lipid binding is mediated through a PH domain unless otherwise stated.

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Tumour suppression by PTEN and INPP4 The loss of function of PTEN through mutation and other mechanisms in a large fraction of epithelial derived cancers has been reviewed elsewhere (Carracedo et al., 2011; Leslie and Foti, 2011) and as discussed above, evidence for the loss of INPP4B function, largely through loss of a single gene copy and reduced protein expression is accumulating from several solid tumour types (Fedele et al., 2010; Gewinner et al., 2009; Hodgson et al., 2011; Taylor et al., 2010). However, loss of phosphatase function is only one mechanism by which downstream PI3K dependent functions can be promoted. The core components of the PI3K signalling pathway are almost all targets for mutations that drive the development of human cancers (Liu et al., 2009). Activating mutations in the p85a and p85b regulatory subunits and most frequently in the p110a catalytic subunit of PI3K have been identified in a range of tumour types (Urick et al., 2011; Vogt et al., 2007). Similarly, activating mutations in AKT1 and less frequently AKT3 have also been reported (Carpten et al., 2007; Davies et al., 2008) as have amplifications of the genes encoding several PI3K components, PDK1 and all 3 isoforms of AKT (Liu et al., 2009). When alterations in the upstream activators of PI3K are taken into account, such as the receptor tyrosine kinases, EGFR, HER2 and MET and the RAS GTPases, a broader PI3K signalling network appears to be the target of oncogenic activation in most patients with tumour types such as prostate, lung, breast and glioblastoma (Greulich, 2010; Korkola and Gray, 2010; Network, 2008; Taylor, et al., 2010). Together these data identify PI3K signalling as one of only a few functionally linked pathways that seem to be dysregulated in many and perhaps most tumours. The existence within the pathway of several protein and lipid kinases, that are considered to be druggable, has driven great interest in developing drugs targeting PI3K signalling. Many such compounds, specifically targeting class I PI3K itself, AKT, mTOR or PDK1 as well as upstream receptors, are now in clinical use or trials (Arteaga, 2010). Despite the wealth of evidence for the dysregulation of PI3K pathway components in cancer, there is still a good deal of uncertainty surrounding the downstream mechanisms by which these changes drive tumorigenesis. This is potentially problematic as several drug programmes target signalling components downstream of PI3K that may drive only specific tumours at best and at worst may further drive tumour development if inhibited, through relief of pathway feedback (Carracedo and Pandolfi, 2008). The identification of INPP4B as a tumour suppressor strongly implies a role for PtdIns(3,4)P2 signalling in the promotion of these tumours and would seem to be in accord with the role of this lipid in AKT activation. However, we are very much at the early stages of our understanding of mechanisms of tumour suppression by INPP4B. Analysis of cancer genomes shows that in general, co-occurrence in one tumour of multiple mutations within the same functional pathway is unusual (Copeland and Jenkins, 2009). However, within the PI3K network, multiple activating events within the same tumour are not uncommon (Yuan and Cantley, 2008). For example, in a glioblastoma genome study, mutations within the same pathway were observed to be mutually exclusive with p-values of 9.3  1010, 2.5  1013 and 0.022, respectively, for the p53, RB and PI3K-RTK pathways (Network, 2008). In agreement with these findings, the effects on tumour formation in vivo of reduced PTEN activity appear highly dose dependent and in contrast to classical models of tumour suppression (Alimonti et al., 2010; Berger et al., 2011). In this regard it seems relevant to consider that according to the current models of PI3K signalling discussed in this review, any reduction in a potential cancer cell of PTEN’s activity to remove PIP3 would be expected to require more PIP3 metabolism to be directed via 5-phosphatases to PI(3,4)P2 and the INPP4 enzymes. In this case, loss of INPP4 function would be expected to have a greater effect to elevate levels of PtdIns(3,4)P2 than in a cell with full PTEN activity and could in some cases contribute to a selection for the simultaneous loss of both activities. Data investigating whether loss of PTEN and INPP4B are related or independent have been reported for breast and prostate tumours (Fedele et al., 2010; Hodgson et al., 2011) and can be derived from publicly available data for other tumours such as glioblastoma and ovarian cancer (Table 2). The results of such analysis are unusual in that there is no evidence that loss of the two tumour suppressors is mutually exclusive, rather their losses appear either to occur independently, or even to be significantly associated as is found in two analyses of breast cancer (Table 1 and (Fedele et al., 2010; Gewinner et al., 2009; Hodgson et al., 2011)). The work reviewed here has started to provide us with an understanding of how PI3K signalling can co-ordinately control the many and diverse cellular processes that it influences. In the future it is to be

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Table 2 Loss of INPP4 and PTEN in tumour samples. Tumour

Low PTEN

Low INPP4

Low INPP4 Low PTEN

Expected if independent

Statistical significance

Ovarian (TCGA gene expressiona) Ovarian (TCGA LOHb) Glioma (TCGA gene expression) Breast (TCGA gene expression) Breast (IHCc Fedele et al., 2010)

242/506 67/494 312/424 42/185 73/267

299/506 119/494 353/424 46/185 71/267

150/506 24/494 272/424 17/185 36/267

143/506 16/494 260/424 10/185 19/267

NS

c2 p ¼ 0.024 NS

c2 p ¼ 0.023 c2 p < 0.0001

a

Reduced expression assessed by mRNA hybridisation microarray analysis (<0.5 log2 tumour/normal) (Network, 2008). Reduced gene copy number assessed by array comparative genomic hybridisation (<0.5 log2 tumour/normal) (Network, 2008). c Reduced protein level assessed by immunohistochemistry (Fedele et al., 2010). b

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