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Suppression of Cellular Proliferation and Invasion by the Concerted Lipid and Protein Phosphatase Activities of PTEN

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Suppression of Cellular Proliferation and Invasion by the Concerted Lipid and Protein Phosphatase Activities of PTEN Oncogene (2010) 29, 687–697 & 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 $32.00 www.nature.com/onc ORIGINAL ARTICLE Suppression of cellular proliferation and invasion by the concerted lipid and protein phosphatase activities of PTEN L Davidson1,4, H Maccario1,4, NM Perera1,4, X Yang2,3, L Spinelli1, P Tibarewal1, B Glancy1, A Gray1, CJ Weijer2, CP Downes1 and NR Leslie1 1Division of Molecular Physiology, College of Life Sciences, University of Dundee, Dundee, UK and 2Division of Cell and Developmental Biology, College of Life Sciences, University of Dundee, Dundee, UK PTEN is a tumour suppressor with phosphatase activity in vivo, seems to inhibit cell proliferation, survival and in vitro against both lipids and proteins and other potential growth and, in some cell types, motility (Keniry and non-enzymatic mechanisms of action. Although the Parsons, 2008; Salmena et al., 2008). PTEN is a lipid importance of PTEN’s lipid phosphatase activity in phosphatase that inhibits phosphoinositide 3-kinase regulating the PI3K signalling pathway is recognized, (PI3K)-dependent signalling, by metabolizing the PI3K the significance of PTEN’s other mechanisms of action is product, PtdInsP3. Although PTEN’s PtdInsP3 phos- currently unclear. In this study, we describe the systematic phatase activity appears to mediate many of its cellular identification of a PTEN mutant, PTEN Y138L, with effects, PTEN has additional potential mechanisms of activity against lipid, but not soluble substrates. Using this action, including robust protein phosphatase activity mutant, we provide evidence for the interfacial activation and proposed non-enzymatic mechanisms (Myers et al., of PTEN against lipid substrates. We also show that 1997; Mahimainathan and Choudhury, 2004; Rafto- when re-expressed at physiological levels in PTEN null poulou et al., 2004; Okumura et al., 2005; Shen et al., U87MG glioblastoma cells, the protein phosphatase activity 2007). Several studies have reported lipid phosphatase- of PTEN is not required to regulate cellular PtdInsP3 levels independent effects of PTEN affecting cell migration, or the downstream protein kinase Akt/PKB. Finally, in and also contributing to other functions potentially three-dimensional Matrigel cultures of U87MG cells related to tumour suppression (Park et al., 2002; similarly re-expressing PTEN mutants, both the protein Freeman et al., 2003; Gildea et al., 2004; Raftopoulou and lipid phosphatase activities were required to inhibit et al., 2004; Leslie et al., 2007; Shen et al., 2007; invasion, but either activity alone significantly inhibited Trotman et al., 2007). A wealth of correlative data proliferation, albeit only weakly for the protein phospha- connects the regulation of PI3K signalling by PTEN tase activity. Our data provide a novel tool to address the with its physiological functions and tumour suppressor significance of PTEN’s separable lipid and protein activity (Sansal and Sellers, 2004). However, given the phosphatase activities and suggest that both activities apparent number and diversity of both PTEN’s normal suppress proliferation and together suppress invasion. functions and of the effects of PTEN loss on tumour Oncogene (2010) 29, 687–697; doi:10.1038/onc.2009.384; development in different tissues (Chow and Baker, 2006; published online 16 November 2009 Suzuki et al., 2008), it seems quite possible that other mechanisms of action may account for some of PTEN’s Keywords: PTEN; PI3 kinase; phosphoinositide; cancer; functions and tumour suppressor activities in some TPIP; Akt tissues. Experiments to reveal the mechanisms and signifi- cance of PTEN’s separable activities have been greatly assisted by the use of functionally selective mutants. In Introduction particular, many studies have used PTEN G129E, a mutation that was identified in two Cowden disease PTEN is one of the most frequently lost tumour families (Liaw et al., 1997) and shows remarkable selec- suppressors in human cancers, which in culture and tivity, displaying greatly reduced lipid phosphatase activity while retaining full protein phosphatase activity in vitro (Furnari et al., 1998; Myers et al., 1998). In this Correspondence: Dr NR Leslie, Division of Molecular Physiology, University of Dundee, Dow Street, Dundee, Scotland DD1 5EH, UK. study we describe the systematic generation of a PTEN E-mail: [email protected] mutant with the converse specificity, that is, retaining 3Current address: Key Laboratory for Regenerative Medicine of the lipid phosphatase while lacking significant protein phos- Ministry of Education, Medical College, Jinan University, Guangzhou phatase activity. We then describe experiments using 510632, PR China. 4These authors contributed equally to this work. both mutants to reveal the contribution of these two Received 1 April 2009; revised 29 September 2009; accepted 6 October separable activities to PTEN’s activity in several cell- 2009; published online 16 November 2009 based assays of proliferation, invasion and migration. PTEN’s lipid and protein phosphatase activities L Davidson et al 688 Results tyrosine/glutamic acid polymer polyGluTyr-P. Second, despite its extremely close sequence similarity with The identification of a PTEN mutant with lipid but not TPIP, TPTE-reactivated displayed robust activity protein phosphatase activity against both the lipid PtdInsP3 and this peptide The proteins encoded in the human genome that are substrate polyGluTyr-P. This identification of a most closely related to PTEN are TPIP and TPTE. PTEN-related phosphatase lacking protein phosphatase These are expressed almost exclusively in the testis activity suggests that protein phosphatase activity may (Walker et al., 2001; Tapparel et al., 2003), although have been selected for during evolution. The protein their physiological functions are unclear. Our previous phosphatase activity displayed by TPTE-reactivated has studies of the lipid phosphatase activity of TPIP and interesting implications for TPTE function, but given its TPTE revealed that TPIP has similar PtdInsP3 phos- close relationship to TPIP, suggests a systematic phatase activity to PTEN, and that while TPTE lacks approach for the mutation of PTEN to produce a detectable activity in the assays used, it could be mutant enzyme with only lipid phosphatase activity. reactivated through mutation, producing an active Therefore, at each of the 11 residues at which TPIP phosphatase termed ‘TPTE-reactivated’ (Walker et al., differs from TPTE and PTEN through the phosphatase 2001; Leslie et al., 2007). However, when the protein domain and N-terminal region of the C2 domain, we phosphatase activity of these proteins was investigated, mutated the PTEN sequence to the corresponding we obtained two surprising results (Figure 1). First, in amino acid in TPIP (See Supplementary Figure S1). contrast to PTEN, TPIP was found to lack detectable In an initial screen, these 11 mutants were expressed protein phosphatase activity against the phosphorylated in bacteria as GST–PTEN fusion proteins, purified PTEN 121 AIHCKAGKGRTGVMICA TPIP 240 AIHCKGGKGRTGTMVCA TPTE 297 AIHCKGGTDRTGTMVCA TPTE React 297 AIHCKGGKGRTGTMVCA PIP3 polyGluTyr-P 12 40 10 8 30 6 20 4 10 2 Phosphatase activity Phosphatase activity (% substrate conversion) 0 0 (% substrate conversion) TPIP TPTE PTEN TPIP TPTE- React TPTE PTEN TPTE- React dePIP3 fP-Poly(Glu,Tyr) 35 35 30 30 PTEN wt PTEN wt 25 Y138L 25 Y138L 20 20 15 15 PTEN PTEN 10 10 wt Y138L 5 5 % Substrate conversion % Substrate conversion 0 0 0 20 40 60 80 100 120 140 0 20 40 60 80 100 120 140 Time (min) Time (min) Figure 1 Both TPIP and PTEN Y138L have lipid but not protein phosphatase activity. (a) An alignment of the active site P-loop of PTEN, TPIP and TPTE is shown, with the PTEN catalytic cysteine residue boxed in red. The black boxing shows the threonine and aspartic acid residues at which TPTE differs from the active phosphatases PTEN and TPIP in this region, and the mutation of these residues performed in reactivated TPTE (called TPTE-R) (Leslie et al., 2007). (b and c) PTEN and the cytosolic regions of TPIP and TPTE (wild-type and reactivated) were expressed as GST-fusion proteins and purified. The activity of the indicated proteins was 33 assayed using either P radiolabelled PtdIns(3,4,5)P3 (b) or phosphorylated polyGluTyr (c) as substrate. Data are shown as mean activity and s.d. from triplicate assays. (d) Wild-type and Y138L PTEN proteins were expressed in bacteria as GST-fusion proteins, affinity purified and the tag removed by proteolysis before analysis of these preparations by electrophoresis and staining with 33 Coomassie. (e and f) These wild-type and Y138L PTEN proteins were assayed against P radiolabelled PtdIns(3,4,5)P3 (e)or phosphorylated polyGluTyr (f) for the indicated times and activity shown as the mean activity and s.d. from triplicate assays. Oncogene PTEN’s lipid and protein phosphatase activities L Davidson et al 689 and assayed against both PtdInsP3 and polyGluTyr-P PTEN phosphatase and C2 domains, interfering with in vitro. This screen identified one mutant, PTEN membrane binding (Vazquez et al., 2001; Das et al., 2003; Y138L, which displayed similar PtdInsP3 phosphatase Ning et al., 2006; Gil et al., 2007). We therefore tested the activity to wild-type PTEN, but lacked detectable effects of phosphorylation of PTEN with CK2, and also activity against polyGluTyr-P (Supplementary Figure with GSK3, on the activity of wild-type PTEN and S2). Further kinetic analysis of PTEN Y138L revealed PTEN Y138L against both PtdInsP3 and Ins(1,3,4,5)P4. 33 that the lipid phosphatase activity against 3- P PtdInsP3 These experiments showed, as might be predicted from is essentially indistinguishable from wild-type PTEN, previous experiments using mutant proteins (Ning et al., whereas activity against polyGluTyr-P is extremely 2006), that phosphorylation of bacterially expressed weak, being approximately 3% of wild-type activity, PTEN with CK2 caused a modest reduction in activity and frequently undetectable (Figures 1, 2, Supplemen- against PtdInsP3 andalargerincreaseinactivityagainst tary Figure S2 and S3). the soluble headgroup Ins(1,3,4,5)P4.
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