ARTICLE A Small-Molecule Inhibitor for Phosphatase and Tensin Homologue Deleted on Chromosome 10 (PTEN)
Erika Rosivatz†,*, Jonathan G. Matthews†, Neil Q. McDonald‡,§, Xavier Mulet†,‡‡, Ka Kei Ho†, Nadine Lossi†,‡‡,¶, Annette C. Schmid†,¶¶, Marianna Mirabelli储,††,‡‡, Karen M. Pomeranz**, Christophe Erneux§§, Eric W.-F. Lam**, Ramón Vilar储,††, and Rüdiger Woscholski†,‡‡,* † Division of Cell and Molecular Biology, Imperial College London, Exhibition Road, London SW7 2AZ, U.K., ‡Structural Biology Laboratory, The London Research Institute, Cancer Research UK, 44 Lincolns Inn Fields, London WC2A 3PX, U.K., §School of Crystallography, Birkbeck College, Malet Street, London WC1E 7HX, U.K., 储The Institute of Chemical Research of Catalonia (ICIQ), Avgda. Paisos Catalans 16, 43007 Tarragona, Spain, **Cancer Research UK Labs, Department of Oncology, Imperial College London, Du Cane Road, London W12 0NN, U.K., †† Department of Chemistry, Imperial College London, Exhibition Road, London SW7 2AZ, U.K., ‡‡Chemical Biology Centre, Imperial College London, Exhibition Road, London SW7 2AZ, U.K., §§Interdisciplinary Research Institute (IRIBHM), Université libre de Bruxelles, Campus Erasme, 808 Route de Lennik, 1070 Brussels, Belgium, ¶Current address: Division of Investigative Science, Faculty of Medicine, Imperial College London, Exhibition Road, London SW7 2AZ, U.K., ¶¶Current address: DoNatur GmbH, Am Klopferspitz 19, 82152 Martinsried, Germany
A B S T R A C T Phosphatase and tensin homologue deleted on chromosome 10 (PTEN), a phosphoinositide 3-phosphatase, is an important regulator of insulindependent signaling. The loss or impairment of PTEN results in an antidiabetic impact, which led to the suggestion that PTEN could be an important target for drugs against type II diabetes. Here we report the design and validation of a smallmolecule inhibitor of PTEN. Compared with other cysteine-based phosphatases, PTEN has a much wider active site cleft enabling it to bind the PtdIns(3,4,5)P3 substrate. We have exploited this feature in the design of vanadate scaffolds complexed to a range of different organic ligands, some of which show potent inhibitory activity. A vanadyl complexed to hydroxypicolinic acid was found to be a highly potent and specific inhibitor of PTEN that increases cellular PtdIns(3,4,5)P3 levels, phosphorylation of Akt, and glucose uptake in adipocytes at nanomolar concentrations. The findings presented here demonstrate the applicability of a novel and specific chemical inhibitor against PTEN in research and drug development.
*Corresponding authors,
[email protected] or
[email protected].
Received for review August 11, 2006 and accepted November 14, 2006. Published online December 8, 2006 10.1021/cb600352f CCC: $33.50 © 2006 by American Chemical Society
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hosphatase and tensin homologue deleted on chromosome 10 (PTEN) shares the CX5R motif, present at the bottom of the active site pocket, with the large family of tyrosine phosphatases (PTPs) and dual specificity protein phosphatases but has little sequence homology to these phosphatase families outside this motif (1). Although there is evidence that PTEN displays protein tyrosine phosphatase activity, it is its lipid phosphatase activity that is associated with PTEN’s cellular impacts, such as inhibition of proliferation, survival, and regulation of insulin signaling (2). In particular, the dephosphorylation of the D3 position of the inositol head group of PtdIns(3,4,5)P3 will counteract phosphoinositide 3-kinase (PI3K) action (3), explaining why the loss of PTEN’s function is linked with the development and progression of tumors (4–7). On the other hand, its deregulation and hyperactivation have been implicated in type II diabetes mellitus due to compromised insulin signaling (8–10). Furthermore, in a muscle-specific knock-out of PTEN, a reduction in the development of high-fat-induced insulin resistance and diabetes could be observed (11). PTEN deficiency in mouse adipose tissue leads to decreased production of the hormone resistin, which positively correlates with insulin resistance (12). It has been suggested that impaired control of physiological PTEN inhibition might www.acschemicalbiology.org
ARTICLE a
1 SopB 2 PTEN 3 PTP-1B 4 MTM1 5 SAC Consensus
M L A H E I D A − − V P A WN C K S G K D R WL S E D D N H − − V A A I H CK AGKGR L S N P P S AG − − P I V V H C S AGAGR V A D K V S S G K S S V L V H C S D GW D R YG F F Y F NG − − S E V QR CQ S G T V R h . . . . . s t . . s . h . p Cp sGh sR
TG− − TG− − TGC Y T AQ L TN− − Ts . .
− − I T − .
− − V − − .
− − I − − .
− − D − − .
− MM D S E I K R E H I S L H Q T − H M L S A P G − − − VM I C A Y − − − − − L L HRG − K F L K AQE − − I M L DMA E R E G V V D I Y N C V K A L R S R R I − − S L AM L M L D S F Y R S I E G F E I L V Q K EW I − C L DC L DR T NS VQA F LG L EML AKQ L − − . . h . . . . . . . . . . . h . s . chL . t . . . .
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Figure 1. CPB active sites a) Alignment of the CX5R motif surroundings. b) Molecular surface around the active site of PTP1B (1), PTEN (2), and MTM (3), drawn using the program Pymol (38). Side chains for the active site cysteine and arginine from the CX5R motif are also shown with the ribbon representation of the peptide backbone. The PTP-1B structure is of a substrate-trapping mutant active site C215S mutation and the position of a phosphotyrosine is also indicated.
be a factor in conferring leptin resistance and obesity in animals and humans (13). In addition, Nakashima et al. (14) report that microinjection of an anti-PTEN antibody increased basal and insulin-stimulated PtdIns(3,4,5)P3 levels and the translocation of glucose transporter type 4 (GLUT4) to the plasma membrane, where its exocytosis enables the uptake of glucose (15). While the role of PTEN on PI3K-dependent PtdIns (3,4,5)P3 generation seems to be well supported, it remains to be seen whether GLUT4 translocation and glucose uptake are controlled by PTEN’s PI 3-phosphatase activity. For example, Tang et al. (16) show that knock-down of PTEN by small interfering RNA enhances insulin-dependent glucose uptake into adipocytes, whereas Mosser et al. (17) propose that PTEN activity does not influence GLUT4 translocation and other metabolic functions of insulin. They found that overexpression of wild-type PTEN abolishes GLUT4 translocation to the same extent as overexpression of PTEN mutants without lipid phosphatase activity. Nevertheless, they do find that overexpression of wild-type PTEN antagonizes the metabolic actions of insulin in a PI3Kdependent fashion, because overexpression of wildtype PTEN significantly decreased Elk-1 phosphorylation in response to chronic insulin treatment. Based on the current understanding of PTEN’s role in diabetes, the idea has recently gained credibility that the inhibition of its lipid phosphatase activity might www.acschemicalbiology.org
increase glucose uptake triggered by the increase of PtdIns(3,4,5)P3 (8, 18). Therefore, small molecules that inhibit PtdIns(3,4,5)P3 phosphatase activity have the potential of enhancing insulin sensitivity and overcoming insulin resistance, which would be beneficial for the development of diabetes therapeutics. Because of its role in insulin-dependent signaling, as demonstrated by knock-out studies, PTEN has been proposed as a suitable drug target for antidiabetic treatment (8). It has been shown that vanadate complexes are able to mimic a variety of insulin-like effects in both in vitro and in vivo systems (19–21). For example, these complexes enhance glucose uptake (22–26), which has been attributed to their broad inhibitory potency on PTPs (27–30). However, we recently discovered that these compounds are able to inhibit PTEN’s phosphatase activity with much higher potency (31). This led us to design, synthesize, and test a PTEN-specific inhibitor, judged by the increase in cellular PtdIns(3,4,5)P3 and PtdIns(3,4)P2 levels, Akt phosphorylation, FoxO translocation, and glucose uptake, providing evidence that the insulin-mimetic properties correlate with PTEN inhibition. RESULTS AND DISCUSSION The PI 3-phosphatase PTEN is a member of the large family of cysteine-based phosphatases (CBPs), which also contains the tyrosine and dual-specificity phosVOL.1 NO.12 • 780–790 • 2006
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phatases. Not surprisingly, PTEN was initially thought to act on proteins. This notion was subsequently corrected by Maehama et al. (3), who discovered that PTEN is in fact a very effective inositol lipid 3-phosphatase. This discovery facilitated the elucidation of PTEN’s influence on PI3Kdependent signaling pathways. In particular, this phosphatase controlled the Akt-dependent cell survival pathway, which correlated well with its antitumor properties (32). However, more recently, PTEN knock-outs demonstrated a role in insulin signaling and the development of diabetes and obesity, suggesting that PTEN would be a good target for antidiabetic drugs (8). The homology of PTEN with tyrosine phosphatases led us to reason that inhibitors of the latter could bind to the former, if the inhibitors were of sufficiently broad specificity within the tyrosine phosphatase family. In this context, bisperoxovanadates (bpVs) were the obvious choice because they possessed generic tyrosine phosphatase potencies and had antidiabetic properties (33). Our initial characterization revealed that some of these bpVs were more potent PTEN inhibitors than PTP inhibitors (31) but did target most CBPs with similar affinity. This lack of specificity prompted us to exploit the wide substrate binding site present in the PTEN phosphatase to design a specific vanadium-based inhibitor. Design and Synthesis of a Library of Small Molecules as Potential PTEN Inhibitors. Vanadium complexes are well known for their ability to mimic phosphoesters and phosphates and thus inhibit a broad range of phosphatases. In particular, bpVs and vanadyl complexed to a variety of organic ligands possess good inhibitory potency against many members of the large tyrosine phosphatase family. While the phosphoinositide 3-phosphatase PTEN is highly homologous to other members of the family of CBPs, including tyrosine phosphatases (Figure 1, panel a), its wide catalytic pocket (⬃8 Å deep) with an elliptical opening (⬃5 Å ⫻ 11 Å) (34, 35) distinguishes it from all other CBPs (Figure 1, panel b). Therefore, we synthesized (see Methods for details) a range of vanadates and bpV complexes with general formulas V(⫽ O)(L–L=)2 (abbreviated herein as VO-ligand) and
Figure 2. Residual PTEN activity after treatment with 50 nM vanadium compounds in vitro. Recombinant PTEN was preincubated with eight vanadium compounds for 5 min and then incubated with 3 nmol of PtdIns(3,4,5)P3 for 30 min. PTEN activity was measured by colorimetric determination of inorganic PO43– levels and is shown relative to the activity of uninhibited PTEN ⴞ standard error (SE) of triplicate experiments. 0) solvent H20, 1) VO-OHpic, 2) bpV-OHpic, 3) bpV-pic, 4) VO-pic, 5) bpV-biguan; 6) VObiguan, 7) bpV-phen, 8) bpV-isoqu.
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Kn[V(⫽ O)(O2)2(L–L=)] (abbreviated herein as bpVligand), respectively, and subsequently test their PTEN inhibitor potency. VO-OHpic Is a Potent and Specific Inhibitor in Vitro. In order to determine the most potent inhibitor, eight vanadium complexes with ligands of different sizes were tested for their ability to inhibit the PtdIns(3,4,5)P3-
a % PTEN activity
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Figure 3. BpV activity and structure a) Inhibition of recombinant PTEN by VO-OHpic in vitro. Recombinant PTEN was pre-incubated with between 10 nM and 10 M VO-OHpic for 5 min and then incubated with 3 nmol of PtdIns(3,4,5)P3 for 30 min. PTEN activity was measured by colorimetric determination of inorganic PO43– levels and is shown ⴞSE of triplicate experiments. b) Schematic representation of the different compounds under study (NOTE: only the bpVs were prepared with phenanthroline (phen) and 1-isoquinoline (isoqu)). c) Representation of the X-ray structure of VO-OHpic previously reported by Nakai (36). www.acschemicalbiology.org
ARTICLE phosphatase activity a TABLE 1. In vitro specificity of VO-OHpic for recombinant PTEN compared with other CBPs of recombinant PTEN at a concentration of PTP (PNPP) M SAC (PI4P) M MTM (PI3P) M SopB (PI4,5P2) nM 50 nM. The weakest b VO-OHpic 57.5 ⫾ 9.4 ⬎10 4.03 ⫾ 0.04 588 ⫾ 16 inhibitor is the phebpV-OHpic 4.9 ⫾ 0.9 0.06 ⫾ 0.01 0.35 ⫾ 0.02 33 ⫾ 7 nylbiguanide vanadic bpV-pic 12.7 ⫾ 3.2 0.99 ⫾ 0.01 0.24 ⫾ 0.01 um(IV) complex VOVO-pic 589 ⫾ 33 0.34 ⫾ 0.07 6.35 ⫾ 3.92 125 ⫾ 2 biguan (Figure 2), bpV-biguan 640 ⫾ 32 1.00 ⫾ 0.01 1.81 ⫾ 0.81 798 ⫾ 41 whereas the 3-hyVO-biguan 112 ⫾ 5 0.77 ⫾ 0.03 4.37 ⫾ 0.94 811 ⫾ 88 droxypicolinate c bpV-phen 0.24 ⫾ 0.01 0.08 ⫾ 0.01 0.41 ⫾ 0.04 vanadium(IV) bpV-isoqu 349 ⫾ 27 0.09 ⫾ 0.01 0.87 ⫾ 0.19 80 ⫾ 10 complex VO-OHpic a is the most potent Enzyme activity was measured by colorimetric determination of inorganic PO43– levels in the presence of 4 mM magnesium inhibitor (IC50 ⫽ 35 chloride and 50 M lipid substrate (in parenthesis). IC50 values are shown ⫾SE of duplicate experiments. bThe IC50 value for VO-OHpic was not determined because there was ⬍50% inhibition at 10 M concentration. cNot determined. nM; Figure 3) of the PTEN lipid phosA wider active site cleft arises from the position of the phatase activity; therefore it was further characterized with respect to its specificity. We determined the inhibi- TI loop unique to the PTEN phosphatase (34). The active site dimensions described by Lee et al. (34) are consistory potency of VO-OHpic, as well as the seven other vanadium compounds, against the enzyme activities of tent with those of the VO-OHpic compound even in the four other recombinant CBPs, PTP-, SAC1, myotubula- absence of any induced fit in the phosphatase. In contrast, other PTPs require selectivity for phosphotyrosine/ rin (MTM1), and SopB, in vitro. The IC50 values of VOphosphothreonine and therefore have much narrower OHpic for the tested mammalian CBP family members catalytic clefts unable to accommodate the bulkier (Table 1) are all in the micromolar range, whereas the VO-OHpic (38). IC50 for the bacterial SopB is in the high nanomolar This proves that range. Since the latter enzyme is not of mammalian a Full stimulation the spatial differ0.2 µg/mL insulin origin and needs ⬎16 ⫻ higher doses, one can con10 20 40 75 150 500 VO-OHpic (nM) ences in the catalytic clude that VO-OHpic is highly specific for PTEN in Phospho-Akt (Ser473) pockets can be Phospho-Akt (Thr308) mammals. The other vanadium compounds possess Total Akt exploited to generate broader specificity. Interestingly, MTM seems to prefer a potent inhibitor b vanadium(V) complexes with small ligands, whereas Full stimulation 0.2 µg/mL insulin with a promising tyrosine phosphatases seem to prefer V-phenanthroline 10 20 40 75 150 500 VO-OHpic (nM) specificity, thus valiPhospho-Akt (Ser473) (phen), which is in agreement with earlier observations Phospho-Akt (Thr308) dating our approach (31). In comparison, VO-OHpic is an encouragingly speTotal Akt of designing vanacific (Table 1) and potent PTEN inhibitor (Figure 2). dates with spacec Full stimulation VO-OHpic is a water-soluble vanadium(IV) complex in intensive ligands as 0.2 µg/mL insulin 10 20 40 75 150 500 VO-OHpic (nM) which the metal center is coordinated to two OHpic potential specific Phospho-Akt (Ser473) Phospho-Akt (Thr308) ligands, a water molecule, and an oxo ligand, yielding a PTEN inhibitors. Total Akt complex with asymmetric octahedral geometry. Recently, Since SopB is a bacNakai et al. (36) have shown by X-ray crystallography terial enzyme, one Figure 4. Dose-dependent inhibition of PTEN by VO-OHpic that in the solid state this compound contains one N,O- can conclude that in vivo. Starved a) NIH 3T3 and b) L1 fibroblasts and c) coordinated and one O,O-coordinated OHpic ligand (see the designed inhibiUmUc-3 epithelial cells were prestimulated with 0.2 g mLⴚ1 insulin for 5 min, and indicated concentrations of the X-ray structure in Figure 3, panel c). A complex of tor (VO-OHpic) is VO-OHpic were applied for 15 min. For comparison, full these dimensions would fit well into the catalytic pocket selectively targeting stimulation of cells was carried out by addition of 10 g of PTEN but would be too large for the narrow active site mammalian PTEN in mLⴚ1 insulin. Cell lysates were analyzed by Western pockets of the other tested phosphatases, such as the nanomolar conblotting for Akt phosphorylation on both Ser473 and Thr308 and equal loading (total Akt). centration range. PTB-1 and MTM (34, 37). www.acschemicalbiology.org
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VO-OHpic Treatment Increases Akt Phosphorylation in a PTEN-Dependent Fashion in Vivo. The data presented above confirm that VO-OHpic is a specific inhibitor for PTEN in vitro. To assess the applicability of this inhibitor in the cellular environment (NIH 3T3 and L1 fibroblasts, as well as the PTEN-negative UmUc-3 carcinoma cell line), we investigated the influence of VO-OHpic on the PTEN effector Akt. The activation of Akt, which can be monitored by detecting phosphorylation at two sites (Thr308 and Ser473), is strictly PtdIns(3,4,5)P3-dependent (39, 40). The Akt phosphorylation will thus be proportional to the changes of the cellular levels of this lipid, which is the major substrate of PTEN in vivo (32, 41). However, because PTEN is downstream of PI3K, it was necessary to prestimulate quiescent cells with low amounts of insulin (0.2 g mL–1), which would slightly raise the activity of PI3K above its basal levels and thus produce sufficient levels of the PTEN substrate, without stimulating Akt phosphorylation (Figure 4, lane 1). Consequently, when VO-OHpic was applied on starved cells without insulin, Akt phosphorylation is not increased (data not shown). However, under PI3K primed conditions most of the generated 3-phosphorylated lipids are quickly metabolized by the PI phosphatases such as PTEN, thus producing an environment where the phosphatase activity is dominant. The PTEN inhibitor increased the phosphorylation of Akt on both sites (Figure 4, panels a and b). This effect is detectable at 40 nM inhibitor concentration and reaches saturation at 75 nM VO-OHpic with a 2-fold increased phosphorylation of Akt as compared with the control (Supplementary Table 1). Not surprisingly, this effect is absent in the PTEN-negative UmUc-3 cells (Figure 4, panel c), providing the proof that this PTEN inhibitor is also very specific in vivo (5). The similarity of the dose responses on the tested fibroblasts to the IC50 values obtained with recombinant PTEN implies that VO-OHpic possesses cell permeability and potency. As expected, VO-OHpic had no effect on insulin-stimulated tyrosine phosphorylation at concentrations up to 10 M, which is well below its IC50 for the tyrosine phosphatase PTP- (data not shown). It was not possible to test concentrations above the IC50 for the PTP- due to cytotoxic effects at concentrations at or higher than 100 M (data not shown). Taking all these observations together, one can conclude that this compound is a specific inhibitor of PTEN in vivo. 784
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VO-OHpic Causes the Translocation of Phospho-Akt to the Plasma Membrane. Inhibiting PTEN in cells should not only increase Akt phosphorylation, which is a prerequisite for activating downstream targets of Akt, but also induce translocation of this protein kinase to the plasma membrane and subsequently the perinuclear/ nuclear region (42). Therefore we employed immunofluorescence microscopy to investigate the spatial distribution of phospho-Akt in response to VO-OHpic. Applying the PTEN inhibitor resulted in an Akt translocation and Ser473 phosphorylation that is comparable to insulin-stimulated cells (Figure 5, panel a). Low amounts of insulin do not cause Akt phosphorylation or translocation, whereas higher amounts of insulin or inhibition of PTEN by VO-OHpic increases the Ser473 phosphorylation of Akt localized on the plasma membrane and the nucleus, which is in agreement with the Western blotting experiments shown above (see Figure 4). The VOOHpic-induced Akt phosphorylation is PI3K-dependent, since it is abolished by pretreatment with the PI3K inhibitor wortmannin (Figure 5, panel a). These data confirm that the PTEN inhibitor is activating Akt in a similar fashion to growth factors as judged by the ability to induce phosphorylation and translocation to membrane compartments. About 90% of the cells on the coverslip reacted in the same way (data not shown), indicating that the potential of the inhibitor in cell lines is independent of cell cycle, because the cells have not been synchronized. In addition, the cells looked healthy and showed no obvious morphological changes as compared with untreated cells. VO-OHpic Treatment Increases PtdIns(3,4,5)P3 Levels in Fibroblasts. The inhibition of PTEN results in the translocation and activation of Akt as shown above, which indicates that cellular levels of 3-phosphorylated inositol lipids must be elevated. The activation of Akt is known to rely on the increase of cellular PtdIns(3,4,5)P3 or PtdIns(3,4)P2 levels or both (43). These lipids can be readily identified by Pleckstrin homology (PH) domains (44), such as the one present in Akt, which recognizes PtdIns(3,4,5)P3 and PtdIns(3,4)P2 (45). While it is possible to image the relative amount and localization of these lipids in the living cell by overexpression of a fluorescently labeled Akt PH domain (46, 47), we preferred to employ this lipid recognition domain in a similar fashion to an antibody in fixed fibroblasts. This postfixation procedure avoids interference by the recognition domain on PtdIns(3,4,5)P3-dependent signaling pathwww.acschemicalbiology.org
ARTICLE PI3K. Taken together, these results prove that VO-OHpic targets PTEN’s lipid phosphatase activity in vivo. They also demonstrate the advantage of this compound for inositol Starved Insulin [1 µg/ml] Starved Insulin [1 µg/ml] lipid research, enabling the exploration of specific PtdIns(3,4,5)P3 signaling pathways and their downstream effects without altering PI3K Insulin [0.2 µg/ml] Insulin [0.2 µg/ml] Insulin [0.2 µg/ml] Insulin [0.2 µg/ml] VO-OHpic [100 nM] VO-OHpic [100 nM] activity or expression, thus complementing the already well-employed PI3K inhibitors wortmannin and LY294002. VO-OHpic Treatment Wortmannin [100 nM] Wortmannin [100 nM] Insulin [0.2 µg/ml] Insulin [0.2 µg/ml] Reduces Akt-Dependent VO-OHpic [100 nM] VO-OHpic [100 nM] VO-OHpic [100 nM] VO-OHpic [100 nM] Transcriptional Activity of FoxO3a. Having established Figure 5. PTEN inhibition leads to increased PtdIns(3,4,5)P3 levels and Akt that VO-OHpic inhibits PTEN translocation. a) Immunofluorescence of Ser473 phospho-Akt. Starved NIH 3T3 fibroblasts were treated as indicated. Fixed cells were immunostained with a activity and Akt function Ser473-phospho-specific antibody labeled with an Alexa Fluor 488 dye (green). in vivo, we investigated whether Fluorescence was imaged in the FITC channel using a Nikon TE2000 inverted increased phosphorylation of fluorescence microscope showing that VO-OHpic induced the phosphorylation of Akt upon PTEN inhibition results Akt (green) under PI3K primed conditions using a low concentration of insulin in a corresponding change in (0.2 g mL–1 insulin), which was not sufficient to activate Akt on its own in contrast to the positive control (1 g mLⴚ1 insulin). Wortmannin treatment abolished any Akt effectors. FoxO3a is known Akt phosphorylation, and VO-OHpic alone had no effect on Akt phosphorylation. to translocate from the nucleus Results are representative of at least three independent experiments carried out in to the cytoplasm upon phosduplicates. b) Detection of PtdIns(3,4,5)P3 and PtdIns(3,4)P2 levels postfixation. phorylation by Akt (50) and was Starved NIH 3T3 cells were treated as indicated, fixed, and probed with a recomtherefore employed as a binant PH[Akt] domain labeled with Alexa Fluor 488 dye (green). Treatment with 100 nM VO-OHpic inhibits PTEN, as indicated by the increase of PtdIns(3,4,5)P3 readout for VO-OHpic’s ability to and PtdIns(3,4)P2 levels. propagate signaling downstream of Akt. Immunofluorescence of FoxO3a in NIH 3T3 cells (Figure 6, panel a) ways, which includes Akt activation and could cause reveals that FoxO3a accumulates in the cytosol after compensation by other mechanisms (48, 49). EmployPTEN inhibitor treatment or insulin stimulation (1 g ing this procedure, we found that PtdIns(3,4,5)P3 and mL–1), whereas in quiescent cells, FoxO3a is localized PtdIns(3,4)P2 levels on the plasma membrane in VO-OHpic-treated cells are comparable to those in fully in the nucleus (0.2 g mL–1 insulin). We corroborated insulin-stimulated cells. Inhibition with wortmannin or these findings by investigating the bim promoter activprestimulation with low amounts of insulin results in ity, which is known to be under the control of the FoxO3a residual or negligible staining (Figure 5, panel b), which transcriptional activator (51). Due to translocation from demonstrates that VO-OHpic is changing cellular inosi- the nucleus to the cytosol upon phosphorylation of Akt, tol lipid levels in a fashion compatible with PTEN inhibi- FoxO3a becomes inactive as a transcription factor, tion. The uniform distribution of lipid staining on the which should therefore result in less bim promoter activplasma membrane indicates that the inhibitor does not ity (51). As expected, VO-OHpic treatment reduces bim localize to certain compartments in cells but targets promoter activity (Figure 6, panel b, left) up to 6-fold, PTEN evenly throughout the cell, resulting in an increase whereas a bim promoter with a mutated consensus in the cellular levels of its substrates, the products of sequence for FoxO3a (Figure 6, panel b, right) has no
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effect. Taken together, these results provide evidence that increased levels of PtdIns(3,4,5)P3 caused by the inhibition of PTEN lead to the functional activation of Akt, demonstrated by a corresponding reduction of the transcriptional activity of FoxO3a. The relocation of FoxO3a to the cytosol and the decrease of transacFigure 6. VO-OHpic treatment on the Akt downstream tivation of the bim target Foxo3a. a) FoxO3a localization in NIH 3T3 fibroblasts. promoter upon Starved cells were treated as indicated and stained with a primary anti-FoxO3a antibody and a secondary Cy5-labeled VO-OHpic treatment antibody. Nuclei were stained with dapi (blue). Fluores(51, 52) verify the cence was imaged in TRITC and dapi channels using a Nikon physiological relTE2000 inverted fluorescence microscope. The upper panel evance of the PTEN shows that Akt substrate FoxO3a (false color magenta) is inhibitor-induced accumulated in the cytosol after VO-OHpic (100 nM) treatment or insulin stimulation (1 g mLⴚ1) due to enhanced Akt activation. Akt activity. In quiescent cells, FoxO3a is localized in the To test whether nucleus (0.2 g mLⴚ1 insulin). Lower panel shows nuclei VO-OHpic acts as a of the same cells (false color blue). Transactivational nonspecific inhibiactivity of Foxo3a. b) bim promoter activity was assessed tor of transcription, using a bim promoter sequence containing the consensus for FoxO3a fused to a luciferase reporter gene. Cells transwe tested two fected with construct and a reference Renilla luciferase were luciferase-coupled treated with low doses of insulin (0.2 ug mLⴚ1) and 100 nM promoters that are VO-OHpic and promoter activity was measured in a luminesnot regulated downcence assay 2 and 4 h after treatment. RLU ⴝ relative lumistream of Akt. The nescence units. VO-OHpic treatment reduces bim promoter activity (black) up to 6-fold after 4 h, whereas a bim proB-myb promoter is moter with a mutated consensus sequence for FoxO3a regulated by the E2F (bim mut, gray) does not change its activity. transcription factor through a consensus-binding site (53), while the cyclin D2 promoter is indirectly repressed by FOXO through a Bcl6/STAT5 site (54). VO-OHpic has little effect on the transactivational activity of the wild-type and mutant cyclin D2 and B-myb promoters (data not shown). Together these results show that VO-OHpic decreases bim promoter activity specifically via a FoxO3a-dependent mechanism, rather than by a nonspecific reduction in transcriptional activity. 786
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Inhibition of PTEN Stimulates Glucose Uptake. The results presented above indicate that VO-OHpic is a potent and specific PTEN inhibitor suitable for in vivo applications aimed to investigate PtdIns(3,4,5)P3dependent signaling. Recent findings suggest that PTEN has a role to play in impaired glucose uptake in diabetes (14, 17, 55–57). While there is evidence that insulinmediated glucose uptake into most cells is dependent on the activation of PI 3-kinases, it is less clear which glucose transporters are responsible for the PtdIns (3,4,5)P3-mediated glucose uptake and to what extent, prompting us to evaluate the effectiveness of VO-OHpic on insulin-mediated glucose uptake. We therefore investigated whether treatment with the PTEN inhibitor VOOHpic causes an increase of glucose uptake in L1 adipocytes. Employing a fluorescent glucose analogue made it possible to image glucose uptake. D-Glucose successfully competes with 2-[N-(7-nitrobenz-2-oxa-1,3-diazol4-yl)amino]-2-deoxy-D-glucose (NBDG-glucose) (Figure 7), indicating that the fluorescent analogue is specific for endogenous glucose transporters. VO-OHpic treatment strongly stimulates glucose uptake as compared with control cells (0.1 g mL–1 insulin), demonstrating a clear role of endogenous PTEN in glucose uptake.
Figure 7. Differentiated L1 adipocytes were glucose- and serum-starved for 4 h and then treated as indicated and supplemented with fluorescent NBDG-glucose-containing medium. After 10 min of incubation, cells were washed, stained with dapi, and imaged in FITC and dapi channels using a Nikon TE2000 inverted fluorescence microscope. Clearly, VO-OHpic treatment stimulates glucose uptake in a dose-dependent manner as inhibitor-treated cells show significantly higher intracellular glucose amount (false color yellow). D-Glucose successfully competed with the fluorescent analogue, as cells show almost only nuclear dapi counterstain, indicating that the uptake of the NBDG-glucose is specific. www.acschemicalbiology.org
ARTICLE Employing this small-molecule chemical inhibitor could be advantageous because the enzyme activity can be reduced in a dose-dependent manner without affecting its scaffolding function and ability to interact with other proteins such as MAGI-2 (58), which complements the results obtained from genetic knock-out studies. When all these observations are taken together, it is evident that VO-OHpic is a potent small-molecule compound that specifically inhibits PTEN’s cellular enzymatic activity, which in turn activates downstream targets such as Akt and FoxO3a. Importantly, the data provide evidence that glucose uptake into adipocytes is dramatically enhanced upon PTEN inhibition with VO-OHpic (Figure 7), suggesting that endogenous PTEN has an important regulatory role in glucose uptake and thus would be an interesting drug target. However, it remains to be seen whether the specific PTEN inhibitor can overcome insulin insensitivity and protect from developing type II diabetes, in particular, since it is not known whether PTEN or SH2-containing 5'-inositol phosphatase (SHIP2) controls glucose transporter translocation (16, 59, 60). The here presented PTEN inhibitor, which does not significantly reduce SHIP2 phosphatase activity in vitro (IC50 ⬎ 10 M; data not shown), could
be a useful tool to unravel the contributions of these two diabetes drug targets (18). Inhibiting PTEN will also cause changes in the migratory and invasive properties of cells (4, 61, 62), a feature that has been linked to its well-documented tumor suppressor function. Our own tests reveal that the PTEN inhibitor accelerates wound healing in fibroblasts (data not shown), indicating that it has the potential to alter these PTEN functions as well. While these observations would cause some caution toward the applicability of PTEN inhibitors in general, it does not rule out a tissue-specific delivery system targeting PTEN. This latter notion is supported by recent reports demonstrating that PTEN loss in pancreatic  cells is not tumorigenic (63) and that selective PTEN deletion in skeletal muscle protects against the development of insulin resistance without the development of cancer (11). A targeted tissue-specific drug could therefore be considered for the treatment of diabetes without causing malignant cell growth. The here presented chemical PTEN-specific inhibitor will be an important and probably decisive research tool in unraveling these issues, since it will facilitate investigation of PTEN’s role in primary tissues as well as immortalized cancer cell lines.
METHODS
tate was filtered through a glass microfiber filter paper, washed with cold water and diethyl ether, and dried overnight under reduced pressure (yield 1.127 g, 58.1%). Elemental analyses calculated for VOC16H20N10 (MW 419 g mol⫺1): C, 45.83; H, 4.81; N, 33.40. Found: C, 45.74; H, 4.57; N, 33.06. FAB(⫹) MS m/z: 419 amu (M⫹). IR (KBr) in cm⫺1: (NH2) 3175, 3300, 3056; (C ⫽ N) 1626; (V ⫽ O) 958. Synthesis of K[V(ⴝ O)(O2)2(phenylbiguanide)] (bpV-biguan). V2O5 (1.43 g, 7.84 mmol) was dissolved in a solution of KOH (1.04 g, 18.53 mmol) in water (15 mL) and stirred for 5 min. To the resulting green solution, 2 mL of a 30% (w/v) aqueous solution of H2O2 was added with production of effervescence. The mixture was stirred for 25 min with a progressive change in color to orange and with complete dissolution of all solids. A further 12 mL of a 30% (w/v) aqueous solution of H2O2 was added, and the reaction mixture was stirred for 15 min. At this point, a solution of 1-phenylbiguanide (2.78 g, 15.68 mmol) in a mixture of water (20 mL) and ethanol (5 mL) was added to the reaction mixture, which was stirred at RT for 30 min, after which consistent cloudiness was noted. The resulting pale yellow precipitate was filtered through glass microfiber filter paper and dried under reduced pressure (yield 6.33 g, 91%). IR (KBr) in cm⫺1: (NH2) 3180; (CN) 1641; (VO) 942; (OO) 868; (VO) 616 and 526 cm⫺1. FAB(⫹) MS m/z: 348 amu ([M ⫹ H]⫹). Elemental analyses calculated for C8H11N5KVO5 (MW ⫽ 347 g mol⫺1): C, 27.67; H, 3.19; N, 20.17. Found: C, 27.53; H, 3.33; N, 19.90. Plasmids and Protein Expression. The coding region of the respective DNA sequences (of human PTEN, MTM, or rat Sac (amino acids 1–1042), mouse Akt PH domain (amino acids 5–108, SwissProt P31750), or bacterial SopB) was cloned into a
Chemistry Experimental Details. General Procedures and Materials. IR spectra were recorded on a Perkin Elmer FT–IR1720 Research Series spectrometer using KBr disks in the range 4000 –500 cm–1. Fast atom bombardment (FAB(⫹)) mass spectra were recorded by J. Barton at Imperial College London on a VG AutoSpec-Q using 3-nitrobenzyl alcohol as matrix. 1,10Phenanthroline, picolinic acid, 3-hydroxypicolinic acid, 1-isoquinoline, 1-phenylbiguanide, and V2O5 (99.99%) were purchased from Sigma Aldrich Chemical Co. and used as received unless otherwise stated. The following complexes were prepared according to previously reported literature procedures (33, 36, 64, 65): V(⫽ O)(pic)2, K2[V(⫽ O)(O2)2(pic)],V(⫽ O)(H2O) (OHpic)2, K2[V(⫽ O)(O2)2(OHpic)], K[V(⫽ O)(O2)2(phen)] and K2[V(⫽ O) (O2)2(isoq)] (where pic ⫽ picolinic acid, OHpic ⫽ 3-hydroxypicolinic acid, phen ⫽ 1,10-phenanthroline, and isoqu ⫽ 1-isoquinolinecarboxylic acid). LY294002 was purchased from Calbiochem. All other chemicals were purchased from Sigma unless otherwise stated in the respective section. Synthesis of V(ⴝ O)(phenylbiguanide)2 (VO-biguan). This reaction was carried out under an atmosphere of argon. A solution of VOSO4·3H2O (1.00 g, 4.60 mmol) in distilled and degassed H2O (5 mL) was mixed with a solution containing 2 equiv of 1-phenylbiguanide (1.63 g, 9.20 mmol) in 5 mL of distilled and degassed H2O. This was followed by adjustment of the pH to 12 by slow addition of 11 mL of a 2 M solution of NaOH (0.90 g, 23.0 mmol). After complete addition of NaOH, the solution was stirred overnight resulting in precipitation of a light purple solid. The precipi-
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pGEX-4T2 expression vector (Pharmacia). Protein expression was induced overnight in the Escherichia coli DH5␣ strain using 100 M isopropyl--D-thiogalactopyranoside at 18 °C. Glutathione S-transferase (GST)-fusion protein was purified according to the manufacturer’s manual using glutathione–Sepharose 4B (Pharmacia). Protein integrity was confirmed by Western blot using GST antibody (Novagen). PTP- was purchased from Sigma. Phosphate Release Assay. Phosphatase activities were determined using a phosphate release assay (31). For the detection of PTEN and Sac1 activities, bismuth was added to the phosphate release assay in order to improve its stability and sensitivity (66). All enzyme preparations were tested for linearity to ensure that suitable amounts of enzyme were employed in the inhibitor assays. Enzymes were incubated with inhibitors at various concentrations prior to starting the phosphatase reaction by adding the corresponding substrates presented in octylglucoside mixed micelles as described before (31). Antibodies. The antibody used for immunostaining of phosphorylated Akt/PKB was a mouse monoclonal anti-phospho-Akt (Ser473) antibody from the IgG2b isotype (Cell Signaling 587F11) or anti-phospho-Akt (Thr308) (Cell Signaling 4G10). The antibody for immunodetection of total Akt/PKB was a rabbit antiAkt antibody (Cell Signaling). Horseradish peroxidase (HRP)conjugated goat anti-mouse or -rabbit antibody was purchased from BioRad. Mouse GST antibody was from Novagen. The antiFoxO3a antibody is a rabbit polyclonal antiserum raised against a C-terminal-specific peptide of human FoxO3a. Antibody Labeling. Antibodies were labeled with Alexa Fluor dyes (488 and 595) with a Zenon IgG Labeling Kit (Molecular Probes) according to manufacturer’s instructions. Tissue Culture. NIH 3T3 fibroblasts (mouse fibroblasts), 3T3-L1 (mouse fibroblasts or fibroblasts differentiated to adipocytes as described previously by Volchuk et al. (67), and UmUc-3 cells (human urinary bladder carcinoma) were purchased from ATCC. All cells were grown in Dulbecco’s modified Eagle’s medium (Sigma) supplemented with 10% (v/v) newborn calf serum (Invitrogen) in an atmosphere of 5% (v/v) CO2 at 37 °C. Immunofluorescence of Total Akt and Ser473 Phosphorylated Akt. If not otherwise indicated, cells were seeded on poly-L-lysine (Sigma) coated glass coverslips and starved over night. Cells were preincubated with inhibitors and stimulated as indicated in the Results and Discussion section. Cells were fixed in 4% (w/v) paraformaldehyde (PFA) for 10 min, quenched with 50 mM NH4Cl/phosphate-buffered saline (PBS), permeabilized with 0.25% (v/v) Triton X-100 for 7 min and blocked with 1% (w/v) bovine serum albumin/PBS (fatty acid free; Sigma) for 1 h at RT. Cells were then incubated with the fluorescently labeled antibody (1:200 diluted in blocking solution) for 1 h followed by three extensive PBS washes and a second fixation step with 4% (w/v) PFA for 10 min. Where applicable, washed coverslips were incubated with a second antibody (1:200) for 1 h, washed extensively, and treated with 300 nM 4=,6-diamidino-2-phenylindole (dapi) for nuclear counterstaining. Thoroughly washed coverslips were mounted on glass slides using Mowiol supplemented with 0.6% (w/v) 1,4-diazabicyclo-[2.2.2]octane. Detection of PtdIns(3,4,5)P3. The dialyzed GST-tagged PH domain of Akt/PKB (amino acids 5–108, SwissProt P31750) was used for detection of PtdIns(3,4,5)P3 and PtdIns(3,4)P2 on fixed cells applying the same method as described above and in Byrne et al. (68) for immunofluorescence with slight modification. The recombinant peptide was diluted 1:1000 in blocking buffer and detected with an Alexa Fluor labeled anti-GST antibody (1:5000). Cell preparations were observed under a Nikon TE 2000 fluorescence microscope using a 100⫻ Fluor oil lens. Filters used in
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the fluorescence experiments were bandpass for dapi, FITC, and tetramethyl rhodamine iso-thiocyanate (TRITC) with excitation wavelengths of 340 –380, 465– 495, and 540 –580, respectively, and with emission wavelengths of 435– 485, 515–555, and 572– 605, respectively. Images were digitally acquired with a CCD camera (Hamamatsu) for each fluorophore separately and processed using IPLab software, v 3.65a. and ImageJ (National Institutes of Health, Bethesda, MD). Western Blotting. Quiescent cells were treated as indicated and lysed in SDS sample buffer. Protein samples were separated by SDS-PAGE and blotted on polyvinylidene difluoride membrane. After blocking in 5% (w/v) milk, membranes were probed with the mouse monoclonal anti-phospho-Akt (Ser473) antibody (1:1000) or anti-phospho-Akt (Thr308) antibody and detected with an HRP-conjugated anti-mouse antibody. Stripped membranes were reprobed with a rabbit anti-Akt antibody and detected with an HRP-conjugated anti-rabbit antibody. Transfection and Luciferase Assays. The wild-type and mutant bim promoter/reporter constructs have previously been described (51). NIH 3T3 cells were transfected using the calcium phosphate coprecipitation method as described previously (51). Briefly, calcium phosphate precipitates containing 1 g of wildtype bim promoter firefly-luciferase reporter plasmid (pGL2hBim) or mutant plasmid (pGL2-mutant-hBim), together with 0.2 g of a Renilla luciferase transfection control (pRL-TK; Promega), were incubated overnight with subconfluent cell cultures in each well of a 24-well plate. The cells were then washed twice in PBS, treated, and harvested for firefly/Renilla luciferase assays using the Dual-Luciferase Reporter Assay System (Promega). Glucose Uptake. Differentiated mouse adipocytes growing on coverslips were serum- and glucose-starved for 4 h. After preincubation with VO-OHpic and insulin, cells were treated with 500 M 2-NBDG fluorescent glucose analogue (Molecular Probes) for 10 min at 37 °C. A negative control preincubated with 10 mM D-glucose was included. Coverslips were washed extensively in PBS and counterstained with dapi. Cell preparations were viewed under a Nikon TE 2000 fluorescence microscope using a band pass filter for FITC in order to analyze the extent of glucose uptake. Accession Codes: PTEN, AAD13528; MTM1, Q13496; PTP-1B, NP_002818; PTP-, AAB26530; SopB, AAS76429; Sac, O15056. Acknowledgments: This work was partially supported by EPSRC, MRC, and Cancer Research U.K. Thanks to D. Briggs for assistance in preparing Figure 1, panel b, to M. Serrano (ICIQ) for his help in preparing some of the previously reported vanadium complexes, to E. Galyov (Institute for Animal Health, U.K.) for providing the SopB plasmid, and to M. Clague (University of Liverpool, U.K.) for providing the MTM1 protein. Supporting Information Available: This material is free of charge via the Internet.
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