MOLECULAR AND CELLULAR BIOLOGY, Oct. 2009, p. 5377–5388 0270-7306/09/$08.00⫹0 doi:10.1128/MCB.01649-08 Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 29, No. 19
p85 Associates with Unphosphorylated PTEN and the PTEN-Associated Complex䌤†‡ Rosalia Rabinovsky,1 Panisa Pochanard,1 Chontelle McNear,1 Saskia M. Brachmann,1§ Jonathan S. Duke-Cohan,1 Levi A. Garraway,1,2* and William R. Sellers1* Department of Medical Oncology and Center for Cancer Genome Discovery, Dana-Farber Cancer Institute, and Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02115,1 and Broad Institute, Cambridge, Massachusetts 021422 Received 22 October 2008/Returned for modification 27 January 2009/Accepted 20 July 2009
The lipid phosphatase PTEN functions as a tumor suppressor by dephosphorylating the D3 position of phosphoinositide-3,4,5-trisphosphate, thereby negatively regulating the phosphoinositide 3-kinase (PI3K)/ AKT signaling pathway. In mammalian cells, PTEN exists either as a monomer or as a part of a >600-kDa complex (the PTEN-associated complex [PAC]). Previous studies suggest that the antagonism of PI3K/AKT signaling by PTEN may be mediated by a nonphosphorylated form of the protein resident within the multiprotein complex. Here we show that PTEN associates with p85, the regulatory subunit of PI3K. Using newly generated antibodies, we demonstrate that this PTEN-p85 association involves the unphosphorylated form of PTEN engaged within the PAC and also includes the p110 isoform of PI3K. The PTEN-p85 association is enhanced by trastuzumab treatment and linked to a decline in AKT phosphorylation in some ERBB2-amplified breast cancer cell lines. Together, these results suggest that integration of p85 into the PAC may provide a novel means of downregulating the PI3K/AKT pathway. taining proteins and attenuates PTEN enzymatic activity (1, 11, 20, 32, 45, 61–63, 66, 67, 71). Conversely, PTEN function is promoted in large part through its stabilization in unphosphorylated form by incorporation into a high-molecular-weight protein complex (the PTEN-associated complex [PAC]) (66). We first demonstrated the existence of the PAC through gel filtration studies of rat liver extracts, which identified PTEN within a high-molecular-mass peak (⬎600 kDa), as well as a lowmolecular-mass peak (40 to 100 kDa) in which PTEN is monomeric and phosphorylated (66). Subsequently, several PDZ domain-containing proteins were shown to interact with PTEN, including MAGI-1b, MAGI-2, MAGI-3, ghDLG, hMAST205, MSP58/MCRS1, NHERF1, and NHERF2, which mediate indirect binding with platelet-derived growth factor (PDGF) receptor  (25, 36, 42, 57, 66). More recently, LKB1, a serine/threonine kinase tumor suppressor (7), was also found to interact with and phosphorylate PTEN in vitro (36). In aggregate, these data suggest that PTEN functional output is controlled by a complex interplay of protein interactions and regulation of C-terminal phosphorylation. Beyond these interactions, there is also evidence to support additional regulatory mechanisms by which the tumor suppressor function of PTEN is mediated. The herpesvirus-associated ubiquitin-specific protease was shown to interact directly with PTEN and promote its nuclear entry (53). Both ubiquitination and relocalization into the nucleus constitute important PTEN regulatory mechanisms (53, 64). In many tumors, PTEN nuclear exclusion has been associated with poor cancer prognosis and more aggressive cancer development (15, 44, 56). Moreover, successful treatment of acute promyelocytic leukemia was shown to be associated with an increase in monoubiquitinylation and relocation of PTEN into the nucleus (53). Like PTEN, the p85 regulatory subunit of PI3K serves as a prominent modulator of PI3K/AKT signaling. p85, which exists
The phosphoinositide 3-kinase (PI3K)/AKT signaling pathway regulates glucose/nutrient homeostasis and cell survival and plays a central role in both normal metabolism and cancer. The PTEN tumor suppressor gene (29, 30, 54) negatively regulates the PI3K/AKT pathway by dephosphorylating the D3 hydroxyl subunit of phosphoinositide-3,4,5-trisphosphate, a key membrane phosphatidylinositol generated by PI3K (34). PTEN undergoes genetic or epigenetic inactivation in many malignancies, including glioblastoma, melanoma, and endometrial, prostate, and breast cancers, among others (6, 13, 22, 23, 47, 49–51, 55, 68). Similarly, germ line mutations of PTEN are associated with the development of hamartomatous neoplasias such as Cowden disease and Bannayan-Zonana syndrome (17, 21, 41). The tumor suppressor function of PTEN undergoes dynamic regulation involving both C-terminal phosphorylation and protein-protein interactions. Phosphorylation of serine and threonine residues at the PTEN C-terminal tail, mediated by kinases such as CK2 and glycogen synthase kinase 3, alters its conformational structure and association with PDZ domain-con-
* Corresponding author. Mailing address for Levi A. Garraway: Department of Medical Oncology, Dana-Farber Cancer Institute, 44 Binney St., Boston, MA 02115. Phone: (617) 632-6689. Fax: (617) 582-7880. E-mail:
[email protected]. Present address for William R. Sellers: Novartis Institutes for BioMedical Research, Oncology Disease Area, 250 Massachusetts Avenue, Cambridge, MA 02139. Phone: (617) 871-7069. Fax: (617) 871-4130. E-mail: william
[email protected]. † Supplemental material for this article may be found at http://mcb .asm.org/. § Present address: Novartis Institutes for Biomedical Research, Basel CH-4057, Switzerland. 䌤 Published ahead of print on 27 July 2009. ‡ The authors have paid a fee to allow immediate free access to this article. 5377
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in three isoforms (␣, , and ␥), targets the catalytic (110-kDa) PI3K subunit to the membrane, which brings it into proximity with membrane-associated phosphatidylinositol lipids. In the steady state, p85 forms a tight association with the catalytic PI3K subunit, usually p110␣ or p110 in nonhematopoietic cells, with p110␦ predominating in leukocytes (19). Consistent with this notion, p85 and p110 exist in equimolar ratios in a wide variety of mammalian cell lines and tissues (19), although some studies have suggested a role for free p85 in cell signaling (33, 65). Several recent lines of evidence have begun to support a possible regulatory relationship between PTEN and p85 (reviewed in references 3 and 53). For example, liver-specific deletion of PIK3R1, which encodes the p85␣ regulatory subunit, reduces both the activation of PI3K and PTEN enzymatic activity in this context. As a result, p85␣-deficient hepatic cells express elevated levels of phosphoinositide trisphosphate and exhibit prolonged AKT activation (60). In addition, both PTEN and p85 are regulated by small GTPase proteins such as RhoA, but PTEN coimmunoprecipitates with the RhoA effector Rock only in the presence of PI3K (18, 31, 37). Although only correlative in nature, these findings may suggest a possible role for PTEN in p85 regulation or vice versa, in addition to its known function as a direct antagonist of the PI3K/AKT pathway (3, 9, 52, 57, 60). In the present study, we demonstrate an endogenous association between p85 and PTEN. Using newly generated antibodies that selectively recognize the PTEN C-terminal tail in its unphosphorylated form, we demonstrate that this PTENp85 association preferentially involves the unphosphorylated form of PTEN. The specificity of this interaction was confirmed using multiple antibodies and through studies of both human cancer cells and murine embryonic fibroblasts (MEFs) deficient for specific p85 subunits. This association, which also engages p110, is enhanced by trastuzumab treatment and correlates with diminished AKT phosphorylation. These results support a functional role for the PTEN-p85 association that may have important biological and therapeutic implications for PI3K/AKT pathway regulation. MATERIALS AND METHODS Plasmids, cell lines, and cell culture. pGEX-GST-2T, pGEX-GST-PTEN;WT, and pSG5L-HA-PTEN;WT were described previously (46, 67). HeLa, ACHN, HEK293-T (293-T), and 786-0 cells were maintained in Dulbecco’s modified Eagle medium supplemented with 4,500 mg of glucose/ml, 2 mM L-glutamine, and 10% fetal bovine serum (HyClone); BT474, MDA-MB-231, and the indicated MEFs were maintained in RPMI medium supplemented with 2 mM Lglutamine and 10% fetal bovine serum; SKBR3 cells were maintained in McCoy’s medium supplemented with 2 mM L-glutamine and 10% fetal bovine serum. Penicillin-streptomycin (1,000 U) was added to all cell culture media. Antibodies and reagents. Rabbit monoclonal anti-phospho-Akt (Ser-473) (Cell Signaling Technology Ltd.; catalog no. 4058) was used for immunoblotting at a 1:1,000 dilution; conjugated polyclonal anti-p85␣-agarose (Upstate Biotechnology, Inc./Millipore; catalog no. 16-107) was used for immunoprecipitation as directed; monoclonal anti-p85␣ antibodies (U13 and U5) (Abcam Inc.; catalog nos. ab250-1 and ab249, respectively) were used at 1:250 and 1:500 dilutions, respectively, for immunoblotting and at a 1:100 dilution for immunoprecipitation. Polyclonal anti-pan-p85 antibody (Upstate Biotechnology, Inc./Millipore; catalog no. 06-497) was used at a 1:1,000 dilution for immunoblotting and at a 1:100 dilution for immunoprecipitation. Polyclonal anti-pan-p85 antibody, a generous gift from Lewis Cantley, was used at a 1:5,000 dilution for immunoblotting and a 1:500 dilution for immunoprecipitation. Generation and use of the C54 rabbit polyclonal anti-PTEN antibody have been described previously (46).
MOL. CELL. BIOL. 6H2.1 and 11G8 anti-PTEN monoclonal antibodies (Cascade Bioscience; catalog nos. ABM-2052 and AMB-2055, respectively) were used for immunoblotting at a 1:1,000 dilution and for immunoprecipitation at 1:500 and 1:100 dilutions, respectively. S380, T382, T383, S385, and T382/383 polyclonal anti-unphosphorylated PTEN antibodies were generated by immunizing rabbits with a keyhole limpet hemocyanin-coupled PTEN peptide (RYSDTTDSDPENEPFDE) (PTEN residues 378 to 403) containing the S380, T382, T383, and S385 residues in their unphosphorylated form. Immune sera were then split into four aliquots and affinity purified against four monophosphorylated peptides, each phosphorylated on a unique serine or threonine residue. This resulted in S380, T382, T383, and S385 antibodies. Next, the T382/383 antibody was affinity purified against the unphosphorylated PTEN peptide (RYSDTTDSDPENEPFDE). T382, T383, and T382/383 antibodies were used for immunoblotting at a 1:1,000 dilution and for immunoprecipitations at a 1:15 dilution; trastuzumab (Herceptin) (21-mg/ml solution) was purchased internally (Dana-Farber Cancer Institute pharmacy) for research purposes. Immunoblotting and immunoprecipitation. Whole-cell lysates were prepared by incubating cells for 20 min at 4°C, with TNN buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 0.5% Nonidet P-40, 5 mM EDTA, pH 8) containing protease inhibitors (Roche Applied Science; catalog no. 1183617001). Cell extracts were separated by gel electrophoresis and transferred to nitrocellulose membranes. Bound proteins were detected by immunoblotting as previously described (46). Briefly, membranes were blocked in Tris-buffered saline containing 0.05% Triton X-100 (TBS-T) and 4% (wt/vol) powdered milk or in ReliaBlocker buffer (Bethyl Laboratories, Inc.; catalog no. WB120) for 1 h at 25°C (room temperature). Membranes were incubated with primary antibodies diluted in TBS-T–4% (wt/ vol) milk or in ReliaBlocker buffer overnight at 4°C, washed with TBS-T, and incubated with horseradish peroxidase-conjugated secondary antibody diluted 1:3,000 to 10,000 (Pierce, Inc.; Bethyl Laboratories, Inc.) in TBS-T–4% (wt/vol) milk or in ReliaBlocker from the ReliaBLOT kit (Bethyl Laboratories, Inc.; catalog no. WB120). Detection was performed using enhanced chemiluminescence (Pierce Supersignal; catalog nos. 34080 and 34075). For immunoprecipitations, whole-cell extracts (WCEs) were incubated with the relevant antibodies for 12 to 18 h (overnight), followed by incubation with protein A for 4 to 6 h. Bound proteins were washed in TNN lysis buffer, resuspended in 1⫻ Laemmli protein sample buffer (26), separated by gel electrophoresis, transferred to nitrocellulose, and detected by immunoblot analysis as described above. GST fusion protein pulldown assay. Glutathione S-transferase (GST)–PTEN (wild-type) protein was expressed in Escherichia coli and purified over glutathione-agarose beads as described previously (46). 293-T and 786-0 cells were lysed in TNN buffer for 20 min at room temperature and incubated overnight at 4°C with GST-PTEN;WT and GST-2T recombinant proteins bound to beads. After washing, bound proteins were eluted by being boiled in 1⫻ Laemmli sample buffer (26). In vitro translation and transcription. Wild-type phosphorylated PTEN protein was translated from the pLSG5-PTEN;WT construct using the TNT T7/T3 coupled reticulocyte lysate system (Promega; catalog no. L5010), according to the manufacturer’s instructions. The unphosphorylated wild-type PTEN protein was translated from pLSG5-PTEN;WT using the TNT T7/T3 coupled wheat germ extract system (Promega; catalog no. L5040), according to the manufacturer’s instructions. The proteins were labeled with [35S]methionine (GE Healthcare; catalog no. AG1594). Gel filtration. 293-T cells were lysed or fractionated into cytosolic and nuclear fractions. To generate WCEs, cells were resuspended in hypotonic detergent buffer (buffer 2) (10 mM Tris-HCl, pH 7.4, 140 mM NaCl, 3 mM MgCl2, 0.5 mM phenylmethylsulfonyl fluoride [PMSF], 0.5% Triton X-100, 1% Nonidet P-40, and protease inhibitors [Roche Applied Science; catalog no. 11836170001]) and incubated on ice for 1 h (samples were vortexed vigorously for 30 s at 15-min intervals during the incubation), followed by centrifugation at 9,300 ⫻ g for 10 min. For subcellular fractionation, cells were resuspended in hypotonic nondenaturing buffer (buffer 1) (10 mM Tris-HCl, pH 7.4, 140 mM NaCl, 3 mM MgCl2, 0.5 mM PMSF, and protease inhibitors [Roche Applied Science; catalog no. 11836170001]) and incubated on ice for 1 h with intermittent vortexing as described above. Next, nuclei were separated from the cytosol by low-speed centrifugation at 409 ⫻ g for 10 min. Soluble nuclear proteins were extracted from isolated nuclei in hypotonic detergent buffer (buffer 2), followed by centrifugation at 9,300 ⫻ g for 10 min. 293-T extracts were applied to a Superose 6 (Amersham Pharmacia) column and washed with BC350 buffer (20 mM TrisHCl, pH 7.4, 0.2 mM EDTA, 10 mM beta-mercaptoethanol, 350 mM KCl, 0.2 mM PMSF, 10% glycerol, and protease inhibitors [Roche Applied Science; catalog no. 11836170001]) at 0.5 ml/min and 4°C. Fractions (0.5 ml) were collected; after the void volume (fractions 1 to 12 for WCEs and 1 to 22 for cytosolic
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FIG. 1. Coimmunoprecipitation of endogenous p85 and PTEN proteins. (A) WCEs from 293-T, ACHN, HeLa (PTEN-positive), and 786-0 (PTEN-negative) cell lines were immunoprecipitated with anti-PTEN (6H2.1) or anti-p85␣ (U13) antibodies and immunoblotted with independent anti-PTEN (C54) and anti-pan-p85 antibodies. (B) Endogenous p85␣ was coimmunoprecipitated with endogenous PTEN in 293-T cells using either polyclonal (C54) or monoclonal (11G8 and 6H2.1) anti-PTEN. Immunoblotting was performed with 6H2.1, C54 (anti-PTEN), or U13 (anti-p85␣) antibody. IgG, immunoglobulin G. (C) Endogenous p85␣ and p85 coimmunoprecipitated with endogenous PTEN in MEFs derived from compound homo- and heterozygote p85␣ and p85 knockout mice. The 6H2.1 (PTEN) antibody was used for immunoprecipitation, and either C54 (PTEN) or polyclonal anti-pan-p85 antibody was used for immunoblotting. (D) Full-length GST-PTEN was incubated with lysates prepared from 293-T and 786-0 cells. p85␣ was detected by immunoblotting the U13 antibody (p85␣). FT, flowthrough.
and nuclear fractions), fractions 13 through 46 (WCEs) or 23 through 49 (cytosolic and nuclear fractions) were subjected to immunoblotting. In these experiments, fractions corresponding to WCEs were collected after a 5-ml void volume was discarded, whereas for cytosolic and nuclear extracts all of the eluted volume was collected. Immunoaffinity purification. The T382/383 immunoaffinity column was prepared as directed (Pierce, Inc.; catalog no. 44893). For unphosphorylated PTEN purification, 293-T cells were lysed in hypotonic detergent buffer (buffer 2) and incubated on ice for 1 h (samples were vortexed vigorously for 30 s with 15-min intervals, during the incubation), followed by centrifugation at 9,300 ⫻ g for 10 min. Lysates were loaded twice on the column (to ensure maximum binding) and washed with 50 ml TNN buffer followed by 7 ml of low-pH buffer (1 M glycine, pH 2.7) to equilibrate the column and remove additional nonspecifically bound proteins. Proteins that remained bound to the column after this step were eluted with additional 1 M glycine (pH 2.7), and fractions (0.5 ml) were collected for immunoblot analysis.
RESULTS p85␣ and p85 associate with PTEN. Based on recent evidence suggesting engagement of PTEN in the PAC (66) and a possible interrelationship between p85 and PTEN function (3, 52, 57, 60), we investigated whether p85 subunits might interact with PTEN as part of the PAC. To test this possibility, protein extracts were prepared from cell lines expressing PTEN (ACHN, 293-T, and HeLa cells) or lacking an intact PTEN gene (786-0 cells). Immunoprecipitations were performed using an anti-PTEN antibody (6H2.1) followed by detection with either an antibody recognizing p85␣ and p85 (pan-p85) or a polyclonal antiserum recognizing PTEN (C54). In cells harboring intact PTEN (ACHN and 293-T cells), endogenous p85 coimmunoprecipitated with PTEN, whereas p85 coimmunoprecipitation was not observed in cells lacking PTEN (Fig. 1A and B). Similar results were obtained using two additional
anti-PTEN antibodies, 11G8 (murine monoclonal) and C54 (rabbit polyclonal) (Fig. 1B). The reciprocal coimmunoprecipitation failed to identify PTEN, although the direct anti-p85␣ immunoprecipitation itself was not robust (Fig. 1A, left). Together, these results suggested an endogenous association between PTEN and p85␣. To confirm the specificity of the PTEN-p85 interaction, we performed immunoprecipitation experiments using MEFs derived from compound homo- and heterozygote p85␣ and p85 knockout mice (8), together with wild-type controls. Here, immunoprecipitation of PTEN from p85␣⫹/⫹; p85⫹/⫹ (wildtype) cells resulted in coimmunoprecipitation of p85␣ and p85 (Fig. 1C). Similarly, p85␣ was coimmunoprecipitated from p85␣⫹/⫺; p85⫺/⫺ MEFs and p85 associated with PTEN in extracts prepared from the p85␣⫺/⫺; p85⫹/⫺ MEFs (Fig. 1C). In contrast, no cross-immunoreactive antigens were detected following coimmunoprecipitation with PTEN in MEFs lacking an intact p85 gene (p85␣⫺/⫺; p85⫺/⫺). Of note, the MEFs employed herein express only a single copy of either p85␣ or p85 genes (8); thus, both protein expression and coimmunoprecipitation efficiency were reduced in these experiments (Fig. 1C). Nevertheless, these results affirmed an endogenous interaction between PTEN and p85␣ in both human and murine cells. To provide additional verification of the association between PTEN and p85, recombinant GST-PTEN was produced in bacteria and isolated using glutathione-Sepharose beads. Protein lysates from 293-T and 786-0 cells were incubated with beads containing either full-length GST-PTEN or GST protein alone (GST-2T). Bound proteins were eluted and separated by electrophoresis, and p85␣ was examined by immunoblotting as
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shown in Fig. 1D. In these experiments, endogenous p85␣ was detected in 293-T lysates eluted from GST-PTEN but not from GST-2T beads (Fig. 1D). p85␣ was not detected in lysates from 786-0, a PTEN⫺/⫺ cancer cell line, although the expression of p85␣ was notably reduced in 786-0 cells compared to that in 293-T cells (Fig. 1D; flowthrough and WCE samples). These results provided additional support for an interaction between PTEN and p85 in mammalian cells. In addition, as bacterially produced GST-PTEN is likely unphosphorylated, this result also suggested that phosphorylation of PTEN is unnecessary for association with p85. Generation of specific antibodies recognizing unphosphorylated PTEN. Our previous observations that phosphorylated PTEN was primarily monomeric led us to propose that unphosphorylated PTEN might become preferentially incorporated into the PAC, which in turn would promote its tumor suppressor function (66). To study the unphosphorylated form of PTEN more directly and its association with p85 and the PAC, we sought to develop antibodies that selectively recognized the unphosphorylated 380SDTTDS385 epitope at the PTEN C-terminal tail. More specifically, we hoped to generate antibodies whose binding would be blocked by phosphorylation. Toward this end, rabbits were immunized with the PTEN peptide RYSDTTDSDPENEPFDE (residues 378 to 403) containing S380, T382, T383, and S385 in unphosphorylated form. Next, serum aliquots were independently preadsorbed in parallel, using affinity resins harboring the same peptide but retaining a single phosphorylated amino acid residue at the targeted epitope. We refer to these monophosphorylated affinity or preadsorption columns as AD1-pS380, AD2-pT382, AD3-pT383, and AD4-pS385 (Fig. 2). Here, we anticipated that any antibody whose binding to the 380SDTTDS385 antigen was blocked by phosphorylation would fail to bind the resin and hence flow through the column. Conversely, antibodies capable of recognizing a phosphorylated amino acid would be retained and hence adsorbed to the column for subsequent elution. This approach produced four antisera, termed S380, T382, T383, and S385 (Fig. 2, bottom). The specificity of these antibodies was tested by immunoblotting against GST-PTEN. The T382 and T383 antisera (preadsorbed onto AD2-pT382 and the AD3-pT383 columns, respectively) were found to robustly detect recombinant unphosphorylated GST-PTEN, while the S380 and S385 antisera (preadsorbed onto AD1-pS380 and the AD4-pS385 columns, respectively) did not (Fig. 2, bottom). These data strongly suggest that the original, nonadsorbed antiserum primarily if not exclusively recognizes the unphosphorylated 381DTTD384 epitope and is blocked by phosphorylation of either T382 or T383 (Fig. 2, top). This antiserum is hereafter referred to as anti-T382/T383 (Fig. 2, top). Validation of antibodies recognizing unphosphorylated PTEN. The specificity of these new antibodies (T382, T383, and T382/383) was confirmed through a series of immunoblotting and immunoprecipitation studies in comparison to an established PTEN antibody (C54) (46), which recognizes phosphorylated and unphosphorylated PTEN. First, recombinant GST-PTEN (1 mg) was immunoblotted using serial dilutions (ranging from 1:500 to 1:10,000) of C54, T382, T383, and T382/383 antibodies (Fig. 3A and B). In reciprocal experi-
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FIG. 2. Generation of specific anti-unphosphorylated PTEN antibodies. Antisera recognizing unphosphorylated PTEN were generated using an epitope inclusive of S380 through S385 (top, underlined). The initial immunized sera (upper panel) and the resulting preabsorbed antisera (lower panel) were examined by immunoblotting for their ability to detect recombinant GST-PTEN. Numbers (left) are molecular masses in kilodaltons.
ments, various amounts of purified GST-PTEN protein (from 0.1 ng to 1 g) were immunoblotted using a fixed dilution (1:1,000) of these antibodies (Fig. 3C and D). The anti-T382/ 383 antibody recognized GST-PTEN, albeit at a 10- to 50-foldlower sensitivity than that of C54 (Fig. 3B and C). The individual T382 and T383 antibodies were less sensitive than the T382/383 antibody (Fig. 3B and D). Nonetheless, both antibodies showed robust affinity for GST-PTEN protein at 1:1,000 to 1:3,000 dilutions (Fig. 3B) and were able to detect at least 100 ng of the recombinant protein at a 1:1,000 dilution (Fig. 3D). Moreover, the T382/383 antibody immunoprecipitated GST-PTEN as robustly as did the anti-total PTEN (C54) antibody (Fig. 3E) and was able to immunoprecipitate as little as 0.01 g of GST-PTEN (Fig. 3H). Although the T382 and T383 antibodies had relatively similar immunoblotting efficiencies (Fig. 3D), T383 was the more sensitive antibody for immunoprecipitation (Fig. 3F and G). Together, these results indicated that the newly generated, anti-unphosphorylated PTEN (T382, T383, and T382/383) antibodies performed well in both immunoblotting and immunoprecipitation studies of PTEN protein. Specific detection of endogenous, unphosphorylated PTEN. To determine whether the anti-unphosphorylated PTEN antibodies could detect endogenous PTEN, total PTEN protein was immunoprecipitated with a monoclonal antibody (6H2.1) that recognizes both its phosphorylated and unphosphorylated forms. Next, immunoblotting studies were performed using
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FIG. 3. Validation of anti-unphosphorylated PTEN antibodies. Recombinant GST-PTEN (1 mg) was immunoblotted with the indicated dilutions of T382/383 (A) or T382 and T383 (B) antibodies, in comparison to the C54 antibody. Alternatively, recombinant GST-PTEN protein (0.1 ng to 1 g) was separated by gel electrophoresis and immunoblotted with a fixed dilution (1:1,000) of T382/383 (C) or T382 and T383 (D) antibodies, in comparison to the C54 antibody. To evaluate specificity for unphosphorylated PTEN, different quantities of purified GST-PTEN were immunoprecipitated with C54 (E), T382 (F), T383 (G), and anti-T382/383 (H) antibodies, followed by immunoblotting with anti-PTEN (6H2.1) antibodies. Numbers at left of panels A, B, and E to H are molecular masses in kilodaltons.
antibodies recognizing total PTEN (C54), phosphorylated PTEN (p380), and unphosphorylated PTEN (T382/383). As shown in Fig. 4A, the anti-T382/383 antibody readily identified endogenous PTEN, although the PTEN signal identified by the T382/383 antibody was reduced in comparison to that seen with the C54 and p380 antibodies. To confirm that the preadsorbed antibodies do not crossreact with phosphorylated PTEN, rabbit reticulocyte and wheat germ lysate transcription-translation systems were used to generate phosphorylated and unphosphorylated PTEN protein, respectively, in the presence of radioactively labeled methionine. Unlike the rabbit reticulocyte system, wheat germ lysate exhibits very low endogenous posttranslational modifi-
cation activity (2, 27, 38, 39). Thus, the translated PTEN protein produced by the wheat germ lysate system should show minimal if any phosphorylation. Translated proteins were incubated with the preadsorbed sera S380, T382, T383, and S385 alongside the C54 antibody as a positive control, and incorporation of label was evaluated by autoradiography (Fig. 4B). None of the preadsorbed sera recognized the protein produced using rabbit reticulocyte lysates (phosphorylated) compared to anti-total PTEN (C54) antibody. Although production of PTEN protein was less efficient in the wheat germ lysate system (unphosphorylated), the preadsorbed antisera robustly immunoprecipitated PTEN translated by this system, as did antitotal PTEN (C54) antibody (Fig. 4B). These results affirmed
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FIG. 4. Specificity of anti-unphosphorylated PTEN antibodies. (A) Endogenous PTEN was immunoprecipitated from HeLa (PTENpositive) cell lysates using anti-PTEN (6H2.1) antibody and immunoblotted with antibodies recognizing unphosphorylated PTEN (T382/ 383), phosphorylated PTEN (p380), or total PTEN (C54). 786-0 (PTEN-null) cell lysates were used as a negative control. Numbers at left are molecular masses in kilodaltons. (B) pSGL-PTEN was translated in either rabbit reticulocyte or wheat germ in vitro transcriptiontranslation lysate systems in the presence of radioactively 35S-labeled methionine to generate phosphorylated and unphosphorylated PTEN, respectively. Labeled, translated extracts were immunoprecipitated using C54, S380, T382, T383, and S385 antibodies. Bound proteins were separated by gel electrophoresis, and 35S labeling was detected by autoradiography.
the specificity of the newly generated antibodies for unphosphorylated PTEN. p85 exists within the high-molecular-weight PAC. To determine whether unphosphorylated PTEN and p85 are part of the PAC, WCEs and cytosolic and nuclear lysates from 293-T cells were subjected to gel filtration over a Superose 6 column (Fig. 5; see also Fig. S1 in the supplemental material). The specificity of the cytosolic and nuclear preparations was confirmed with a panel of well-established cellular markers (see Fig. S1A in the supplemental material). In keeping with previously reported data (57, 66), both monomeric PTEN (44 to 100 kDa, fractions 36 to 44 [WCEs] and 40 to 49 [cytosolic and nuclear]) and the PAC (⬃670 kDa, fractions 17 to 20 [WCEs] and 26 to 30 [cytosolic and nuclear]) were detected in WCEs (Fig. 5A) and subcellular fractions (Fig. 5B; see also Fig. S1B in the supplemental material). The separation of PTEN into monomeric and high-molecular-weight peaks was particularly striking when cytosolic extracts were examined (Fig. 5B). Interestingly, while we had previously observed that the phosphorylated form of PTEN was found only in the monomeric peak (44 to 100 kDa) (66), the anti-T382/383 antibody detected unphosphorylated PTEN both as a monomer and within the high-molecular-weight complex (Fig. 5A and B; see also Fig. S1B in the supplemental material). In these experiments, the relative intensity of monomeric and high-molecular-weight unphosphorylated PTEN identified by the anti-T382/383 antibody suggested an enrichment of unphosphorylated PTEN in the high-molecular-weight complex compared to the C54 antibody (which recognizes total PTEN). To determine whether p85 is a part of the PAC, gel filtration fractions were also immunoblotted with anti-p85␣ and/or antipan-p85 antibodies. In addition to the p85 monomer, which corresponds to ⬃100-kDa fractions, p85 subunits were also
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present in high-molecular-weight fractions. One p85 fractionation peak comigrated with the PAC in the same elution volume that corresponded to the high-molecular-weight fractions of PTEN (Fig. 5A and B; see also Fig. S1B in the supplemental material), suggesting that p85 might be part of the PAC. The presence of the p85␣ subunit in the PTEN high-molecularweight fractions was also observed in rat liver extracts (data not shown). To confirm that p85 interacts with PTEN within the PAC, fractions from WCEs corresponding to the PAC or low-molecular-weight protein (near 670 kDa or 44 to 100 kDa, respectively) were subjected to immunoprecipitation with the 6H2.1 anti-PTEN monoclonal antibody (Fig. 5C). Notably, both p85␣ and p85 coimmunoprecipitated with PTEN from the highmolecular-weight fractions (Fig. 5C). However, neither p85 subunit was identified when PTEN immunoprecipitation was performed in the low-molecular-weight control fractions (Fig. 5C). In some gel filtration experiments, PTEN migrated as a doublet, with the lower band corresponding to the predicted molecular weight of monomeric PTEN (e.g., Fig. 5; see also Fig. S1B in the supplemental material); the basis for this difference in migration from that found in studies of WCEs is currently unclear. Altogether, these results provided direct evidence that an association between PTEN and p85 occurs in the PAC. p110 associates with p85 and PTEN in the PAC. As noted above, p85 associates tightly with p110 isoforms in the cell, thereby effecting both stabilization and regulation of the catalytic PI3K subunits. To determine if p110 subunits coexist together with p85 and PTEN, the gel filtration fractions described above were immunoblotted for p110␣ and p110 (Fig. 5A and B; see also Fig. S1B and C in the supplemental material). As expected, monomeric p110␣ and p110 were identified in similar cytosolic fractions; these were also highly coincident with monomeric p85␣ (Fig. 5B). Interestingly, an additional p110 peak that migrated parallel to the PAC was detected (Fig. 5B; see also Fig. S1B and C in the supplemental material). A trace amount of p110␣ was also detected in parallel with the PAC in cytosolic but not WCE gel filtration experiments (Fig. 5B). These results suggested that p110 subunits in general and p110 in particular may also associate with p85 and PTEN in the PAC. To confirm an association between p110 and PTEN, wholecell lysates were immunoprecipitated using the 6H2.1 antibody (recognizing total PTEN) and immunoblotted for p110 (Fig. 6). The p110 subunit was faintly detected in 293-T cell lysates under immunoprecipitation conditions optimized for coimmunoprecipitation of PTEN and p85 (Fig. 6A). Increasing the stringency of the washing conditions resulted in a robust coimmunoprecipitation of p110 with PTEN. The p85␣ signal remained detectable, albeit more weakly, under these conditions (Fig. 6B). Together, these data suggested that PTEN associates with the physiologically relevant p85-p110 heterodimer, thereby raising the possibility of a functional role for this interaction in mediating the PTEN tumor suppressor function. p85 associates with unphosphorylated PTEN. To determine if p85 interacts with endogenous, unphosphorylated PTEN, we generated an immunoaffinity column by immobilizing the antiT382/383 antibody on agarose beads. Next, WCEs prepared
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FIG. 5. p85 comigrates with a high-molecular-weight PAC. (A and B) WCEs (A) or cytosolic fractions (B) from 293-T cells were separated by gel filtration. Eluted fractions were separated by electrophoresis and immunoblotted for unphosphorylated PTEN (T382/383), total PTEN (C54), and p85 (anti-p85␣ [U13] and anti-pan-p85). WCEs were also immunoblotted for p110␣; cytosolic extracts were immunoblotted for both p110␣ and p110. (C) Gel filtration fractions containing monomeric (“control”) or high-molecular-weight (“complex”) PTEN were pooled, immunoprecipitated with anti-PTEN (6H2.1), and immunoblotted with anti-PTEN (C54) and anti-pan-p85 antibodies.
from 293-T cells were separated using this immunoaffinity column. After extensive washing with TNN lysis buffer to remove unbound protein followed by an additional washing-equilibration step with low-pH buffer (see Materials and Methods), PTEN was eluted with low-pH elution buffer (1 M glycine, pH 2.7) and 0.5-ml fractions were collected. The eluted fractions were separated by gel electrophoresis and immunoblotted with anti-total PTEN (C54), anti-p85␣, and anti-p110 antibodies (Fig. 7). Endogenous (unphosphorylated) PTEN was detected in fractions eluted from the anti-T382/383 immunoaffinity column (Fig. 7, bottom). Notably, the p85␣ subunit was also identified in a subset of fractions eluted from this column (fractions 25 to 32; Fig. 7, middle). Similar results were obtained using an anti-T383 immunoaffinity column (data not shown). Interestingly, p110 was also eluted from this column and showed enrichment in the fractions where p85␣ was detected (Fig. 7, top). These results supported the premise that p85 complexes with unphosphorylated PTEN.
The association between PTEN and p85␣ is enhanced by trastuzumab. Next, we sought to examine the functional importance of the PTEN-p85 interaction. Accordingly, we considered this interaction in relationship to HER2/neu inhibition mediated by trastuzumab, an anti-HER2 humanized antibody (4). The antitumor effects of trastuzumab depend on ERBB2 gene amplification in breast cancer but are modified by both PIK3CA mutation and PTEN expression (5). In particular, PTEN expression has been identified as a candidate biomarker predictive of outcome following trastuzumab treatment in patients with ERBB2-amplified (HER2/neu-positive) breast cancer (5, 10, 16, 40, 43, 48). To investigate whether trastuzumab treatment might modulate formation of the PTEN-p85 complex, protein lysates were prepared from two ERBB2-amplified cell lines (BT474 and SKBR3) together with two ERBB2 “wild-type” counterparts (MDA-MB-231 and MCF7) following 40, 60, and 120 min and 24 h of trastuzumab exposure. These lysates were
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To assess the possible functional importance of the PTENp85 association, we examined AKT phosphorylation (p-AKT) in the setting of trastuzumab treatment. The levels of p-AKT, characteristically high in ERBB2-amplified cell lines (72, 73), showed a measurable decline following short-term (40- to 120min) trastuzumab exposure, as shown previously (40) (Fig. 8, lower panel). The reduction of p-AKT levels in BT474 and SKBR3 (ERBB2-amplified) cells therefore correlated with both trastuzumab exposure and the association of p85/p110 with PTEN. In contrast to earlier studies (40), we found no variations in unphosphorylated PTEN levels in response to trastuzumab treatment (Fig. 8). As a negative control, the same immunoprecipitation was carried out in 293-T cells, an ERBB2 wild-type context in which the p85-PTEN association is present (see Fig. S2 in the supplemental material). Here, coimmunoprecipitation of p85 and p110 with PTEN was detectable regardless of trastuzumab exposure. Interestingly, the PTEN-p85 interaction was not induced when ERBB2-amplified cells were treated with lapatinib, a small-molecule HER2 inhibitor (data not shown). Thus, these results suggest that the PTEN-p85 association may contribute to inhibition of PI3K signaling by PTEN in the setting of monoclonal antibody-based receptor tyrosine kinase inhibition.
FIG. 6. p110 coimmunoprecipitates with PTEN and p85. (A) WCEs from 786-0 (PTEN-negative) and HeLa and 293-T (PTEN-positive) cell lines were immunoprecipitated with anti-PTEN (6H2.1 or C54) and immunoblotted with anti-PTEN (C54), anti-pan-p85, or anti-p11 antibodies. (B) WCEs from 786-0 and 293-T cell lines were immunoprecipitated with anti-PTEN (6H2.1) under more stringent washing conditions (see Materials and Methods) and immunoblotted for PTEN (C54), p85␣, or p110.
subjected to immunoprecipitation with an anti-PTEN antibody (6H2.1). Coimmunoprecipitation of p85 with PTEN was minimally detectable in untreated ERBB2-amplified cell lines; however, both p85 and p110 were readily coimmunoprecipitated with PTEN within 40 to 60 min following exposure to trastuzumab (Fig. 8, upper panel). In contrast, p85 and p110 were poorly detectable in ERBB2 wild-type cell lines regardless of trastuzumab exposure (Fig. 8, upper panel). The p110␣ subunit was virtually undetectable following PTEN immunoprecipitation (Fig. 8). These data suggested that trastuzumab treatment may augment the PTEN-p85 association in ERBB2amplified cells, possibly through integration of p85 into the PAC.
DISCUSSION Engagement of PTEN within a high-molecular-weight complex may promote its stabilization and function as a phosphatase and tumor suppressor, particularly in its unphosphorylated form (45, 66). Here we show that the PI3K regulatory subunit p85 associates with PTEN and is a component of the PAC. The association between PTEN and p85 was observed by direct immunoprecipitation from several different cell lines, by GST affinity purification, and by biochemical purification over gel filtration and immunoaffinity columns. Moreover, this association was enhanced by trastuzumab treatment. Although we were unable to detect the association between p85 and PTEN in the reciprocal experiment, we note that the majority of cellular p85 is tightly associated with one or more p110 catalytic subunits of PI3K. Conceivably, more sensitive antibodies against p85, or those recognizing distinct epitopes, may be needed to identify the p85-PTEN interaction. In total, our results suggest a functionally important regulatory interaction between PTEN and p85 proteins. In cells expressing p85␣ and p85, both subunits could be coimmunoprecipitated together with PTEN. In addition, we
FIG. 7. p85␣ interacts with unphosphorylated PTEN. WCEs (200 mg) from 293-T cells were loaded over a T382/383 antibody column (see Materials and Methods). Eluted fractions were separated by gel electrophoresis and immunoblotted for p110, p85␣ (U13), and PTEN (C54), respectively.
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FIG. 8. Trastuzumab enhances the association between p85␣ and PTEN. BT474, SKBR3 (ERBB2-amplified), MDA-MB-231, and MCF-7 (ERBB2-wt) cells were treated with trastuzumab for 40, 60, and 120 min and 24 h. WCEs were then immunoprecipitated with anti-PTEN (6H2.1) antibodies and immunoblotted for total PTEN (C54), p85 protein (U13), p110␣, and p110. AKT phosphorylation and steady-state levels of the aforementioned proteins were monitored in WCEs from these experiments using a rabbit monoclonal antibody recognizing phosphorylation at Ser473 (pAKT S473).
observed an association with p85 in p85␣⫺/⫺ MEFs. These findings are consistent with prior studies showing that in the absence of p85␣, p85 may become the dominant regulatory isoform to interact with the catalytic p110 subunit (8, 12, 65). However, in HeLa cells, which lack p85␣ expression, p85 was not coimmunoprecipitated with PTEN. This may imply that the interaction occurs preferentially with p85␣, that p85 expression is insufficient for PAC integration in these cells, or that other factors may modulate the interaction in some contexts. The phosphorylation status of PTEN plays a major role in regulating its tumor suppressor function. Alanine mutations of the C-terminal residue T382 or T383 correlate with increased efficacy of PTEN function, based on phenotypes such as cell cycle arrest, accelerated PTEN degradation, increased membrane translocation, and cell migration (11, 20, 45, 61–63, 67).
Conversely, both phosphorylation and protein-protein interactions protect PTEN from degradation (67). Importantly, we show that p85␣ is not only part of the PAC but also associates with the unphosphorylated form of PTEN. Three lines of evidence support this notion. First, p85 was isolated from WCEs by affinity purification using recombinant GST-PTEN, indicating that phosphorylation of PTEN is unnecessary for this interaction. Second, immunoaffinity columns containing antibodies whose binding to PTEN is blocked by phosphorylation (anti-T382/3 and anti-T383) recovered both PTEN and p85. These data suggest that the “active,” unphosphorylated form of PTEN interacts with p85 and may antagonize its function. Third, p85 was coimmunoprecipitated together with PTEN in gel filtration fractions that corresponded to the PAC. Altogether, these data suggest that unphosphor-
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ylated PTEN interacts with p85 to modulate its function as a mediator of PI3K activity. Accumulating evidence suggests that the PTEN tumor-suppressive function is linked specifically to deregulation of the p110 isoform in several contexts. For example, in a mouse model of prostate cancer driven by PTEN loss, genetic ablation of p110 but not p110␣ suppressed the cancer phenotype (24). In other experiments that utilized inducible short hairpin RNAs directed against several p110 isoforms, knockdown of p110 suppressed the growth of PTEN-null cell lines, whereas p110␣ knockdown had no effect (69). In light of these observations, it is intriguing that the PTEN-p85 association described here occurred preferentially with the p110 subunit in both gel filtration and immunoprecipitation assays. This observation also suggests that p85 retains a 1:1 heterodimeric association with p110 even when complexed with PTEN in the PAC (e.g., we did not find evidence for “free” p85 in this context) (19). Overall, these findings raise the possibility that PTEN exerts a critical tumor-suppressive role in p85/p110 activity through specific protein-protein interactions as well as its established lipid phosphatase activity. The results of this study also imply that only a fraction of total cellular p85 and PTEN engages the PAC. The GSTPTEN affinity purification studies demonstrated that the majority of p85␣ protein remains in the flowthrough component. Along these lines, it is possible that an additional component(s) may be rate limiting for complex formation. Toward this end, several proteins that interact with PTEN have been identified (25, 36, 42, 57, 66) and therefore may also exist in the PAC. Gel filtration experiments performed on mouse brain lysates showed that NHERF2, PDGF receptor , and a portion of MEGI-1 migrate in parallel with PTEN high-molecularweight fractions (57). Also, our immunoprecipitation experiments suggest that unphosphorylated PTEN is present at a much lower abundance in the cell than is phosphorylated PTEN, consistent with prior observations (66). Moreover, sizeexclusion chromatography experiments showed that significant amounts of monomeric, unphosphorylated PTEN are present in resting cells. Thus, the relative roles of the p85 interaction and the PAC compared to monomeric PTEN in exerting its cellular and tumor suppressor function require additional clarification. Toward this end, prior studies have established that both PTEN expression and aberrant p110 activation may modulate trastuzumab resistance in cancer cells (5, 40). In support of the functional relevance of a PTEN-p85 association, we observed that this interaction is induced by trastuzumab in some ERBB2-amplified cancer cell lines and coincides with a decrease in AKT phosphorylation. Thus, the PTEN-p85 interaction may be linked to PI3K/AKT down-modulation in settings where this pathway is aberrantly active, such as ERBB2 amplification. A similar decline in pAKT levels following trastuzumab treatment was reported previously by Nagata and colleagues (40). Collectively, these results raise the possibility that PTEN promotes the uncoupling of p85 from membrane receptor tyrosine kinases in addition to its known lipid phosphatasedependent tumor suppressor function. The role of the PAC in modulating sensitivity or resistance to trastuzumab and other tyrosine kinase inhibitors will provide an interesting avenue for further study.
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The precise inhibitory mechanism of the PAC remains incompletely elucidated; however, the relationship between PTEN and p85 may parallel that seen with PTEN and p53. The PTEN-p53 association stabilizes p53 while simultaneously upregulating PTEN transcription, thereby triggering cell cycle arrest and/or apoptosis (28, 35). On the other hand, activation of the PI3K/AKT pathway can result in p53 inactivation followed by downregulation of PTEN gene expression and cell proliferation (14, 59). We note that the PTEN-p85 complex may also be involved in additional regulatory functions associated with both proteins, including cell migration and cytoskeletal rearrangement. Toward this end, Raftopoulou and colleagues have suggested a novel function for PTEN as a regulator of cell migration. In particular, their evidence implicates the unphosphorylated C-terminal tail in this process (45). Notably, the same MEFs, derived from p85␣ and p85 knockout mice, that were used in this study were found to be defective in PDGF-beta-polypeptide chain-induced membrane ruffling and in PDGF-dependent actin remodeling, responsible ultimately for flawed cell migration (8). This indicates that p85 subunits, like PTEN, may also play a role in cell migration. Moreover, both PTEN and p85 have been biochemically identified at the cadherin junctional complexes, where they were coimmunoprecipitated with E-cadherin and catenins. Further, p85 interacts directly with -catenin (70). Interestingly, MAGI-1b (an established PTEN-interacting protein) has been shown to associate with both PTEN and -catenin through its PDZ domain (25). The cadherin junctional complex is actively involved in the regulation of cell proliferation, survival, and differentiation (58). These data suggest that the unphosphorylated PTEN/p85 complex may have a role in the regulation or sensing of the migration/adhesion state of the cell. The interaction between PTEN and p85 may be either direct or mediated by other proteins engaged in the ⬃670-kDa PAC. Further studies will be necessary to fully understand the nature of the PTEN/p85 complex and its involvement in PI3K/AKT pathway regulation and cellular functions such as adhesion. These results, however, imply that the p85 and PTEN complex is linked to the inhibition of pathway signaling and is likely linked to the function of PTEN as a tumor suppressor. In conclusion, our results indicate that unphosphorylated PTEN and p85 associate within the PAC and that this association may promote PI3K/AKT pathway downregulation. These findings may therefore point to a distinct mechanism by which PTEN exerts its tumor suppressor function and may carry important implications for biological and therapeutic understanding of this key cell signaling pathway. ACKNOWLEDGMENTS This work was supported by DOD W81XWH-05-1-0029 (R.R.) and R01CA085912-09 (W.R.S. and L.A.G.) and the Novartis Institute for Biomedical Research (W.R.S. and L.A.G.). REFERENCES 1. Al-Khouri, A. M., Y. Ma, S. H. Togo, S. Williams, and T. Mustelin. 2005. Cooperative phosphorylation of the tumor suppressor phosphatase and tensin homologue (PTEN) by casein kinases and glycogen synthase kinase 3beta. J. Biol. Chem. 280:35195–35202. 2. Anderson, C. W., J. W. Straus, and B. S. Dudock. 1983. Preparation of a cell-free protein-synthesizing system from wheat germ. Methods Enzymol. 101:635–644. 3. Barber, D. F., M. Alvarado-Kristensson, A. Gonzalez-Garcia, R. Pulido, and
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