Abstract
The mTOR (mammalian target of rapamycin) protein kinase is an important regulator of cell growth and is a key target for therapeutic intervention in cancer. Two complexes of mTOR have been identified: complex 1 (mTORC1), consisting of mTOR, Raptor (regulatory associated protein of mTOR) and mLST8 (mammalian lethal with SEC13 protein 8) and complex 2 (mTORC2) consisting of mTOR, Rictor (rapamycin-insensitive companion of mTOR), Sin1 (stress-activated protein kinase-interacting protein 1), mLST8 and Protor-1 or Protor-2. Both complexes phosphorylate the hydrophobic motifs of AGC kinase family members: mTORC1 phosphorylates S6K (S6 kinase), whereas mTORC2 regulates phosphorylation of Akt, PKCα (protein kinase Cα) and SGK1 (serum- and glucocorticoid-induced protein kinase 1). To investigate the roles of the Protor isoforms, we generated single as well as double Protor-1- and Protor-2-knockout mice and studied how activation of known mTORC2 substrates was affected. We observed that loss of Protor-1 and/or Protor-2 did not affect the expression of the other mTORC2 components, nor their ability to assemble into an active complex. Moreover, Protor knockout mice display no defects in the phosphorylation of Akt and PKCα at their hydrophobic or turn motifs. Strikingly, we observed that Protor-1 knockout mice displayed markedly reduced hydrophobic motif phosphorylation of SGK1 and its physiological substrate NDRG1 (N-Myc downregulated gene 1) in the kidney. Taken together, these results suggest that Protor-1 may play a role in enabling mTORC2 to efficiently activate SGK1, at least in the kidney.
- mammalian target of rapamycin (mTOR)
- mammalian target of rapamycin complex 2 (mTORC2)
- N-Myc down-regulated gene 1 (NDRG1)
- phosphoinositide 3-kinase (PI3K)
- Protor/PRR5
- serum- and glucocorticoid-induced protein kinase 1 (SGK1)
INTRODUCTION
mTOR (mammalian target of rapamycin) protein kinase plays a key role in controlling cell growth and is likely to be over-activated in the majority of cancers. mTOR exists in two evolutionarily conserved complexes, mTORC1 (mTOR complex 1) and mTORC2 (reviewed in [1]). mTORC1 consists of mTOR bound to Raptor (regulatory associated protein of mTOR) and mLST8 (mammalian lethal with SEC13 protein 8) and is inhibited by rapamycin [2–4]. mTORC1 is activated downstream of growth factors via the PI3K (phosphoinositide 3-kinase) pathway, involving Akt-mediated phosphorylation of PRAS40 (proline-rich Akt substrate of 40 kDa) and TSC2 (tuberous sclerosis complex 2), leading to activation of the Rheb GTPase [5,6]. In addition, mTORC1 activity is stimulated by amino acids through a pathway including Rag GTPases and MAPKKKK3 (mitogen-activated protein kinase kinase kinase kinase 3) [7–9]. Although mTORC2 also contains mLST8, Raptor is replaced by Rictor (rapamycin-insensitive companion of mTOR), Sin1 (stress-activated protein kinase-interacting protein 1) and one of the closely related isoforms of Protor-1 or Protor-2 [10–17]. mTORC2 is insensitive to acute rapamycin treatment, although it is also activated by growth factors through an ill-defined PI3K-dependent mechanism.
Some of the best-characterized substrates of mTORCs are the Akt, PKC (protein kinase C), SGK (serum- and glucocorticoid-induced protein kinase) and S6K (S6 kinase) protein kinases. These belong to the AGC (protein kinase A/protein kinase G/PKC) kinase family and typically members of this family are activated by phosphorylation of two key sites: the T-loop and the hydrophobic motif [18]. mTORC1 phasphorylates the hydrophobic motif of p70 ribosomal S6K [3,4], and mTORC2 regulates the equivalent site in Akt [19], PKCα [11] and SGK1 [20], accounting for the ability of mTOR to control protein synthesis, cell proliferation, survival, metabolism and reorganization of the actin cytoskeleton. Akt (Thr450) and PKCα (Thr638) are also stabilized by phosphorylation of their turn-motif, an event which is mediated by mTORC2 and that probably occurs during translation [21–23]. SGK1 was originally identified as a serum- and glucocorticoid-inducible gene and it is activated downstream of insulin and growth factors (reviewed in [24]). The generation of PtdIns(3,4,5)P3 by PI3K triggers a signalling cascade that leads to the phosphorylation of the hydrophobic motif (Ser422 in SGK1) by mTORC2 [20]. This does not directly activate SGK1, but instead creates a docking site for PDK1 (phosphoinositide-dependent kinase 1), which can subsequently phosphorylate SGK1 at the T-loop residue (Thr256), resulting in its activation [25,26]. One of the best-characterized substrates of SGK1 is NDRG1 (N-Myc down-regulated gene 1) and the phosphorylation of this protein is frequently used as a marker for SGK1 activity [20,27].
Protor-1 and Protor-2 isoforms are encoded by different genes and interact with Rictor through a conserved N-terminal region [15–17]. This may stabilize Protor, as total protein levels of Protor are vastly reduced in Rictor knockout fibroblasts [15]. As Protor isoforms have no other obvious functional domains it is hard to predict their physiological function. To address how Protor isoforms influence mTORC2, we generated single Protor-1−/− and Protor-2−/−, as well as Protor-1−/−Protor-2−/− double-knockout mice. Although phosphorylation of the hydrophobic and turn motifs of both Akt and PKCα was unaffected in Protor-1−/− mice, we observed a significant reduction in phosphorylation of the hydrophobic motif of SGK1 in kidney, a tissue in which SGK1 phosphorylation and activity is high. Protor-1-deficient mice also displayed a marked reduction in phosphorylation of the SGK1 T-loop and also that of NDRG1, a specific SGK1 substrate. Our results indicate that Protor-1 plays a critical role in mediating the phosphorylation of SGK1 by mTORC2 in the kidney.
MATERIALS AND METHODS
Materials
Glutathione–Sepharose, Protein G–Sepharose and Protein A–agarose were purchased from Amersham Bioscience. IGF1 (insulin-like growth factor 1) was from Cell Signaling Technology. Tween 20, DMSO and dimethyl pimelimidate were from Sigma, and CHAPS was from Calbiochem.
Antibodies
The following antibodies were raised in sheep and affinity purified on the appropriate antigen: anti-NDRG1 (S276B, 3rd bleed; raised against the full-length human protein, used for immunoblotting), anti-phospho-NDRG1 which recognizes NDRG1 phosphorylated at Thr346, Thr356 and Thr366 (S911B, 2nd bleed; raised against CRSRSHTpSEG, used for immunoblotting), anti-Akt1 (S742B, 3rd bleed; raised against the full-length human protein, used for immunoblotting), anti-PRAS40 [S115B, 1st bleed; residues 238–256 of human PRAS40 (DLPRPRLNTSDFQKLKRKY), used for immunoblotting], anti-phospho-PRAS40, which recognizes PRAS40 phosphorylated at Thr246 [S114B, 2nd bleed; raised against residues 240–251 of human PRAS40 (CRPRLNTpSDFQK), used for immunoblotting], anti-Protor-1 (S020C, 3rd bleed; raised against the full-length human protein, used for immunoprecipitation and immunoblotting), anti-Protor-2 (S136C, 3rd bleed; raised against the full-length human protein, used for immunoprecipitation and immunoblotting), anti-Rictor [S274C, 1st bleed; raised against residues 6–20 of mouse Rictor (RGRSLKNLRIRGRND), used for immunoblotting], anti-Sin1 (S008C, 2nd bleed; raised against full-length human Sin1, used for immunoprecipitation and immunoblotting) and anti-SGK1 (S515A, 2nd bleed; raised against full-length human SGK1, used for immunoprecipitation and immunoblotting). The total SGK1 (#5188) and the γ-ENaC (γ-epithelial sodium channel) antibodies were purchased from Sigma. The antibody recognizing the α-subunit of ENaC was a gift from Dr S. Wilson (Division of Maternal & Child Health Sciences, Dundee University, Dundee, Scotland, U.K.) and Dr R.C. Boucher (School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, U.S.A.). The total anti-mTOR (#sc-1549), anti-(phospho-SGK1 Ser422) (#sc16745) and anti-flotillin-1 (#sc-25506) antibodies were purchased from Santa Cruz Biotechnology. The anti-phospho-Akt Ser473 (#9271), Thr308 (#4056), anti-phospho-IGF1/insulin receptor (#3024), anti-IGF1 receptor (#3027), anti-total FoxO1 (forkhead box O1; #2880), anti-phospho-FoxO1/3 (#9464) and anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (#2118) antibodies were purchased from Cell Signaling Technology. For immunoblotting of the phosphorylated T-loop of SGK1, we employed the pan-PDK1 site antibody from Cell Signaling Technology (#9379).
General methods
Tissue culture, immunoblotting, restriction enzyme digests, DNA ligations and other recombinant DNA procedures were performed using standard protocols. DNA constructs used for transfection were purified from Escherichia coli DH5α using a Qiagen plasmid Mega or Maxi kit according to the manufacturer's protocol. All DNA constructs were verified by DNA sequencing, which was performed by DNA Sequencing and Services (MRCPPU, College of Life Sciences, University of Dundee, Scotland; http://www.dnaseq.co.uk) using Applied Biosystems Big-Dye Ver 3.1 chemistry on an Applied Biosystems model 3730 automated capillary DNA sequencer.
Generation, genotyping and maintenance of Protor-1−/−, Protor-2−/− and Protor-1−/−Protor-2−/− mice
Protor-1fl/+ and Protor-2fl/+ mice were generated by TaconicArtemis as described in Figure 1. Protor-1+/fl mice were crossed with Bal1Cre+/− mice which express Cre recombinase in all tissues [28], enabling the generation of mice containing one Protor-1-knockout allele (Protor-1+/−). These heterozygous mice were then crossed together in order to obtain whole-body Protor-1 knockout mice (Protor-1−/−). Whole-body Protor-2−/− mice were generated using the same strategy. The mice were maintained on a C57BL/6J background and genotyping was carried out by PCR using genomic DNA isolated from ear or tail biopsies. For Protor-1 mice, Primer 1 (5′-GAGTTCATCTTCAAACCCAAGC-3′), Primer 2 (5′-CCCGTGCCAGATTAACATGG-3′) and Primer 6 (5′-TCAACATGACGAAGTCAAGTGTC-3′) were used to detect the wild-type, heterozygous and knockout alleles. For Protor-2 mice, Primer 1 (5′-AACCTGGGAAAGGAAGAAGC-3′), Primer 2 (5′-GCACTCAAAATCTCTGTGGC-3′) and Primer 4 (5′-TCTGATTCTCCCACCTGAAGT-3′) were utilized. The PCR programme consisted of 5 min at 95°C, 35 cycles of 30 s at 95°C, 30 s at 60°C and 1 min at 72°C, followed by 10 min at 72°C on a PTC-200 Peltier Therma Cycler DNA engine. Mice were maintained under specific pathogen-free conditions and routine animal ear notching for genotyping was carried out by staff in the College of Life Science Transgenic Unit (University of Dundee). All procedures were carried out in accordance with the regulations set by the University of Dundee and the United Kingdom Home Office.
(A) Diagram depicting the Protor-1 targeting construct, the endogenous Protor-1 allele, the targeted allele obtained following homologous recombination, the Protor-1 conditional knockout allele with the neomycin cassette removed by Flp recombinase and the constitutive knockout allele obtained following Cre-mediated recombination. The light grey rectangles represent exons, black rectangles indicate the targeted exons and the dark grey and light grey triangles represent loxP and FRT sites respectively. The positions of the 5′ and 3′ probes used for Southern blot analysis and the Protor-1 PCR primers (P1, P2 and P6) used for genotyping are indicated. The translation initiation sites for Protor-1α/β and Protor-1γ are within exons 2 and 4 respectively. (B) Genomic DNA purified from the targeted ES (embryonic stem) cells of the indicated genotypes was digested with either BamHI or AseI and subjected to Southern blot analysis with the corresponding DNA probes. In the case of the 5′ probe, the wild-type allele produces a 4.9 kb fragment whereas the conditional knockout allele generates a 7 kb fragment. The 3′ probe detects a fragment of 25.5 kb from the wild-type allele and a 15.1 kb fragment from the conditional knockout allele. (C) Genomic DNA was PCR amplified with Protor-1 primers P1, P2 and P6. The wild-type allele (detected using P1 and P2) generates a 290 bp product, whereas the knockout allele (detected using P1 and P6) generates a 559 bp product. (D) Protor-1 was immunoprecipitated (IP) from each of the indicated mouse tissue lysates and HEK-293 cell lysate and immunoblotting (IB) was carried out using the same antibody. (E) Diagram depicting the Protor-2 targeting construct, the endogenous Protor-2 allele, the targeted allele obtained following homologous recombination, the Protor-2 conditional knockout allele and the constitutive knockout allele. The positions of the Protor-2 PCR primers (P1, P2 and P4) used for genotyping are indicated with arrows. The translation initiation site for Protor-2 is within exon 2. (F) Genomic DNA purified from the targeted ES cells of the indicated genotypes was digested with either EcoRI or HindIII and subjected to Southern blot analysis with the corresponding DNA probes. In the case of the 5′ probe, the wild-type allele produces a 6.8 kb fragment whereas the conditional knockout allele generates a 9.8 kb fragment. The 3′ probe detects a fragment of 12 kb from the wild-type allele and a 10.6 kb fragment from the conditional knockout allele. (G) Genomic DNA was PCR amplified with the Protor-2 primers P1, P2 and P4. The wild-type allele (detected using P1 and P2) generates a 221 bp product, whereas the knockout allele (detected using P1 and P4) generates a 527 bp product. (H) Protor-2 was immunoprecipitated (IP) from each of the indicated mouse tissue lysates and immunoblotting (IB) was carried out using the same antibody.
Blood glucose and plasma insulin measurement
Blood glucose levels were measured using the Ascensia Breeze 2 blood glucose monitoring system (Bayer) following tail incision. For plasma insulin measurements, blood was collected in haematocrit capillary tubes (Hawksley) from mice following tail incision and incubated on ice. Blood was centrifuged at 3000 g for 15 min to obtain plasma. Insulin levels were measured using a rat/mouse insulin ELISA kit (Millipore; EZRMI-13K). Assays were carried out according to the manufacturer's instructions, typically this involved the use of 5 μl of plasma per assay and mouse insulin standards from 0 to 10 ng/ml were used to generate a standard curve. Samples were assayed in duplicate.
Glucose tolerance test
Mice were deprived of food overnight (16 h), weighed and basal blood glucose levels were measured following tail incision. Mice were injected intraperitoneally with 2 mg of D-glucose solution/g of body weight and blood glucose levels were measured at 15, 30, 45, 60 and 120 min post-injection.
Insulin tolerance test
Mice were allowed to feed overnight ad libitum and then food was removed for 3 h before the experiment. Mice were weighed and the basal blood glucose level was determined following tail incision. Mice were then injected intraperitoneally with 1 m-unit of insulin/g of body weight and blood glucose levels were measured at 15, 30, 45, 60 and 120 min post-injection.
Injection of mice with IGF1
Mice were starved for 3 h before being anaesthetized using sodium pentobarbital (80–90 μg/g of body weight intraperitoneally injected) and placed on a heating pad to maintain body temperature. This was followed by an intravenous injection through the inferior vena cava of either PBS or 0.5 μg of IGF1/g of body weight dissolved in PBS [29]. Tissues were rapidly excised 5 min after injection and frozen in liquid nitrogen.
Buffers
The following buffers were used: Tris-CHAPS lysis buffer (50 mM Tris/HCl, pH 7.5, 1 mM EGTA, 1 mM EDTA, 0.3% CHAPS, 1 mM sodium orthovanadate, 10 mM sodium β-glycerophosphate, 50 mM sodium fluoride, 5 mM sodium pyrophosphate, 0.27 M sucrose, 0.15 M NaCl, 0.1% 2-mercaptoethanol, 1 mM benzamidine and 0.1 mM PMSF); Buffer A (50 mM Tris/HCl, pH 7.5, 0.1 mM EGTA and 0.1% 2-mercaptoethanol); Hepes-CHAPS lysis buffer (40 mM Hepes, pH 7.5, 120 mM NaCl, 1 mM EDTA, 0.3% CHAPS, 10 mM sodium pyrophosphate, 10 mM sodium β-glycerophosphate, 50 mM sodium fluoride, 0.5 mM sodium orthovanadate, 1 mM benzamidine and 0.1 mM PMSF); Hepes kinase buffer (25 mM Hepes, pH 7.5, and 50 mM KCl); TBS (Tris-buffered saline)-Tween buffer (50 mM Tris/HCl, pH 7.5, 0.15 M NaCl and 0.1% Tween 20) and sample buffer [50 mM Tris/HCl, pH 6.8, 6.5% (v/v) glycerol, 1% (w/v) SDS and 1% (v/v) 2-mercaptoethanol].
Preparation of tissue lysates
Mouse tissues were rapidly excised, snap-frozen in liquid nitrogen and stored at −80°C until use. Frozen tissues were weighed and homogenized in a 10-fold excess of ice-cold Tris-CHAPS or Hepes-CHAPS lysis buffer using a Polytron homogenizer. Lysates were centrifuged at 3000 g for 15 min at 4°C and supernatants were then further centrifuged at 15000 g for 20 min at 4°C to remove all insoluble material. The protein concentration of each lysate was determined before being snap-frozen in aliquots and stored at −80°C.
Purification of inactive GST (glutathione transferase)–Akt1 and GST–ΔN-SGK1 (61–431) from HEK-293 cells
HEK (human embryonic kidney)-293 cells (10-cm-diameter dishes) were cultured and each dish was transfected with 5 μg of either GST–Akt1 or GST–ΔN-SGK1-(61–431) using the PEI (polyethyleneimine) method. At 24 h post-transfection, HEK-293 cells that had been transfected with GST–Akt1 or GST–ΔN-SGK1-(61–431) were serum-starved for 16 h. Cells were treated with 1 μM of the inhibitor PI-103 for 30 min and harvested in Tris-CHAPS lysis buffer. Lysate (3 mg) was affinity purified on 10 μl of glutathione–Sepharose for 1 h at 4°C on a rotating wheel. The resulting precipitates were washed twice with Tris-CHAPS lysis buffer, twice with Buffer A and twice with Buffer A containing 0.27 M sucrose. GST-tagged proteins were eluted from the resin by resuspension in an equal amount of Buffer A containing 0.27 M sucrose and 10 mM glutathione (pH 7.5–8) for 1 h on ice. Supernatants were filtered through a 0.22 μm spin column and aliquots were snap-frozen and stored at −80°C.
mTORC2 activity assays
Mouse tissues or MEFs (mouse embryonic fibroblasts) were lysed in Hepes-CHAPS lysis buffer and 1–4 mg of lysate was pre-cleared by incubation with 5 μl of Protein G–Sepharose conjugated to pre-immune IgG. The lysates were then incubated with 5 μl of Protein G–Sepharose covalently conjugated to either 5 μg of anti-Sin1 antibody or 5 μg of pre-immune IgG for 2 h at 4°C on a vibrating platform. The immunoprecipitates were washed four times with Hepes-CHAPS lysis buffer, followed by two washes with Hepes kinase buffer. Assays were carried out in a final volume of 40 μl of Hepes kinase buffer containing 1 μg of GST–Akt1 or 1 μg of GST–ΔN-SGK1-(61–431) and kinase reactions were initiated upon addition of 0.1 mM ATP and 10 mM MgCl2. Reactions were carried out for 30 min at 30°C on a vibrating platform and stopped by the addition of SDS sample buffer. Samples were heated to 70°C for 5 min, passed through a Spin-X column and subjected to electrophoresis and immunoblot analysis.
Immunoprecipitation of endogenous Protor-1 and Protor-2
Lysate (1 mg for Protor-1 immunoprecipitates or 2 mg for Protor-2 immunoprecipitates) from each of the indicated tissues was pre-cleared by incubation with 5 μl of Protein G–Sepharose for 30 min. The lysates were then incubated with 5 μg of anti-Protor-1 or anti-Protor-2 antibody covalently coupled to Protein G–Sepharose for 1.5 h at 4°C. Immunoprecipitates were washed four times with Tris-CHAPS lysis buffer lacking 2-mercaptoethanol and twice with Buffer A in which reducing agent was omitted. Immunoprecipitates were resuspended in 20 μl of SDS sample buffer (lacking reducing agent) and filtered through a Spin-X column to remove the Sepharose resin. NuPAGE reducing agent (1×; Invitrogen) was added to the eluted samples. These were then subjected to electrophoresis and immunoblot analysis as described below.
Kidney membrane preparations for ENaC immunoblots
Kidney membrane preperations were undertaken as described previously [30]. Harvested mouse kidneys were snap-frozen immediately after isolation and homogenized in detergent-free lysis buffer containing 50 mM Tris/HCl, pH 7.5, 1 mM EDTA, 1 mM EGTA, 1 mM sodium orthovanadate, 5 mM sodium fluoride, 5 mM sodium pyrophosphate, 0.27 M sucrose, 0.1% 2-mercaptoethanol and protease inhibitors (Roche, 1 tablet per 50 ml) with a Polytron homogenizer. Nuclei and debris were pelleted by centrifugation at 1500 g for 5 min. The procedure was repeated at 3000 g for 10 min and the low-speed supernatants were next centrifuged at 100000 g for 1 h. The pellet was washed with 1 vol. of lysis buffer without reducing agent and centrifuged again at 100000 g for 15 min to remove any remaining cytosolic particles. The pellet was then resuspended in lysis buffer including 1% (v/v) Nonidet P40 and centrifuged at 16500 g for 10 min. The protein content of the membrane preparations was quantified and membranes were resuspended in SDS sample buffer.
Immunoblotting
Tissue or cell lysates (10–20 μg), or immunoprecipitated samples were heated at 70°C for 5 min in sample buffer and subjected to PAGE and electrotransfer on to nitrocellulose membranes. Membranes were blocked for 1 h in TBS-Tween buffer containing 5% (w/v) non-fat dried skimmed milk. The membranes were probed with the indicated antibodies in TBS-Tween containing 5% (w/v) non-fat dried skimmed milk or 5% (w/v) BSA for 16 h at 4°C. Detection was performed using horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence reagent.
Generation and stimulation of MEFs
MEFs isolated from mouse embryos at E13.5 (embryonic day 13.5) were generated and cultured as described previously [31]. Cells were serum-starved in DMEM (Dulbecco's modified Eagle's medium) for 16 h prior to stimulation with 50 ng/ml IGF1 for 30min. Cells were subsequently lysed and the lysates were centrifuged at 18000 g for 15 min at 4°C. Supernatants were snap-frozen and stored at −80°C. Lysates (10 μg) were analysed by immunoblotting using the antibodies indicated in the Figures.
RESULTS
Generation of Protor-1−/−, Protor-2−/− and Protor-1−/−Protor-2−/− mice
To study the physiological role of the Protor isoforms, we generated Protor-1−/− and Protor-2−/− mice as described in the Materials and methods section and Figure 1. The breeding strategy for the Protor-1−/− and Protor-2−/−, as well as the Protor-1−/−Protor-2−/− mice is summarized in Table 1. Mice were generated and maintained on a pure C57BL/6J background. The single- and double-knockout mice were viable, born at the expected Mendelian frequency (Table 1) and did not display any overt phenotype. The genotype of the Protor-1−/− and Protor-2−/− mice was verified by PCR (Figures 1C and 1G). In order to confirm the ablation of protein expression, Protor-1 was immunoprecipitated from the tissues of the Protor-1 mice and, as expected, there was a reduction in Protor-1 expression of approximately 50% in the Protor-1+/− mice and Protor-1 could not be detected in the homozygous knockout mice (Figure 1D). A similar result was also obtained in the Protor-2−/− mice (Figure 1H). There was no significant difference in the body weight of male or female Protor-1−/− or Protor-2−/− or Protor-1−/−Protor-2−/− mice compared with wild-type mice up to 4 months of age (results not shown). In addition, the Protor-1−/− animals displayed normal blood glucose and plasma insulin levels under both fasted and fed conditions, and there was no detectable change in whole-body glucose or insulin tolerance compared with wild-type animals (see Supplementary Figure S1 at http://www.BiochemJ.org/bj/436/bj4360169add.htm).
Homozygous Protor-1−/− or Protor-2−/− as well as Protor-1−/−Protor-2−/− mice were bred using the depicted strategy. The number and the percentage of each genotype are indicated followed by its expected Mendelian frequency. In the case of the Protor-1−/−Protor-2−/− mice, as wild-type littermate and double-knockin mice can only be obtained at a ratio of 1:16 in the same cross (Table 1), we interbred Protor-1−/−Protor-2−/− mice as well as Protor-1+/+Protor-2+/+ animals.
NDRG1 phosphorylation is diminished in the kidney of Protor-1−/− mice
We initially examined the phosphorylation state of Akt, its substrates PRAS40 and FoxO1, as well as NDRG1, an SGK1 substrate, in various tissues from fed littermate Protor-1+/+, Protor-1+/− and Protor-1−/− mice. This revealed no significant difference in Akt, PRAS40 or FoxO1 phosphorylation in any of the tissues examined among the three genotypes (Figure 2). The only tissue in which significant phosphorylation of NDRG1 was observed was in the kidney of Protor-1+/+ animals (Figure 2A). Consistent with this, the NDRG1 protein migrated as several electrophoretic species in kidney and only as a single species in other tissues. Interestingly, there was a partial reduction in the phosphorylation of NDRG1 in the kidney of Protor-1+/− animals and this was more pronounced in the Protor-1−/− mice (Figure 2A).
Tissue lysates from (A) kidney, (B) adipose, (C) brain, (D) heart, (E) liver (F) lung (G) muscle, (H) spleen or (I) testis were generated from fed mice of the indicated genotype and subjected to immunoblotting (IB) with the indicated antibodies.
We next investigated NDRG1 phosphorylation in kidney extracts derived from single and double Protor-1- and Protor-2-knockout animals. Although there was no change in NDRG1 phosphorylation in the kidney of Protor-2−/− mice (Figure 3B), the Protor-1−/−Protor-2−/− mice displayed reduced NDRG1 phosphorylation, similar to that seen in the Protor-1−/− mice (Figure 3C). In all cases, Akt (Thr308 and Ser473) and PRAS40 phosphorylation was unaffected by the loss of Protor-1 and/or Protor-2. In addition, the phosphorylation of Akt and PKCα at the turn motif (Thr450 and Thr638 respectively) and the PKCα hydrophobic motif (Ser657) remained unchanged in the kidney of the Protor isoform knockout mice (Figures 3A–3C). We also investigated phosphorylation of mTORC2 targets in the lung and liver tissues of the Protor-2−/− mice as well as the Protor-1−/−Protor-2−/− mice. This confirmed that there was no substantial phosphorylation of NDRG1 detected in these tissues and that phosphorylation of Akt and its substrates PRAS40 and FoxO1 was not reduced in the Protor-deficient animals (see Supplementary Figure S2 at http://www.BiochemJ.org/bj/436/bj4360169add.htm).
(A) Kidney lysates were generated from fed Protor-1+/+, Protor-1+/− and Protor-1−/− mice and subjected to immunoblotting (IB) with the indicated antibodies. (B) As in (A), except lysates were from Protor-2+/+, Protor-2+/− and Protor-2−/− mice. (C) As in (A), except lysates were from Protor-1+/+Protor-2+/+ and Protor-1−/−Protor-2−/− mice.
IGF1-induced SGK1 phosphorylation is reduced in the kidney of Protor-1−/− mice
To investigate further the role that Protor-1 plays in regulating SGK1 activation in the kidney, single and double Protor-deficient mice were injected intravenously with 0.5 μg of IGF1/g of body weight for 5 min to activate the PI3K pathway in a wide variety of tissues (instead of limited insulin-sensitive tissues). Tissue extracts derived from these animals were probed using antibodies that recognize SGK1 phosphorylated at its hydrophobic motif (Ser422) as well as its T-loop residue (Thr256). Although SGK1 hydrophobic motif phosphorylation was stimulated upon IGF1 treatment in the wild-type control mice, phosphorylation of this site was barely detectable in the Protor-1−/− mice (Figure 4A) or Protor-1−/−Protor-2−/− mice (Figure 4C). Consistent with phosphorylation of Ser422 being required for the subsequent phosphorylation of Thr256 [25], there was also no phosphorylation of Thr256 in these animals. The loss of Ser422 and Thr256 phosphorylation would result in a lack of SGK1 activation, which accounts for the much-reduced phosphorylation of NDRG1. In contrast, SGK1 and NDRG1 were phosphorylated to the same extent in kidney extracts derived from wild-type and Protor-2−/− knockout mice (Figure 4B). There was also no significant difference in the IGF1-induced phosphorylation of Akt at either Ser473 or Thr308 or that of PRAS40 in the kidney of Protor-1- and/or Protor-2-deficient mice (Figure 4).
(A) Protor-1+/+ and Protor-1−/− mice were deprived of food for 3 h and intravenously injected with 0.5 μg of IGF1/g of body weight (+) or the equivalent amount of PBS as a negative control (−). After 5 min, tissues were rapidly removed and frozen in liquid nitrogen. Kidney lysates were subjected to immunoblotting (IB) with the indicated antibodies and each lane represents a sample derived from a different mouse. (B) As in (A), except Protor-2+/+ and Protor-2−/− mice were injected with IGF1. (C) As in (A) except Protor-1+/+Protor-2+/+ and Protor-1−/−Protor-2−/− mice were injected with IGF1.
NDRG1 phosphorylation was also assessed in response to IGF1 stimulation in the liver of the single-knockout mice and Protor-1−/−Protor-2−/− mice (see Supplementary Figure S3 at http://www.BiochemJ.org/bj/436/bj4360169add.htm). We observed that IGF1 did not lead to a marked increase in NDRG1 phosphorylation and, consistent with this, did not induce an electrophoretic band-shift of the total NDRG1 protein under conditions in which Akt and its substrate PRAS40 were phosphorylated (see Supplementary Figure S3). We also generated MEFs derived from wild-type and Protor-1−/−Protor-2−/− mice and found that IGF1 induced a significant phosphorylation and an electrophoretic band-shift of NDRG1 protein in both wild-type and Protor-1−/−Protor-2−/− MEFs (Figure 5). This observation is considered further in the Discussion.
The wild-type and Protor-1−/−Protor-2−/− MEF cells were serum-starved overnight and stimulated with 50 ng/ml of IGF1 for 30 min prior to lysis and immunoblot (IB) analysis with the indicated antibodies. Similar results were obtained in two separate experiments.
mTORC2 formation is unaffected by the loss of Protor-1 and Protor-2
Previously it was reported that loss of the mTORC2 component Sin1 affects the ability of mTOR and Rictor to interact [12–14]. As reduced mTORC2 complex formation could potentially account for the decreased phosphorylation of SGK1, the ability of mTOR, Rictor and Sin1 to bind to one another was investigated in the Protor-1−/− and Protor-2−/− single- and double-knockout animals. mTOR and Rictor still co-immunoprecipitated with Sin1, irrespective of the presence of either Protor-1 or Protor-2 in kidney (Figures 6A–6C). This provides evidence that Protor-1 and Protor-2 are not essential for mTORC2 complex assembly.
(A–C) Sin1 was immunoprecipitated (IP) from kidney lysates obtained from mice of the indicated genotype and subjected to immunoblotting (IB) with the indicated antibodies. Similar results were obtained in two separate experiments.
Protor isoforms are not required for mTORC2 kinase activity in vitro
We next investigated whether Protor isoforms are required for the kinase activity of mTORC2. mTORC2 was immunoprecipitated using the Sin1 antibody from either fed (Figures 7A and 7B) or IGF1-injected (Figures 7C and 7D) wild-type or double Protor-1−/− and Protor-2−/− knockout mouse kidney or liver lysates, and its ability to phosphorylate GST–Akt1 or GST–SGK1 was assessed. These studies revealed that loss of Protor isoforms did not have a significant impact on the ability of immunoprecipitated mTORC2 to phosphorylate GST–Akt1 or GST–SGK1 in cell free-assays. Similar results were obtained when mTORC2 activity was assayed in the Protor-1−/−Protor-2−/− MEFs (Figures 7E and 7F). These findings indicate that Protor isoforms are dispensable for regulating intrinsic mTORC2 kinase activity.
(A) Immunoprecipitates were prepared using either pre-immune IgG (negative control) or Sin1 antibodies from fed wild-type or Protor-1−/−Protor-2−/− mouse kidney or liver lysates. Immunoprecipitates (IP) were incubated with GST–Akt1 in the presence of 0.1 mM ATP and 10 mM MgCl2 for 30 min at 30°C. Kinase reactions were analysed by immunoblotting (IB) with the indicated antibodies. (B) As in (A), except GST–ΔN-SGK1-(61–431) was used as the substrate in the mTORC2 kinase assay. (C) As in (A), except immunoprecipitates were generated from kidney or liver lysates from mice injected with IGF1 for 5 min. (D) As in (C), except GST–ΔN-SGK1-(61–431) was used as the substrate in the mTORC2 kinase assay. (E) as in (A), except immunoprecipitates were prepared from wild-type or Protor-1−/−Protor-2−/− MEF cells. (F) as in (E), except GST–ΔN-SGK1-(61–431) was used as the substrate in the mTORC2 kinase assay. Similar results were obtained in two separate experiments.
DISCUSSION
The results of the present study indicate that, in contrast with the other mTORC2-specific components Sin1 and Rictor, Protor subunits are not essential for mouse viability, growth, mTORC2 complex assembly or phosphorylation of the hydrophobic and turn motifs of Akt. Studies using tissue-specific knockouts of Rictor in both muscle and adipose tissue have demonstrated the key role mTORC2 plays in insulin signalling and glucose homoeostasis. Muscle-specific Rictor−/− mice have defective insulin-stimulated glucose uptake in muscle and are glucose intolerant [32], and adipose-specific knockouts of Rictor display impaired glucose uptake and reduced whole-body insulin sensitivity [33,34]. However, we did not observe any significant defects in glucose clearance and insulin sensitivity, consistent with Akt Ser473 phosphorylation being unaffected in the single-knockout mice or Protor-1−/−Protor-2−/− mice. These observations indicate that the role of Protor-1 is distinct from that of Rictor and is not required for activation of Akt, at least under the conditions that we have studied.
Instead, our results from the present study suggest that Protor-1 is required for efficient activation of SGK1 in the kidney, the only tissue in which we were able to measure significant endogenous hydrophobic and T-loop motif phosphorylation of SGK1 and that of its substrate NDRG1. Interestingly, Protor-1 expression was previously shown to be high in the kidney, perhaps indicating the key role Protor-1 plays in this tissue [15]. Our results also indicate that Protor-1 rather than Protor-2 is critical for efficient activation of SGK1 in the kidney, as defects in SGK1 and NDRG1 phosphorylation were not observed in the Protor-2-knockout animals. Moreover, the Protor-1−/−Protor-2−/− mice displayed identical reductions in SGK1 and NDRG1 phosphorylation to the Protor-1−/− mice, emphasizing that Protor-1 rather that Protor-2 is key for regulating SGK1 activation in the kidney.
In contrast with what was observed in kidney tissues, IGF1 induced a significant phosphorylation and an electrophoretic band-shift of NDRG1 protein in both wild-type and Protor-1−/−Protor-2−/− MEFs, suggesting that the effects of Protor on SGK1 activation and NDRG1 phosphorylation may be specific to the kidney. In our hands, endogenous SGK1 protein in MEFs is undetectable using antibodies that readily detect SGK1 in the kidney. It is possible that when SGK1 levels are low, much higher levels of Akt or a distinct kinase contribute to observed NDGR1 phosphorylation in MEFs. Moreover, SGK2 or SGK3, which we did not assess, may not be regulated by Protor isoforms and if this was the case, might account for observed NDGR1 phosphorylation in Protor-deficient MEFs.
The best-characterized role of SGK1 in vivo is in the kidney where it stimulates sodium transport into epithelial cells by enhancing the stability and expression of ENaC (reviewed in [35]). The expression of ENaC is typically maintained at a low level by ubiquitination and degradation. SGK1 phosphorylates the HECT (homologous with E6-associated protein C-terminus) E3 ubiquitin ligase, Nedd4.2, preventing its interaction with ENaC and hence preventing ENaC degradation [36–38]. Consistent with this, SGK1−/− mice have a salt wasting phenotype, excreting more Na+ than wild-type mice when on a low-salt diet, leading to reduced blood pressure [39,40]. SGK1−/− mice are also resistant to dexamethasone-induced increases in blood pressure [41] and display reduced glucocorticoid-induced intestinal glucose transport [42]. As SGK1 activity is reduced in the Protor-1−/−Protor-2−/− mice, ENaC expression may also be lowered. However, we observed similar levels of ENaCα and ENaCγ subunits in membrane fractions derived from the kidney tissue of wild-type and Protor-1−/− Protor-2−/− mice (see Supplementary Figure S4 at http://www.BiochemJ.org/bj/436/bj4360169add.htm), suggesting that Protor isoforms are not essential for normal regulation of basal ENaC expression in otherwise healthy unstressed animals. It would be interesting to test whether under conditions of salt challenge or in hypertensive models, Protor isoforms play a more dominant role in the control of ENaC expression.
It will also be necessary to address the mechanism by which Protor regulates efficient activation of SGK1 in the kidney. Our studies suggest that loss of Protor isoforms does not have an impact on the ability of immunoprecipitated mTORC2 to phosphorylate SGK1 or Akt. This raises the possibility that Protor-1 may instead act as a scaffolding component to localize SGK1 to a particular location within the cell and/or to present SGK1 to mTORC2. In Saccharomyces cerevisiae TORC2, there are two non-essential components termed Bit61 and AVO2 [2,43], that have been implicated in binding two substrates of TORC2 (Slm1 and Slm2) [44,45]. Although there is no obvious sequence similarity between Protor isoforms and Bit61 and AVO2, it would be interesting to investigate whether Protor isoforms performed a similar function. Thus far we have been unable to demonstrate a convincing co-immunoprecipitation of endogenous Protor-1 and SGK1 derived from kidney extracts. However, we cannot rule out the possibility that SGK1 and Protor-1 may interact with one another with low micromolar affinity. This would not be observed in co-immunoprecipitation studies, as the subunits would dissociate under the conditions and timeframe of such experiments.
In conclusion, our results from the present study establish that Protor-1 is required for SGK1 phosphorylation and signalling in the kidney. Further work is required to delineate the mechanism by which Protor-1 regulates efficient activation of SGK1 in the kidney and whether Protor isoforms are required for the activation of SGK1 and other SGK isoforms in other tissues and cell types. In addition, our results indicate that Protor-1 and Protor-2 play distinct roles and further work will be required to uncover the function of Protor-2. It is also possible that Protor isoforms play a key role in regulating mTORC2 function under conditions that we have not investigated. Furthermore, mTORC2 may phosphorylate substrates other than Akt, SGK and perhaps PKC isoforms, so in the future it will be important to investigate the role of Protor isoforms in controlling these, as yet uncharacterized, functions of mTORC2. The availability of Protor-knockout mouse models and MEFs should prove useful for further studies to define the roles of Protor subunits in regulating mTORC2.
AUTHOR CONTRIBUTION
Laura Pearce performed most of the experiments with the help of Eeva Sommer, and Kei Sakamoto performed IGF1 injections and provided critical advice. Laura Pearce and Stephan Wullschleger performed the experiments shown in Supplementary Figure S1. Laura Pearce, Eeva Sommer and Dario Alessi planned experiments, analysed the data and wrote the manuscript.
FUNDING
L.R.P. is funded by an MRC UK Studentship and E.M.S. is supported by an AstraZeneca supported BBSRC-CASE PhD Studentship. We thank the Medical Research Council, and the pharmaceutical companies supporting the Division of Signal Transduction Therapy Unit (AstraZeneca, Boehringer-Ingelheim, GlaxoSmithKline, Merck KgaA and Pfizer) for financial support.
Acknowledgments
We thank Gail Fraser for genotyping of the mice, the Sequencing Service (College of Life Sciences, University of Dundee, Dundee, Scotland, U.K.) for DNA sequencing, and the protein production and antibody purification teams [Division of Signal Transduction Therapy (DSTT), University of Dundee] co-ordinated by Hilary McLauchlan and James Hastie.
Abbreviations: ENaC, epithelial sodium channel; FoxO, forkhead box O; GST, glutathione transferase; HEK, human embryonic kidney; IGF1, insulin-like growth factor 1; MEF, mouse embryonic fibroblast; mLST8, mammalian lethal with SEC13 protein 8; mTOR, mammalian target of rapamycin; mTORC, mTOR complex; NDRG1, N-Myc down-regulated gene 1; PDK1, phosphoinositide-dependent kinase 1; PI3K, phosphoinositide 3-kinase; PKC, protein kinase C; PRAS40, proline-rich Akt substrate of 40 kDa; Raptor, regulatory associated protein of mTOR; Rictor, rapamycin-insensitive companion of mTOR; S6K, S6 kinase; SGK, serum- and glucocorticoid-induced protein kinase; Sin1, stress-activated protein kinase-interacting protein 1
- © The Authors Journal compilation © 2011 Biochemical Society
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May 2011
Volume: 436 Issue: 1
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