USP 1 deubiquitinates Akt to inhibit PI 3K‐Akt‐FoxO signaling in muscle during prolonged starvation
2020; Springer Nature; Volume: 21; Issue: 4 Linguagem: Inglês
10.15252/embr.201948791
ISSN1469-3178
AutoresDana Goldbraikh, Danielle Neufeld, Yara Eid‐Mutlak, Inbal Lasry, Jennifer E. Gilda, Anna Parnis, Shenhav Cohen,
Tópico(s)Genetics and Neurodevelopmental Disorders
ResumoArticle5 March 2020free access Source Data USP1 deubiquitinates Akt to inhibit PI3K-Akt-FoxO signaling in muscle during prolonged starvation Dana Goldbraikh Faculty of Biology, Technion Institute of Technology, Haifa, Israel Search for more papers by this author Danielle Neufeld Faculty of Biology, Technion Institute of Technology, Haifa, Israel Search for more papers by this author Yara Eid-Mutlak Faculty of Biology, Technion Institute of Technology, Haifa, Israel Search for more papers by this author Inbal Lasry Faculty of Biology, Technion Institute of Technology, Haifa, Israel Search for more papers by this author Jennifer E Gilda Faculty of Biology, Technion Institute of Technology, Haifa, Israel Search for more papers by this author Anna Parnis Faculty of Biology, Technion Institute of Technology, Haifa, Israel Search for more papers by this author Shenhav Cohen Corresponding Author [email protected] orcid.org/0000-0001-8307-466X Faculty of Biology, Technion Institute of Technology, Haifa, Israel Search for more papers by this author Dana Goldbraikh Faculty of Biology, Technion Institute of Technology, Haifa, Israel Search for more papers by this author Danielle Neufeld Faculty of Biology, Technion Institute of Technology, Haifa, Israel Search for more papers by this author Yara Eid-Mutlak Faculty of Biology, Technion Institute of Technology, Haifa, Israel Search for more papers by this author Inbal Lasry Faculty of Biology, Technion Institute of Technology, Haifa, Israel Search for more papers by this author Jennifer E Gilda Faculty of Biology, Technion Institute of Technology, Haifa, Israel Search for more papers by this author Anna Parnis Faculty of Biology, Technion Institute of Technology, Haifa, Israel Search for more papers by this author Shenhav Cohen Corresponding Author [email protected] orcid.org/0000-0001-8307-466X Faculty of Biology, Technion Institute of Technology, Haifa, Israel Search for more papers by this author Author Information Dana Goldbraikh1,‡, Danielle Neufeld1,‡, Yara Eid-Mutlak1, Inbal Lasry1, Jennifer E Gilda1, Anna Parnis1 and Shenhav Cohen *,1 1Faculty of Biology, Technion Institute of Technology, Haifa, Israel ‡These authors contributed equally to this work *Corresponding author. Tel: +972 4 8294214; E-mail: [email protected] EMBO Rep (2020)21:e48791https://doi.org/10.15252/embr.201948791 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract PI3K-Akt-FoxO-mTOR signaling is the central pathway controlling growth and metabolism in all cells. Ubiquitination of the protein kinase Akt prior to its phosphorylation is required for PI3K-Akt activity. Here, we found that the deubiquitinating (DUB) enzyme USP1 removes K63-linked polyubiquitin chains on Akt to restrict PI3K-Akt-FoxO signaling in mouse muscle during prolonged starvation. DUB screening platform identified USP1 as a direct DUB for Akt, and USP1 depletion in mouse muscle increased Akt ubiquitination, PI3K-Akt-FoxO signaling, and glucose uptake during fasting. Co-immunoprecipitation and mass spectrometry identified disabled homolog-2 (Dab2), the tuberous sclerosis complex TSC1/TSC2, and PHLPP1 as USP1 bound proteins. During starvation, Dab2 is essential for Akt recruitment to USP1-TSC1-PHLPP1 complex, and for PI3K-Akt-FoxO inhibition. Surprisingly, USP1 limits TSC1 levels to sustain mTOR-mediated basal protein synthesis rates and maintain its own protein levels. We propose that Dab2 recruits Akt to USP1-TSC1-PHLPP1 complex to efficiently terminate the transmission of growth signals when cellular energy level is low. Synopsis The deubiquitinating enzyme USP1 restricts PI3K-Akt-FoxO signaling in muscle atrophy by removing K63-linked polyubiquitin chains on Akt. During fasting, Dab2 promotes the recruitment of Akt to USP1, which together with the phosphatase PHLPP1 inhibits Akt, thereby suppressing PI3K-Akt signaling when cellular energy level is low. USP1 directly deubiquitinates and inhibits Akt to sustain PI3K-Akt-FoxO signaling low in the muscle during prolonged starvation (low insulin condition). During fasting, Dab2 promotes the recruitment of Akt to USP1, which also binds the tuberous sclerosis complex TSC1/TSC2, and the phosphatase PHLPP1. USP1-mediated deubiquitination of Akt reduces Akt phosphorylation at T308 in fasting, but loss of Akt phosphorylation at S473 requires PHLPP1. To maintain its own protein levels high, USP1 limits TSC1 levels to sustain mTOR-mediated basal protein synthesis rates in fasting. Introduction Phosphoinositide 3-kinase (PI3K)–Akt–mammalian target of rapamycin (mTOR) signaling is the central pathway controlling cell growth, proliferation, and metabolism 1, 2. Activation of this pathway by IGF-I or insulin promotes cell division, and in non-dividing muscle cells, it promotes growth by stimulating overall protein synthesis and inhibiting protein degradation 3-5. Conversely, inhibition of this pathway reduces cell survival and, in muscle, causes atrophy. A critical player at the core of PI3K-Akt signaling is the serine/threonine kinase Akt, which serves as an indispensable conduit for transmission of growth and survival signals from cell surface receptors. Because dysregulation of Akt results in various pathologies including cancer, insulin resistance (as occurs in type-2 diabetes or obesity), neurological disorders, and muscle atrophy, its activity must be tightly regulated in all cells. Akt1 (referred to as Akt in this study) is a member of the protein kinase B (PKB) family of kinases, which comprises three isoforms in mammalian cells, Akt1/PKBα, Akt2/PKBβ, and Akt3/PKBγ. These isoforms are composed of an N-terminal pleckstrin homology (PH) domain, a central catalytic domain containing a T308 phosphorylation site, and a C-terminal regulatory domain containing a S473 phosphorylation site 6. The kinases PDK-1 and mTORC2 phosphorylate Akt at T308 and S473, respectively 7, downstream of PI3K. Phosphorylation of Akt at these residues is considered rate limiting and obligatory for maximal activation of Akt. Once activated, Akt inhibits the transcription factor FoxO by phosphorylation, preventing its nuclear translocation and stimulation of expression of atrophy-related genes (“atrogenes”) 3, 8. Akt also inhibits the negative regulator of mTORC1, the tuberous sclerosis complex 1 and 2 (TSC1/2) 9, consequently leading to mTORC1-mediated phosphorylation of Ribosomal protein 6 Kinase β-1 (S6K1) 10, 11 and activation of protein synthesis and cell growth 12. In addition, Akt activates glycogen synthesis by phosphorylating and inactivating glycogen synthase kinase β (GSK3-β), which under low insulin conditions inhibits glycogen synthesis 13. When blood glucose and insulin levels rise, insulin promotes glucose uptake by enhancing trafficking of the glucose transporter GLUT4 to the plasma membrane 14. However, during fasting and catabolic diseases, PI3K–Akt–mTOR signaling decreases, and consequently, glucose uptake and protein synthesis fall; simultaneously, proteolysis increases largely through the FoxO-mediated expression of the atrogene program 1. In fact, activation of FoxO3 alone is sufficient to trigger proteolysis via the ubiquitin proteasome system 15 and autophagy 5, 16, and to cause substantial atrophy 15. Overproduction of insulin-like growth factor 1 (IGF1) or Akt in mice, either through transgenic expression or by electroporation into muscles, was sufficient to reduce muscle weight loss, and induce systemic hypertrophy 17, 18. These anabolic effects are mediated by Akt; however, the aberrant activation of this kinase and PI3K-Akt signaling may relay proliferative and pro-survival signals that are often associated with solid and hematological malignancies in humans. Therefore, many new anticancer treatments currently in use or in clinical trials are potent suppressors of Akt signaling and can efficiently inhibit growth of various tumors 19. Because Akt controls diverse biological processes, from cell growth and proliferation to survival and migration, its activity must be tightly regulated in normal cells. Previous studies in mouse embryonic fibroblasts proposed a new mode of regulation of Akt by ubiquitination, which appears to be required for Akt phosphorylation at T308 and S473 and activation 20. The present studies were undertaken to identify the enzyme that deubiquitinates Akt and suppresses PI3K-Akt signaling. These studies have identified a novel regulator of Akt activity, the ubiquitin-specific protease 1 (USP1). USP1 is a member of the USP family of cysteine proteases, which process ubiquitin chains to reverse protein modification by ubiquitination. Mammalian cells contain over 50 members of this family, all of which have a catalytic core composed of conserved N-terminal Cys box motif and C-terminal His box motif 21. USP1 is best characterized functions are in the nucleus as a regulator of DNA damage response, mainly in the Fanconi anemia pathway 22, and as a negative regulator of certain transcription factors to prevent cell differentiation 23. Recent evidence indicates an additional role for this enzyme in diabetes by promoting apoptosis of insulin-secreting pancreatic β-cells 24. Previously, a USP1 knockout mouse has been described that exhibits multiple developmental defects including osteopenia 23, perinatal lethality, male infertility, chromosome instability, and a Fanconi anemia phenotype 25. Although USP1's important roles in DNA repair mechanisms are well documented, its precise functions in regulating metabolism and growth are not yet known. We demonstrate here that during fasting, USP1 suppresses the major anabolic pathway in all cells, PI3K-Akt signaling, through effects on the key component in this pathway, Akt. Activation of USP1 requires association with the WD40 repeat-containing protein USP1-associated factor 1 (UAF1) 26, and its inhibition is mediated by phosphorylation 27, by decreasing its protein levels via reduced gene expression 23, or by autocleavage at an internal diglycine motif (Gly670-Gly671) that promotes USP1 degradation by the proteasome 28. USP1 levels are elevated in several human cancers 23, where it seems to sustain DNA repair mechanisms; thus, inhibition of this enzyme may help hypersensitize cancer cells to chemotherapy-induced DNA damage 29. Interestingly, mutations in USP1 have also been identified in certain human cancers 30, but the functional consequences of these mutations on USP1 activity remain elusive. We demonstrate here a new role for USP1 in the inhibition of signaling by the PI3K-Akt cascade. Surprisingly, we show that although ubiquitination of Akt is important for phosphorylation at both T308 and S473 20, the direct deubiquitination of Akt by USP1 results in suppression of Akt phosphorylation only at T308. Loss of Akt phosphorylation at S473, however, requires the function of PHLPP1, which is found within an intact complex with USP1 to completely inhibit Akt during fasting. Recruitment of Akt to USP1-PHLPP1 complex is mediated by the tumor suppressor disabled homolog 2 (Dab2), whose ectopic expression in cancer cell lines is sufficient to inhibit cell proliferation 31-33. During fasting, protein synthesis rates are low. However, we surprisingly show that USP1 maintains its own protein levels high by limiting the protein content of TSC1 and sustaining basal rates of protein synthesis. Thus, USP1 is a novel inhibitor of Akt, and by binding to PHLPP1 and TSC1, this DUB ensures a complete inhibition of Akt. Recruitment of Akt to USP1-TSC1-PHLPP1 complex by Dab2 appears critical for regulation of PI3K-Akt signaling and maintenance of cellular homeostasis. Results USP1 is a deubiquitinating enzyme for Akt Because PI3K-Akt-FoxO-mTOR pathway is the primary regulator of cell growth and metabolism, and given the importance of Akt ubiquitination for its activity 20, we set out to identify the specific DUB that reduces Akt ubiquitination and consequently inhibits PI3K-Akt signaling. To initially confirm that Akt is actually deubiquitinated when PI3K-Akt-FoxO-mTOR falls, we studied mouse muscles under the physiological condition of fasting, when blood glucose and insulin levels are low and PI3K-Akt-FoxO-mTOR signaling in all cells is inhibited. Analysis of skeletal muscle extracts from fed and fasted (2d) mice by SDS–PAGE and immunoblotting showed that Akt is ubiquitinated under normal conditions, but its ubiquitination levels are reduced during fasting, while the unmodified form of Akt accumulates (Fig 1A). Furthermore, Akt immunoprecipitation from muscles from fed or fasted mice and immunoblotting with anti-ubiquitin also indicated that during fasting Akt ubiquitination is reduced (Fig 1B). Interestingly, upon fasting, high molecular weight ubiquitinated species of all three Akt isoforms, Akt1, Akt2, and Akt3, were reduced (Fig EV1A). These findings suggested that Akt is deubiquitinated in vivo during fasting. Figure 1. USP1 is a deubiquitinating enzyme for Akt Akt is deubiquitinated during fasting. Left: Soluble fractions of TA muscles from fed and fasted mice were analyzed by SDS–PAGE and immunoblot using Akt antibody. Right: densitometric measurement of presented blots (n = 3). *P < 0.05 versus fed by one-tailed t-test. Data are represented as mean ± SEM. Akt was immunoprecipitated from the soluble fraction of muscles from fed or fasted (2 days) mice. Precipitated were analyzed by immunoblotting using an anti-ubiquitin conjugates. Mouse IgG was used as a control for non-specific binding. Scheme for DUBs screening experiment to identify the enzyme that directly deubiquitinates Akt in vitro. Ubiquitinated Akt is a substrate for USP1 in vitro. Top: Akt was immunoprecipitated from the soluble fraction of TA muscle from fed mice and was subjected to an in vitro deubiquitination by a panel of purified DUBs arrayed in a multi-well plate. The dividing line indicates the removal of an intervening lane for presentation purposes. Bottom: Densitometric measurements of presented blots. Data are presented as the ratio between ubiquitinated Akt to total Akt in each well. shRNA-mediated knockdown of USP1 in HeLa cells. Soluble extracts were analyzed by immunoblotting. USP1 deubiquitinates Akt during fasting in vivo. Left: Soluble fractions of TA muscles transfected with shLacz control or shUSP1 from fed and fasted mice were analyzed by SDS–PAGE and immunoblotting using anti-Akt. The actin blot serves as a loading control. Right: densitometric measurements of presented blots (n = 3). Data are presented as ratio of ubiquitinated Akt to total Akt normalized to actin. *P < 0.05 versus fed by one-tailed t-test. Data are represented as mean ± SEM. USP1 removes K63-linked polyubiquitin chains on Akt in vivo. Akt was immunoprecipitated from the soluble fraction of muscles expressing USP1(C90S) or control plasmids. Mouse IgG was used as a control for non-specific binding. Protein precipitates were subjected to immunoblotting with an antibody against K63-linked polyubiquitin conjugates. USP1 removes polyubiquitin chains linked to K8 on Akt. Akt was immunoprecipitated from the soluble fraction of TA muscles transfected with USP1(C90S), HA-Akt(K8R), or control from fasted mice, and protein precipitates were analyzed by immunoblotting with anti-Akt antibody. Mouse IgG was used as a control for non-specific binding. Right: Densitometric measurements of presented blots. Data are presented as the ratio between ubiquitinated Akt to total Akt in each lane (n = 2). Source data are available online for this figure. Source Data for Figure 1 [embr201948791-sup-0004-SDataFig1.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. USP1 promotes deubiquitination of all three Akt isoforms in fasting Akt was immunoprecipitated from the soluble fraction of muscles from fed or fasted (2d) mice. Precipitates were analyzed by immunoblotting using specific antibodies against Akt1, Akt2, Akt3, and anti-ubiquitin conjugates. Mouse IgG was used as a control for non-specific binding. Akt was immunoprecipitated from the soluble fraction of muscles expressing shUSP1 or shLacz from fasted (2d) mice. Protein precipitates were subjected to immunoblotting with an antibody against K63-linked polyubiquitin conjugates. Mouse IgG was used as a control for non-specific binding. shRNA-mediated knockdown of USP11 in HEK293 cells. mRNA preparations from transfected cells were analyzed by RT–PCR using primers for USP11. Data are plotted as the mean fold change relative to control ± SEM. n = 5. *P < 0.005 by one-tailed t-test. Data are represented as mean ± SEM. shRNA-mediated knockdown of OTUB2 in mouse muscle during fasting. Soluble extracts were analyzed by immunoblotting. The actin blot serves as a loading control. USP11 and OTUB2 deubiquitinate Akt during fasting in vivo. Soluble fractions of TA muscles transfected with shLacz control, shUSP11, or shOTUB2 from fed and fasted mice were analyzed by SDS–PAGE and immunoblotting using anti-Akt1. Akt1 is known to control skeletal muscle homeostasis 17. The actin blot serves as a loading control. Source data are available online for this figure. Download figure Download PowerPoint To identify the DUB that can directly deubiquitinate Akt in vitro, we immunoprecipitated Akt from normal muscle homogenate and added the precipitates to a DUB screening plate containing an array of pure active DUBs (Fig 1C). Five DUBs, USP1/UAF1, USP11, OTUB2, OTUD5 (p177S), and OTUD6A, cleaved the polyubiquitin chain on Akt and reduced its ubiquitination levels compared with ubiquitinated Akt in reactions that did not contain a DUB (Fig 1D). Among these DUBs, we decided to focus on USP1 because it preferentially removes K63-linked chains 34, the type of polyubiquitination that is essential for Akt activation 20, and it is the only DUB among the five that has been linked to diabetes, a pathological condition associated with dysregulated insulin-PI3K-Akt signaling 24. To determine whether USP1 can deubiquitinate Akt in vivo, we downregulated this enzyme in mouse muscle by the electroporation of a specific shRNA (shUSP1) (Fig EV2), which can efficiently reduce USP1 content (Fig 1E), and analyzed the effects on Akt ubiquitination levels during fasting. As shown above (Figs 1A and B), analysis of the soluble fraction from muscles expressing control plasmid (shLacz) from fed and fasted mice indicated that upon fasting there was a decrease in high molecular weight ubiquitinated species of Akt (Fig 1F). However, downregulation of USP1 blocked this decrease in Akt ubiquitination levels, and instead, Akt accumulated as ubiquitinated species (Figs 1F and EV1B). It is noteworthy that downregulation of two other representative enzymes from our screen, USP11 and OTUB2, in mouse muscles also blocked Akt deubiquitination in vivo, further exemplifying the validity of our DUB scan approach and the physiological relevance of our findings (Fig EV1C–E). Similar results were obtained when we inhibited USP1 by the electroporation of a GFP-tagged USP1 dominant-negative encoding plasmid (USP1(C90S)) 35, which lacks the catalytic cysteine and thus can bind substrates but cannot deubiquitinate them (Fig 1G). Immunoprecipitation of Akt from USP1(C90S)-expressing muscles from fasted mice, and analysis of protein precipitates by SDS–PAGE and immunoblotting using anti-K63-linked ubiquitin chains antibody revealed that in the muscles lacking functional USP1 (expressing USP1(C90S)) Akt accumulated as K63-ubiquitinated protein (Fig 1G). In fact, the content of K63-ubiquitinated Akt in these muscles exceeded the amounts observed in muscles of fed mice (P < 0.05), suggesting that K63 polyubiquitination of Akt may increase during fasting, and USP1 catalyzes the deubiquitination of this protein. Click here to expand this figure. Figure EV2. In vivo electroporation of skeletal muscleParaffin-embedded cross sections of TA muscle from mice deprived of food (2 d) were stained with H&E or by immunofluorescence using the indicated antibodies. Scale bar, 150 μm for H&E and 50 μm for immunofluorescence. Download figure Download PowerPoint Akt ubiquitination on K8 within the PH domain is essential for its activation 20. To learn whether USP1 cleaves the ubiquitin chain that is linked to K8 on Akt, we co-electroporated muscles with USP1(C90S) to cause accumulation of ubiquitinated Akt, and either shLacz or a plasmid encoding HA-tagged Akt carrying a K8-to-R mutation (HA-Akt(K8R)) (Fig 1H). By 2d of fasting, HA-Akt(K8R) immunoprecipitated from transfected muscles showed limited ubiquitination, i.e., much less than the endogenous Akt immunoprecipitated from muscles expressing USP1(C90S) alone (Fig 1H, compare lanes 1–2 with 3–4). These findings indicate that on inhibition of USP1 during fasting, Akt is ubiquitinated on K8. Thus, USP1 is essential for Akt deubiquitination in vivo, which most likely leads to inhibition of PI3K-Akt signaling. Deubiquitination by USP1 reduces Akt phosphorylation at T308 and PI3K-Akt-FoxO signaling To determine whether USP1-mediated Akt deubiquitination in fact influences PI3K-Akt-FoxO signaling, we inhibited USP1 by electroporation of USP1(C90S) into mouse muscle during fasting. By 2 days of fasting, phosphorylation of insulin receptor, Akt, and its targets, FoxO3 and GSK3β, was markedly reduced (Fig 2A) 15, 36. However, inhibition of USP1 almost completely blocked this response to fasting. In fact, the levels of phosphorylated Akt (T308), FoxO3, and GSK3β were similar to those in muscles from fed mice, even though the levels of phosphorylated insulin receptor remained low. Surprisingly, although Akt ubiquitination facilitates phosphorylation on both T308 and S473 20, in the muscles where USP1 was inhibited and ubiquitinated Akt accumulated (Figs 1F and G), phosphorylated Akt (S473) was low (Fig 2A). Thus, during fasting, deubiquitination by USP1 is required to prevent Akt phosphorylation at T308, but is dispensable for Akt phosphorylation at S473. It is noteworthy that USP1 overexpression in muscles from fasted mice does not further reduce Akt phosphorylation at T308 (Fig EV3A), which is already low when blood glucose and insulin levels are low and endogenous USP1 is functional. Figure 2. Deubiquitination by USP1 reduces Akt phosphorylation at T308 and PI3K-Akt-FoxO signaling Inhibition of USP1 increases PI3K-Akt-FoxO signaling during fasting. Soluble fractions of normal and atrophying muscles expressing shLacz or USP1(C90S) were analyzed by SDS–PAGE and immunoblot. The black line indicates the removal of intervening lanes for presentation purposes. Right: densitometric measurements of presented pAkt (T308), pAkt (S473), and Akt blots. Data are presented as ratio of phosphorylated Akt to total Akt (n = 3). *P < 0.05 versus fed by one-tailed t-test. Data are represented as mean ± SEM. Downregulation of USP1 reduces MuRF1 and Atrogin1 expression during fasting. Quantitative RT–PCR of mRNA preparations from atrophying and control muscles expressing shLacz or shUSP1 (80% transfection efficiency) using primers for MuRF1 and Atrogin1. Data are plotted as the mean fold change relative to fed control. n = 4. *P < 0.05 versus shLacz in fed. #P < 0.05 versus shLacz in fasting by one-tailed t-test. Data are represented as mean ± SEM. Downregulation of USP1 markedly reduces muscle fiber atrophy. Cross-sectional areas of 500 fibers transfected with shUSP1 (that express GFP, green bars) versus 500 non-transfected fibers (black bars) in the same muscle. n = 5 mice. A representative image is shown; laminin staining is in red; scale bar, 50 μm. Downregulation of USP1 attenuates the loss of muscle mass during fasting. shUSP1 was delivered to more than 60% of muscle fibers. Mean weights of electroporated muscles are plotted as the percent weight loss. n = 6. #P < 0.05 versus shLacz in fed; *P < 0.05 versus shLacz in fasting by one-tailed t-test. Data are represented as mean ± SEM. USP1 inhibition significantly increases glucose tolerance in mice during fasting. Left: mice injected i.p. with specific USP1 inhibitor (12 μg/g body weight) or saline during fasting (2 days) were subjected to glucose tolerance test. Blood glucose levels were measured at the indicated time points following glucose injection (1 mg/g body weight). Data are depicted in a graph as mg/dl glucose (n = 3). *P < 0.05, **P < 0.005, ***P < 0.0005 versus mice injected with saline by one-tailed t-test. Data are represented as mean ± SEM. Right: Soluble fractions of muscles from injected mice were analyzed by SDS–PAGE and immunoblot. Source data are available online for this figure. Source Data for Figure 2 [embr201948791-sup-0005-SDataFig2.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV3. During fasting, downregulation of USP1 in mouse muscles or cultured myotubes enhances PI3K-Akt signaling and TSC1 stability Overexpression of USP1 in mouse muscles does not further reduce phosphorylated-Akt (T308) levels in fasting. Soluble fractions of atrophying muscles expressing shLacz, GFP-USP1(C90S), or GFP-USP1-WT were analyzed by SDS–PAGE and immunoblot. TSC1 was immunoprecipitated from the soluble fraction of muscles expressing shLacz or shUSP1 from fed or fasted mice. Mouse IgG was used as a control for non-specific binding. Precipitates were analyzed by immunoblotting. Quantitative RT–PCR of mRNA preparations from muscles from fed and fasted mice using primers for USP1 and UAF1. Data are plotted as the mean fold change relative to fed control. n = 6 by one-tailed t-test. Data are represented as mean ± SEM. During fasting, downregulation of USP1 reduces rates of protein synthesis. Mice were injected with puromycin, and soluble fractions of muscles expressing shLacz or gUSP1 (in vivo CRISPR) were analyzed by immunoblotting using puromycin antibody. Inhibition of USP1 does not affect TSC1 expression during fasting. Quantitative RT–PCR of mRNA preparations from atrophying and control muscles expressing shLacz or USP1(C90S) using primers for TSC1. Data are plotted as the mean fold change relative to fed control. n = 4. *P < 0.05 versus shLacz in fed by one-tailed t-test. Data are represented as mean ± SEM. USP1 downregulation or proteasome inhibition causes accumulation of ubiquitinated TSC1 in cultured myotubes. Myotubes were infected with control or shUSP1 viruses for 48 h and then serum-starved for 4 h in the presence or absence of Epoxomicin (100 nM). Right lane: Cells infected with shUSP1 and are not starved. Cell extracts were analyzed by SDS–PAGE and immunoblot. Source data are available online for this figure. Download figure Download PowerPoint Normally during fasting, FoxO is activated (dephosphorylated) and stimulates the expression of atrogenes, including the muscle-specific ubiquitin ligases MuRF1 and Atrogin1, which are essential for rapid fiber atrophy (Fig 2B) 37, 38. However, USP1 downregulation with shRNA (shUSP1) resulted in a marked decrease in MuRF1 and Atrogin1 expression in the tibialis anterior (TA) muscles during fasting (Fig 2B). This inhibition of atrogene expression during fasting together with the maintenance of normal PI3K–Akt–mTOR signaling should block muscle wasting 39, 40. Accordingly, the mean cross-sectional area of 500 fibers expressing shUSP1 (and GFP to identify transfected fibers) was bigger than that of 500 non-transfected fibers (Fig 2C). Moreover, by 2 days of fasting, there was a 31% decrease in the mean weight of the control TA muscles below levels in fed mice (Fig 2D), but USP1 downregulation clearly attenuated this wasting, even though ~30% of the fibers were not transfected with the shUSP1 (Fig 2D). In addition to regulating cell size and protein balance, the PI3K-Akt-FoxO pathway also mediates insulin's stimulation of glucose uptake into muscle and adipose tissue. As predicted, inhibition of USP1 by the injection of mice with the USP1-specific inhibitor ML323 41 significantly improved glucose tolerance and stimulated PI3K-Akt-FoxO signaling in muscle during fasting (Fig 2E). Thus, USP1 function is critical in causing the reduction in PI3K-Akt-FoxO signaling during fasting that triggers the decrease in glucose uptake and protein synthesis, the FoxO-mediated expression of the atrogene program, and muscle wasting 39, 40. The Ser/Thr phosphatase PHLPP1 reduces Akt phosphorylation at S473 in fasting Although Akt ubiquitination is required for phosphorylation at both T308 and S473 20, the deubiquitination of this kinase by USP1 in fasting reduces Akt phosphorylation at T308 but not at S473 (Figs 2A and EV3A). Prior studies indicated that the Ser/Thr protein phosphatase PHLPP1 can dephosphorylate Akt at S473 to promote apoptosis and suppress tumor growth 42. To investigate whether Akt phosphorylation at S473 is affected by the levels of PHLPP1 during fasting, we downregulated PHLPP1 by electroporation of a specific shRNA (shPHLPP1) into mouse TA muscle. The resulting fall in PHLPP1 caused an increase in phosphorylated Akt (S473) and S6K (T389), while the levels of phosphorylated Akt (T308) remained low (Fig 3A) because in these muscles USP1 was functional. Consistent with the increase in Akt and S6K phosphorylation, PHLPP1 downregulation also enhanced protein synthesis because puromycin incorporation into newly translated proteins was higher than in shLacz-expressing muscles (Fig 3B). This enhanced protein synthesis can acc
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