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• W2067488166 abstract "Insulin-like growth factor 1 (IGF1) induces skeletal muscle hypertrophy by activating the IGF1R/IRS1/PI3K/Akt pathway. However the effect of IGF1 in differentiated muscle is limited by IRS1 ubiquitination and proteasome-mediated breakdown. In skeletal muscle, IGF1R activation sensitizes IRS1 to degradation, and a screen for the responsible E3 ligase identified Fbxo40 as mediating this rapid turnover of IRS1, since IRS1 loss can be rescued by knockdown of Fbxo40. In biochemical assays, an SCF E3 ligase complex containing Fbxo40 directly ubiquitinates IRS1, and this activity is enhanced by increased tyrosine phosphorylation of IRS1. Fbxo40 is muscle specific in expression and is upregulated during differentiation. Knockdown of Fbxo40 induces dramatic hypertrophy of myofibers. Mice null for Fbxo40 have increased levels of IRS1 and demonstrate enhanced body and muscle size during the growth phase associated with elevated IGF1 levels. These findings establish an important means of restraining IGF1's effects on skeletal muscle. Insulin-like growth factor 1 (IGF1) induces skeletal muscle hypertrophy by activating the IGF1R/IRS1/PI3K/Akt pathway. However the effect of IGF1 in differentiated muscle is limited by IRS1 ubiquitination and proteasome-mediated breakdown. In skeletal muscle, IGF1R activation sensitizes IRS1 to degradation, and a screen for the responsible E3 ligase identified Fbxo40 as mediating this rapid turnover of IRS1, since IRS1 loss can be rescued by knockdown of Fbxo40. In biochemical assays, an SCF E3 ligase complex containing Fbxo40 directly ubiquitinates IRS1, and this activity is enhanced by increased tyrosine phosphorylation of IRS1. Fbxo40 is muscle specific in expression and is upregulated during differentiation. Knockdown of Fbxo40 induces dramatic hypertrophy of myofibers. Mice null for Fbxo40 have increased levels of IRS1 and demonstrate enhanced body and muscle size during the growth phase associated with elevated IGF1 levels. These findings establish an important means of restraining IGF1's effects on skeletal muscle. Fbxo40 mediates IGF1-induced degradation of IRS1 in skeletal muscle Fbxo40 expression is muscle and differentiation specific Knockdown of Fbxo40 induces hypertrophy in skeletal myofibers Fbxo40 knockout enhances growth and muscle mass, associated with higher IRS1 levels The IGF1/IRS1/PI3K/Akt pathway induces skeletal muscle hypertrophy, both by increasing protein synthesis (Coleman et al., 1995Coleman M.E. DeMayo F. Yin K.C. Lee H.M. Geske R. Montgomery C. Schwartz R.J. Myogenic vector expression of insulin-like growth factor I stimulates muscle cell differentiation and myofiber hypertrophy in transgenic mice.J. Biol. Chem. 1995; 270: 12109-12116Crossref PubMed Scopus (503) Google Scholar, Lai et al., 2004Lai K.-M.V. Gonzalez M. Poueymirou W.T. Kline W.O. Na E. Zlotchenko E. Stitt T.N. Economides A.N. Yancopoulos G.D. Glass D.J. Conditional activation of akt in adult skeletal muscle induces rapid hypertrophy.Mol. Cell. Biol. 2004; 24: 9295-9304Crossref PubMed Scopus (302) Google Scholar, Musarò et al., 2001Musarò A. McCullagh K. Paul A. Houghton L. Dobrowolny G. Molinaro M. Barton E.R. Sweeney H.L. Rosenthal N. Localized Igf-1 transgene expression sustains hypertrophy and regeneration in senescent skeletal muscle.Nat. Genet. 2001; 27: 195-200Crossref PubMed Scopus (845) Google Scholar, Pallafacchina et al., 2002Pallafacchina G. Calabria E. Serrano A.L. Kalhovde J.M. Schiaffino S. A protein kinase B-dependent and rapamycin-sensitive pathway controls skeletal muscle growth but not fiber type specification.Proc. Natl. Acad. Sci. USA. 2002; 99: 9213-9218Crossref PubMed Scopus (290) Google Scholar, Rommel et al., 2001Rommel C. Bodine S.C. Clarke B.A. Rossman R. Nunez L. Stitt T.N. Yancopoulos G.D. Glass D.J. Mediation of IGF-1-induced skeletal myotube hypertrophy by PI(3)K/Akt/mTOR and PI(3)K/Akt/GSK3 pathways.Nat. Cell Biol. 2001; 3: 1009-1013Crossref PubMed Scopus (1113) Google Scholar, Izumiya et al., 2008Izumiya Y. Hopkins T. Morris C. Sato K. Zeng L. Viereck J. Hamilton J.A. Ouchi N. LeBrasseur N.K. Walsh K. Fast/Glycolytic muscle fiber growth reduces fat mass and improves metabolic parameters in obese mice.Cell Metab. 2008; 7: 159-172Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar), and by blocking protein degradation (Sandri et al., 2004Sandri M. Sandri C. Gilbert A. Skurk C. Calabria E. Picard A. Walsh K. Schiaffino S. Lecker S.H. Goldberg A.L. Foxo transcription factors induce the atrophy-related ubiquitin ligase atrogin-1 and cause skeletal muscle atrophy.Cell. 2004; 117: 399-412Abstract Full Text Full Text PDF PubMed Scopus (2000) Google Scholar, Stitt et al., 2004Stitt T.N. Drujan D. Clarke B.A. Panaro F. Timofeyva Y. Kline W.O. Gonzalez M. Yancopoulos G.D. Glass D.J. The IGF-1/PI3K/Akt pathway prevents expression of muscle atrophy-induced ubiquitin ligases by inhibiting FOXO transcription factors.Mol. Cell. 2004; 14: 395-403Abstract Full Text Full Text PDF PubMed Scopus (1368) Google Scholar). IGF1 modulation of protein synthesis downstream of IRS1/Akt operates in part by activating mammalian target of rapamycin (mTOR) signaling (Glass, 2005aGlass D.J. A signaling role for dystrophin: inhibiting skeletal muscle atrophy pathways.Cancer Cell. 2005; 8: 351-352Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, Léger et al., 2006Léger B. Cartoni R. Praz M. Lamon S. Dériaz O. Crettenand A. Gobelet C. Rohmer P. Konzelmann M. Luthi F. Russell A.P. Akt signalling through GSK-3beta, mTOR and Foxo1 is involved in human skeletal muscle hypertrophy and atrophy.J. Physiol. 2006; 576: 923-933Crossref PubMed Scopus (258) Google Scholar, McGee et al., 2008McGee S.L. Mustard K.J. Hardie D.G. Baar K. Normal hypertrophy accompanied by phosphoryation and activation of AMP-activated protein kinase alpha1 following overload in LKB1 knockout mice.J. Physiol. 2008; 586: 1731-1741Crossref PubMed Scopus (81) Google Scholar, Mounier et al., 2009Mounier R. Lantier L. Leclerc J. Sotiropoulos A. Pende M. Daegelen D. Sakamoto K. Foretz M. Viollet B. Important role for AMPK{alpha}1 in limiting skeletal muscle cell hypertrophy.FASEB J. 2009; 23: 2264-2273Crossref PubMed Scopus (90) Google Scholar, Park et al., 2005Park I.H. Erbay E. Nuzzi P. Chen J. Skeletal myocyte hypertrophy requires mTOR kinase activity and S6K1.Exp. Cell Res. 2005; 309: 211-219Crossref PubMed Scopus (62) Google Scholar); its inhibition of protein degradation is via IRS1/Akt-mediated inhibition of the Forkhead, or Foxo, family of transcription factors (Sandri et al., 2004Sandri M. Sandri C. Gilbert A. Skurk C. Calabria E. Picard A. Walsh K. Schiaffino S. Lecker S.H. Goldberg A.L. Foxo transcription factors induce the atrophy-related ubiquitin ligase atrogin-1 and cause skeletal muscle atrophy.Cell. 2004; 117: 399-412Abstract Full Text Full Text PDF PubMed Scopus (2000) Google Scholar, Stitt et al., 2004Stitt T.N. Drujan D. Clarke B.A. Panaro F. Timofeyva Y. Kline W.O. Gonzalez M. Yancopoulos G.D. Glass D.J. The IGF-1/PI3K/Akt pathway prevents expression of muscle atrophy-induced ubiquitin ligases by inhibiting FOXO transcription factors.Mol. Cell. 2004; 14: 395-403Abstract Full Text Full Text PDF PubMed Scopus (1368) Google Scholar), which are required for the upregulation of the E3 ubiquitin ligases MuRF1 and MAFbx, which are, in turn, required for skeletal muscle atrophy (Bodine et al., 2001Bodine S.C. Latres E. Baumhueter S. Lai V.K. Nunez L. Clarke B.A. Poueymirou W.T. Panaro F.J. Na E. Dharmarajan K. et al.Identification of ubiquitin ligases required for skeletal muscle atrophy.Science. 2001; 294: 1704-1708Crossref PubMed Scopus (2417) Google Scholar, Glass, 2005bGlass D.J. Skeletal muscle hypertrophy and atrophy signaling pathways.Int. J. Biochem. Cell Biol. 2005; 37: 1974-1984Crossref PubMed Scopus (750) Google Scholar, Glass, 2010Glass D.J. Signaling pathways perturbing muscle mass.Curr. Opin. Clin. Nutr. Metab. Care. 2010; 13: 225-229Crossref PubMed Scopus (260) Google Scholar). The IGF1 pathway can be inactivated by targeting IRS1 for ubiquitin-mediated degradation. This has been reported in settings of prolonged insulin-mediated cell stimulation, in which IRS1 was degraded in a PI3K-sensitive fashion, in various cell lines (Haruta et al., 2000Haruta T. Uno T. Kawahara J. Takano A. Egawa K. Sharma P.M. Olefsky J.M. Kobayashi M. A rapamycin-sensitive pathway down-regulates insulin signaling via phosphorylation and proteasomal degradation of insulin receptor substrate-1.Mol. Endocrinol. 2000; 14: 783-794Crossref PubMed Scopus (322) Google Scholar, Lee et al., 2000Lee A.V. Gooch J.L. Oesterreich S. Guler R.L. Yee D. Insulin-like growth factor I-induced degradation of insulin receptor substrate 1 is mediated by the 26S proteasome and blocked by phosphatidylinositol 3′-kinase inhibition.Mol. Cell. Biol. 2000; 20: 1489-1496Crossref PubMed Scopus (107) Google Scholar, Tzatsos and Kandror, 2006Tzatsos A. Kandror K.V. Nutrients suppress phosphatidylinositol 3-kinase/Akt signaling via raptor-dependent mTOR-mediated insulin receptor substrate 1 phosphorylation.Mol. Cell. Biol. 2006; 26: 63-76Crossref PubMed Scopus (316) Google Scholar, Xu et al., 2008Xu X. Sarikas A. Dias-Santagata D.C. Dolios G. Lafontant P.J. Tsai S.-C. Zhu W. Nakajima H. Nakajima H.O. Field L.J. et al.The CUL7 E3 ubiquitin ligase targets insulin receptor substrate 1 for ubiquitin-dependent degradation.Mol. Cell. 2008; 30: 403-414Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, Zhande et al., 2002Zhande R. Mitchell J.J. Wu J. Sun X.J. Molecular mechanism of insulin-induced degradation of insulin receptor substrate 1.Mol. Cell. Biol. 2002; 22: 1016-1026Crossref PubMed Scopus (176) Google Scholar). However, these reports differ in terms of the pathways downstream of PI3K that modulate IRS1 turnover (Haruta et al., 2000Haruta T. Uno T. Kawahara J. Takano A. Egawa K. Sharma P.M. Olefsky J.M. Kobayashi M. A rapamycin-sensitive pathway down-regulates insulin signaling via phosphorylation and proteasomal degradation of insulin receptor substrate-1.Mol. Endocrinol. 2000; 14: 783-794Crossref PubMed Scopus (322) Google Scholar, Tzatsos and Kandror, 2006Tzatsos A. Kandror K.V. Nutrients suppress phosphatidylinositol 3-kinase/Akt signaling via raptor-dependent mTOR-mediated insulin receptor substrate 1 phosphorylation.Mol. Cell. Biol. 2006; 26: 63-76Crossref PubMed Scopus (316) Google Scholar, Xu et al., 2008Xu X. Sarikas A. Dias-Santagata D.C. Dolios G. Lafontant P.J. Tsai S.-C. Zhu W. Nakajima H. Nakajima H.O. Field L.J. et al.The CUL7 E3 ubiquitin ligase targets insulin receptor substrate 1 for ubiquitin-dependent degradation.Mol. Cell. 2008; 30: 403-414Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, Zhande et al., 2002Zhande R. Mitchell J.J. Wu J. Sun X.J. Molecular mechanism of insulin-induced degradation of insulin receptor substrate 1.Mol. Cell. Biol. 2002; 22: 1016-1026Crossref PubMed Scopus (176) Google Scholar). Some suggest that the PI3K/Akt/mTOR pathway activates IRS1 degradation in MCF-7 (Xu et al., 2008Xu X. Sarikas A. Dias-Santagata D.C. Dolios G. Lafontant P.J. Tsai S.-C. Zhu W. Nakajima H. Nakajima H.O. Field L.J. et al.The CUL7 E3 ubiquitin ligase targets insulin receptor substrate 1 for ubiquitin-dependent degradation.Mol. Cell. 2008; 30: 403-414Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar) and 293 cells (Tzatsos and Kandror, 2006Tzatsos A. Kandror K.V. Nutrients suppress phosphatidylinositol 3-kinase/Akt signaling via raptor-dependent mTOR-mediated insulin receptor substrate 1 phosphorylation.Mol. Cell. Biol. 2006; 26: 63-76Crossref PubMed Scopus (316) Google Scholar), while other reports suggest that activation of PI3K but not mTOR signaling is required for IRS1 degradation (Zhande et al., 2002Zhande R. Mitchell J.J. Wu J. Sun X.J. Molecular mechanism of insulin-induced degradation of insulin receptor substrate 1.Mol. Cell. Biol. 2002; 22: 1016-1026Crossref PubMed Scopus (176) Google Scholar). Several distinct E3 ubiquitin ligases have been demonstrated to serve as IRS1 ligases, including SOCS1 and SOCS3, which were shown to promote degradation of IRS1 and IRS2, and which may mediate inflammation-induced insulin resistance (Rui et al., 2002Rui L. Yuan M. Frantz D. Shoelson S. White M.F. SOCS-1 and SOCS-3 block insulin signaling by ubiquitin-mediated degradation of IRS1 and IRS2.J. Biol. Chem. 2002; 277: 42394-42398Crossref PubMed Scopus (671) Google Scholar). Cbl-b was reported to degrade IRS1 in settings of muscle atrophy (Nakao et al., 2009Nakao R. Hirasaka K. Goto J. Ishidoh K. Yamada C. Ohno A. Okumura Y. Nonaka I. Yasutomo K. Baldwin K.M. et al.Ubiquitin ligase Cbl-b is a negative regulator for IGF1 signaling during muscle atrophy caused by unloading.Mol. Cell. Biol. 2009; 29: 4798-4811Crossref PubMed Scopus (130) Google Scholar). The cullin 7 complex, containing the E3 ligase Fbxw8, was shown to be activated by an mTOR-dependent negative feedback loop after which it could degrade IRS1 (Xu et al., 2008Xu X. Sarikas A. Dias-Santagata D.C. Dolios G. Lafontant P.J. Tsai S.-C. Zhu W. Nakajima H. Nakajima H.O. Field L.J. et al.The CUL7 E3 ubiquitin ligase targets insulin receptor substrate 1 for ubiquitin-dependent degradation.Mol. Cell. 2008; 30: 403-414Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). In this study, we demonstrate that the E3 ubiquitin ligase Fbxo40 induces IRS1 ubiquitination and breakdown specifically in skeletal muscle cells and only upon IGF1 stimulation, thus identifying a regulator of IGF1-induced skeletal muscle signaling. Because IGF1 has been demonstrated to be sufficient to induce hypertrophy in adult skeletal muscle, we were interested to know whether IRS1 is degraded upon IGF1 treatment of differentiated muscle cells, or myotubes, since this could limit the utility of IGF1 as an anabolic agent. We also checked IRS1 stability during an atrophy stimulus—after treatment with the glucocorticoid dexamethasone (DEX)—to determine whether settings in which protein breakdown was enhanced would affect IRS1 stability. To discriminate between degradation of IRS1 and its synthesis, a protein synthesis inhibitor, emetin (Eme), was used to determine the degradation rate of IRS1. Differentiated C2C12 myotubes were treated with increasing concentrations of IGF1 or DEX, plus or minus Eme. In C2C12 myotubes, IRS1 is degraded upon IGF1 but not DEX treatment, in a dose-dependent manner (see Figure S1A available online). In contrast, the upstream signaling molecule, the insulin-like growth factor 1 receptor (IGF1R), is not affected by either treatment. Based on these data, we chose 10 nM IGF1 for our further experiments, since 10 nM was sufficient to induce the effect. In C2C12 myotubes, IRS1 is rapidly degraded upon IGF1 treatment, with a half-life of around 2 hr (Figure 1A ). Note that in Figure S1B, IRS1 protein levels also decreased in cells treated with IGF1 alone, without Eme added, though with slower kinetics, suggesting that its rapid degradation outstrips active protein synthesis upon IGF1 stimulation. IRS1 is degraded through a proteasome-dependent pathway, based on the observation that the proteasome inhibitor MG132 substantially stabilizes IRS1 in the setting of IGF1 stimulation (Figure 1A). In order to determine whether ubiquitinated IRS1 protein could be detected after IGF1 stimulation, C2C12 myotubes were infected with an adenovirus overexpressing his-myc tagged ubiquitin, and then these myotubes were incubated with IGF1 along with either MG132 or the isopeptidase inhibitor G5 (to block deubiquitination and thus increase the ability to capture the ubiquitinated species) (Figure 1B). IRS1 was immunoprecipitated with an anti-IRS1 antibody (Figure 1B, lanes 6, 8, 10, 12) or a rabbit IgG as a negative control (Figure 1B, lanes 5, 7, 9, 11). Polyubiquitination was detected with a monoclonal anti-myc antibody against his-myc tagged ubiquitin and was found to be specifically immunoprecipitated with IRS1 (Figure 1B, upper panel). Visualization of the polyubiquitinated IRS1 is more clearly seen with G5 treatment (Figure 1B, lower panel, lanes 10 and 12), demonstrating that deubiquitinating enzymes are active in the myotubes, and thus need to be inhibited in order to stabilize ubiquitin conjugates of IRS1. In agreement with the induced degradation of IRS1 upon IGF1 treatment, polyubiquitinated IRS1 increased upon IGF1 treatment (Figure 1C, left panel), while total ubiquitinated proteins actually decreased slightly (Figure 1C, right panels), as might be expected given IGF1's antiatrophy effects (Glass, 2010Glass D.J. Signaling pathways perturbing muscle mass.Curr. Opin. Clin. Nutr. Metab. Care. 2010; 13: 225-229Crossref PubMed Scopus (260) Google Scholar). Blocking the degradation of IRS1 by MG132 rescues Akt/p70S6K signaling (Figure 1A; Figure S1C). Surprisingly, this rapid degradation of IRS1 in myotubes cannot be rescued using PI3K, Akt, mTOR, MEK1/2, GSK3, p38, or JNK inhibitors (Figure S1C and data not shown), implying that IRS1 turnover in this setting is regulated simply by IGF1/IGF1R-mediated phosphorylation of IRS1. This finding distinguishes the IRS1 degradation seen in the setting of IGF1 stimulation from previous reports of IRS1 turnover. We also examined IRS1 degradation in undifferentiated C2C12 myoblasts. While IRS1 is again degraded upon IGF1 treatment in a dose-dependent manner, the half-life of IRS1 in C2C12 myoblasts is significantly prolonged in comparison to that observed in myotubes—around 6 hours in a myoblast versus 2 hours in a myotube (Figure S2A versus Figure 1A). Note that in Figure S2B, without protein synthesis inhibitor, IRS1 protein level does not decrease with IGF1 treatment, in contrast to the case in myotubes (Figure S1B), due to active protein synthesis and slower protein degradation in the myoblasts. In contrast to what was observed in a differentiated myotube, inhibitors of PI3K can rescue IRS1 degradation in a myoblast setting (Figures S2C and S2D), adding to the impression that the degradation process seen in a myotube is a distinct and stage-specific mechanism of IRS1 regulation in skeletal muscle. This finding was later explained by the demonstration that Fbxo40 expression is differentiation specific. Next, we determined the E3 ligase that is responsible for targeting IRS1 to the proteasome for degradation upon IGF1 stimulation. Since IRS1 is degraded rapidly in C2C12 myotubes, and is distinctly regulated in comparison to that seen in C2C12 myoblasts (and other cell types reported so far in the literature), we focused our search for responsible E3s on C2C12 myotubes, reasoning that even if the E3 was one of those previously reported, it would be necessary to see if its regulation was distinct in the setting of differentiated muscle cells. Thus, we started with reported E3s that regulate IRS1 in fibroblast type cell lines, to determine whether they are required for the loss of IRS1 seen in myotubes; cullin 7-Fbxw8 complex (Xu et al., 2008Xu X. Sarikas A. Dias-Santagata D.C. Dolios G. Lafontant P.J. Tsai S.-C. Zhu W. Nakajima H. Nakajima H.O. Field L.J. et al.The CUL7 E3 ubiquitin ligase targets insulin receptor substrate 1 for ubiquitin-dependent degradation.Mol. Cell. 2008; 30: 403-414Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar) (Figures S3A–S3D) and Cbl family members (Suzue et al., 2006Suzue N. Nikawa T. Onishi Y. Yamada C. Hirasaka K. Ogawa T. Furochi H. Kosaka H. Ishidoh K. Gu H. et al.Ubiquitin ligase Cbl-b downregulates bone formation through suppression of IGF-I signaling in osteoblasts during denervation.J. Bone Miner. Res. 2006; 21: 722-734Crossref PubMed Scopus (42) Google Scholar, Nakao et al., 2009Nakao R. Hirasaka K. Goto J. Ishidoh K. Yamada C. Ohno A. Okumura Y. Nonaka I. Yasutomo K. Baldwin K.M. et al.Ubiquitin ligase Cbl-b is a negative regulator for IGF1 signaling during muscle atrophy caused by unloading.Mol. Cell. Biol. 2009; 29: 4798-4811Crossref PubMed Scopus (130) Google Scholar) (Figures S3F and S3G) were examined, and scrambled control siRNA (siCON) was included as control. Neither of these was found to be responsible for the rapid degradation of IRS1 in myotubes. We also checked the elongin B/C protein complex since it was reported that SOCS1 and SOCS3 may regulate IRS1 overexpressed in 293 cells (Rui et al., 2002Rui L. Yuan M. Frantz D. Shoelson S. White M.F. SOCS-1 and SOCS-3 block insulin signaling by ubiquitin-mediated degradation of IRS1 and IRS2.J. Biol. Chem. 2002; 277: 42394-42398Crossref PubMed Scopus (671) Google Scholar). There was no significant rescue of IRS1 degradation demonstrated when elongin B mRNA was decreased to 20% of basa level (data not shown). In addition, a distinct E3 ligase, Nedd4, has been suggested to regulate ligand-induced degradation of IGF1R (Vecchione et al., 2003Vecchione A. Marchese A. Henry P. Rotin D. Morrione A. The Grb10/Nedd4 complex regulates ligand-induced ubiquitination and stability of the insulin-like growth factor I receptor.Mol. Cell. Biol. 2003; 23: 3363-3372Crossref PubMed Scopus (203) Google Scholar), but its knockdown failed to demonstrate a requirement in the IGF1-induced IRS1 degradation observed in C2C12 myotubes (data not shown). There are more than 600 E3 ligases in the mammalian cell (Li et al., 2008Li W. Bengtson M.H. Ulbrich A. Matsuda A. Reddy V.A. Orth A. Chanda S.K. Batalov S. Joazeiro C.A.P. Genome-wide and functional annotation of human E3 ubiquitin ligases identifies MULAN, a mitochondrial E3 that regulates the organelle's dynamics and signaling.PLoS ONE. 2008; 3: e1487Crossref PubMed Scopus (494) Google Scholar), which include two large protein families, the RING-finger domain (RNF) and HECT domain containing E3 ligases. RNF-dependent E3 ligases can be further divided into two categories: the single polypeptide chain RNF E3s and the multisubunit cullin-RING E3 complexes. Within this latter category, there are four different classes; each consists of a RING protein (Rbx1), one of seven different cullin family members and various adaptor proteins. This information allows one to narrow the potential E3 class involved by knocking down Rbx1, since its involvement would point to the Rbx1/cullin-required families of E3s. C2C12 myotubes were therefore transfected with three different siRNAs targeting Rbx1. siRbx1_1 and siRbx1_2 decreased Rbx1 protein to 50% of basal levels, and yet even with this modest knockdown an almost 100% blockade of IGF1-induced IRS1 degradation was observed; as a control, siRbx1_3, which does not knock down protein significantly, had no effect (Figure 2A ). This result narrowed our search to the multisubunit cullin-Ring E3 complexes. We next differentiated the four subtypes, using siRNAs to different cullin family members and adaptor proteins. Knockdown of Skp1 (Figure 2B) and cullin 1 (Figures 2C and 2D) resulted in reduced loss of IRS1, suggesting that the Rbx1 containing Skp1-cullin 1-F-box (SCF) complex comprises the subfamily of E3 ligases targeting IRS1 for proteasomal degradation. As a control, cullin 2-containing cullin-Ring E3 complexes were tested and determined to be not required (Figures S4A and S4B). Of note, Rbx1 can be coimmunoprecipitated with IRS1, demonstrating that these two proteins exist in a complex (Figure 2E). This finding allowed us to focus on genes encoding F-box containing proteins as the likely source of the E3s in this case. Seventy-seven F-box proteins have been identified so far in mice. We used a functional genomic approach to screen 68 of these F-box containing proteins to determine if any could target IRS1 for degradation (siRNAs to the remaining genes were not represented in the library). C2C12 myotubes were transfected with a mouse ubiquitin conjugation siRNA library from Dharmacon (subset 2), which contains siRNAs against SOCS-box and F-box containing proteins. Two days posttransfection, myotubes were treated with 10 nM IGF1 for 16 hr, and IRS1 protein level was analyzed by western blotting. The positive hits from this screen were further corroborated by three different siRNAs to the F-box gene under study, obtained from a different source, QIAGEN. Knockdown efficiency was evaluated by quantitative real-time PCR (qRT-PCR) for the positive hits. The results of the screen are summarized in Table 1. Of the 68 genes encoding F-box proteins screened, we identified one positive hit, Fbxo40. Figure 3A demonstrates the dose-dependent rescue of IRS1 achieved by knocking down Fbxo40. Among the three siRNAs targeting Fbxo40, siFbxo40_7 decreases the protein level to 10% of basal level and has the best efficacy, while siFbxo40_9, which leads to the least effective knockdown, only caused a small increase of IRS1 protein above control level (Figure 3A, left panels). Importantly, none of these three siRNAs caused an increase in IRS1 protein level without IGF1 treatment (Figure 3A, right panels), indicating phosphorylation of IRS1 downstream of IGF1R activation is required for Fbxo40 targeting of IRS1 for degradation. In C2C12 myotubes with knockdown of Fbxo40, the half-life of IRS1 increased to more than 6 hr (Figure 3B), similar to what has been observed in C2C12 myoblasts (Figure S2A). Interestingly, in siFbxo40 transfected cells, phosphorylation of Akt on both S473 and T308 sites was maintained after 4 hr of IGF1 stimulation, demonstrating that sparing of IRS1 can lead to preservation of downstream signaling through Akt (Figure 3B). Furthermore, knockdown of Fbxo40 leads to the rescue of IRS1 upon either of two different doses of IGF1 stimulation (Figure 3C).Table 1Summary of F-Box Proteins Screening ResultF-Box ProteinIn the Library?Rescue IRS1?F-Box ProteinIn the Library?Rescue IRS1?F-Box ProteinIn the Library?Rescue IRS1?F-Box ProteinIn the Library?Rescue IRS1?Fbxw1aGenes were additionally tested by three individual siRNAs from QIAGEN. The knockdown efficiency was verified by qRT-PCR. The siRNAs targeting Fbxw13-19, Fbxl21, Fbxo20, and Fbxo48 are not represented in the ubiquitin conjugation siRNA library from Dharmacon. The expression level of Fbxl21 is low in C2C12 myotubes judged from qRT-PCR data. Three individual siRNAs targeting Fbxo20 were obtained from Qiagen. No inhibition of IRS1 degradation is observed with knockdown of Fbxo20 to 40% of basal mRNA level.+−Fbxl1+−Fbxo1+−Fbxo25+−Fbxw2+−Fbxl2+−Fbxo2+−Fbxo27+−Fbxw4+−Fbxl3+−Fbxo3+−Fbxo28aGenes were additionally tested by three individual siRNAs from QIAGEN. The knockdown efficiency was verified by qRT-PCR. The siRNAs targeting Fbxw13-19, Fbxl21, Fbxo20, and Fbxo48 are not represented in the ubiquitin conjugation siRNA library from Dharmacon. The expression level of Fbxl21 is low in C2C12 myotubes judged from qRT-PCR data. Three individual siRNAs targeting Fbxo20 were obtained from Qiagen. No inhibition of IRS1 degradation is observed with knockdown of Fbxo20 to 40% of basal mRNA level.+−Fbxw5aGenes were additionally tested by three individual siRNAs from QIAGEN. The knockdown efficiency was verified by qRT-PCR. The siRNAs targeting Fbxw13-19, Fbxl21, Fbxo20, and Fbxo48 are not represented in the ubiquitin conjugation siRNA library from Dharmacon. The expression level of Fbxl21 is low in C2C12 myotubes judged from qRT-PCR data. Three individual siRNAs targeting Fbxo20 were obtained from Qiagen. No inhibition of IRS1 degradation is observed with knockdown of Fbxo20 to 40% of basal mRNA level.+−Fbxl4aGenes were additionally tested by three individual siRNAs from QIAGEN. The knockdown efficiency was verified by qRT-PCR. The siRNAs targeting Fbxw13-19, Fbxl21, Fbxo20, and Fbxo48 are not represented in the ubiquitin conjugation siRNA library from Dharmacon. The expression level of Fbxl21 is low in C2C12 myotubes judged from qRT-PCR data. Three individual siRNAs targeting Fbxo20 were obtained from Qiagen. No inhibition of IRS1 degradation is observed with knockdown of Fbxo20 to 40% of basal mRNA level.+−Fbxo4+−Fbxo30aGenes were additionally tested by three individual siRNAs from QIAGEN. The knockdown efficiency was verified by qRT-PCR. The siRNAs targeting Fbxw13-19, Fbxl21, Fbxo20, and Fbxo48 are not represented in the ubiquitin conjugation siRNA library from Dharmacon. The expression level of Fbxl21 is low in C2C12 myotubes judged from qRT-PCR data. Three individual siRNAs targeting Fbxo20 were obtained from Qiagen. No inhibition of IRS1 degradation is observed with knockdown of Fbxo20 to 40% of basal mRNA level.+−Fbxw7aGenes were additionally tested by three individual siRNAs from QIAGEN. The knockdown efficiency was verified by qRT-PCR. The siRNAs targeting Fbxw13-19, Fbxl21, Fbxo20, and Fbxo48 are not represented in the ubiquitin conjugation siRNA library from Dharmacon. The expression level of Fbxl21 is low in C2C12 myotubes judged from qRT-PCR data. Three individual siRNAs targeting Fbxo20 were obtained from Qiagen. No inhibition of IRS1 degradation is observed with knockdown of Fbxo20 to 40% of basal mRNA level.+−Fbxl5+−Fbxo5+−Fbxo31+−Fbxw8aGenes were additionally tested by three individual siRNAs from QIAGEN. The knockdown efficiency was verified by qRT-PCR. The siRNAs targeting Fbxw13-19, Fbxl21, Fbxo20, and Fbxo48 are not represented in the ubiquitin conjugation siRNA library from Dharmacon. The expression level of Fbxl21 is low in C2C12 myotubes judged from qRT-PCR data. Three individual siRNAs targeting Fbxo20 were obtained from Qiagen. No inhibition of IRS1 degradation is observed with knockdown of Fbxo20 to 40% of basal mRNA level.+−Fbxl6+−Fbxo6aGenes were additionally tested by three individual siRNAs from QIAGEN. The knockdown efficiency was verified by qRT-PCR. The siRNAs targeting Fbxw13-19, Fbxl21, Fbxo20, and Fbxo48 are not represented in the ubiquitin conjugation siRNA library from Dharmacon. The expression level of Fbxl21 is low in C2C12 myot" @default.
• W2067488166 created "2016-06-24" @default.
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• W2067488166 date "2011-11-01" @default.
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• W2067488166 title "The SCF-Fbxo40 Complex Induces IRS1 Ubiquitination in Skeletal Muscle, Limiting IGF1 Signaling" @default.
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• W2067488166 doi "https://doi.org/10.1016/j.devcel.2011.09.011" @default.
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