Glucocorticoids differentially regulate degradation of MyoD and Id1 by N-terminal ubiquitination to promote muscle protein catabolism Liping Sun, Julie S. Trausch-Azar, Louis J. Muglia, and Alan L. Schwartz* The Edward Mallinckrodt Department of Pediatrics and Department of Developmental Biology, Washington University School of Medicine and St. Louis Children’s Hospital, 660 South Euclid Avenue, St. Louis, MO 63110
Accelerated protein degradation via the ubiquitin–proteasome pathway is the principal cause of skeletal muscle wasting associated with common human disease states and pharmacological treatment with glucocorticoids. Although many protein regulatory factors essential for muscle development and regeneration are degraded via the ubiquitin system, little is known about the mechanisms and regulation of this pathway that promote wasting muscle. Here, we demonstrate that, in differentiated myotubes, glucocorticoid, via the glucocorticoid receptor, selectively induces a decrease in protein abundance of MyoD, a master switch for muscle development and regeneration, but not that of its negative regulator Id1. This decrease in MyoD protein results from accelerated degradation after glucocorticoid exposure. Using MyoD and Id1 mutants deficient in either N terminus-dependent or internal lysine-dependent ubiquitination, we further show that these ubiquitination pathways of MyoD degradation are regulated differently from those of Id1 degradation. Specifically, glucocorticoid activates the N-terminal ubiquitination pathway in MyoD degradation in myotubes, without concomitant effects on Id1 degradation. This effect of glucocorticoid on MyoD and Id1 protein degradation is associated with the distinct cellular compartments in which their degradation occurs. Taken together, these results support a key role for the N terminus-dependent ubiquitination pathway in the physiology of muscle protein degradation. ubiquitin 兩 proteolysis
S
keletal muscle wasting is a common debilitating condition associated with human disease states, including cancer, AIDS, renal failure and diabetes. A variety of studies, including those in rodent models of human disease, have shown that muscle wasting occurs primarily through accelerated protein degradation via the ubiquitin–proteasome pathway. Evidence has accumulated from studies in wasting skeletal muscle that supports the notion of involvement of the ubiquitin– proteasome pathway, including increased ubiquitin conjugation to muscle proteins, increased sensitivity of muscle proteins to proteasome inhibitors, and up-regulation of several components in the ubiquitin–proteasome pathway. However, our understanding of the molecular mechanisms and their regulation of the ubiquitin system in wasting muscles is still limited. Glucocorticoids, induced during states of physiological stress, such as poorly controlled diabetes mellitus, acidosis, and starvation, are one critical factor involved in the induction of the ubiquitin–proteasome pathway in skeletal muscle wasting. Pharmacological treatment with glucocorticoids is similarly associated with negative muscle protein balance. The catabolic effects of glucocorticoid on overall muscle protein metabolism are well known. For example, glucocorticoid increased overall protein degradation in rat L8 and L6 myotubes and enhanced the degradation of myofibrillar proteins in C2C12 myotubes (1–3). In response to fasting, muscles of adrenalectomized animals, unlike normals, are unable to increase protein breakdown, www.pnas.org兾cgi兾doi兾10.1073兾pnas.0800165105
whereas complete restoration occurs after glucocorticoid administration. mRNA levels for several components of the ubiquitin system are up-regulated by glucocorticoids (2, 4–6). For example, muscle-specific E3 ligases Atrogin-1/MAFbx and Murf1 were both up-regulated upon glucocorticoid administration to skeletal muscle (4). This transcriptional induction may facilitate increased ubiquitin-mediated degradation. Muscle differentiation is regulated by an intricate genetic network of at least three protein families: myogenic regulatory factors (MRFs), E proteins, and Id proteins. MyoD is a tissuespecific MRF that acts as a master transcriptional switch for muscle differentiation and development. MyoD is also required for healthy self-renewing proliferation of the adult skeletal muscle satellite cells (7–9). The transcriptional activities of MyoD are negatively regulated by a family of inhibitors of DNA-binding (Id) proteins. Among the Id protein family, Id1 is most active in terms of MyoD binding. Sequestering ubiquitous E2A proteins allows Id1 to control the transcriptional activity of muscle-specific MyoD (10). Regulated cellular abundance of these important transcription factors, achieved by precisely controlled balance between protein degradation and synthesis, is essential for the maintenance of normal muscle structure and function. However, regulation of the turnover of these muscle regulatory factors in wasting muscles is unknown. MyoD was the first protein identified as a substrate of the N terminus ubiquitination pathway (11). Characterized by the initial site of ubiquitination, there are two subpathways of the ubiquitin system, the internal lysine-dependent pathway and the N terminus-dependent pathway. In most cases, the first ubiquitin moiety is transferred to an -NH2 group of an internal lysine residue of the target protein to generate an isopeptide bond. The first ubiquitin, however, may be covalently attached to the free and exposed N-terminal residue of the substrate (12). These substrates for N terminus ubiquitination include the human papillomavirus 16 (HPV 16) oncoprotein E7 (13), the Arf tumor suppressor (14), and the Id2 developmental regulator (15) as well as naturally occurring lysineless proteins such as HPV-58 E7 and p16INK4␣ (16). However, the biological role of the N terminus ubiquitination pathway remains unknown. Here, we have investigated the ubiquitin–proteasomemediated degradation of MyoD and Id1 in muscle cells exposed to glucocorticoid. We show that in differentiated myotubes glucocorticoid, via the glucocorticoid receptor, selectively reduces protein abundance of MyoD but not that of Author contributions: A.L.S. designed research; L.S., J.S.T.-A., L.J.M., and A.L.S. performed research; L.J.M. contributed new reagents/analytic tools; L.S., J.S.T.-A., L.J.M., and A.L.S. analyzed data; and L.S., L.J.M., and A.L.S. wrote the paper. The authors declare no conflict of interest. *To whom correspondence should be addressed. E-mail:
[email protected]. This article contains supporting information online at www.pnas.org/cgi/content/full/ 0800165105/DC1. © 2008 by The National Academy of Sciences of the USA
PNAS 兩 March 4, 2008 兩 vol. 105 兩 no. 9 兩 3339 –3344
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Communicated by Aaron J. Ciechanover, Technion–Israel Institute of Technology, Haifa, Israel, January 8, 2008 (received for review November 12, 2007)
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myoblasts
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Fig. 1. Protein expression of glucocorticoid receptor (GR) (A) during C2C12 myogenic differentiation and localization of GR (B) in C2C12 myoblasts with or without dexamethasone treatment (1 M for 16 h).
Id1. This decrease in MyoD abundance results from accelerated MyoD degradation via activation of the N terminusdependent ubiquitination pathway. Our findings suggest that the effect of glucocorticoid on MyoD and Id1 protein degradation is related to the role of the N terminus-dependent pathway in their degradation and to the distinct intracellular compartments in which degradation occurs. The results also implicate the N terminus-dependent ubiquitination pathway in the regulation of muscle protein degradation under normal and catabolic conditions. Results Glucocorticoid receptors (GR) mediate the cellular response to glucocorticoids. Upon ligand binding, the cytoplasmic GR becomes activated and translocates to the nucleus, where it can bind to specific DNA elements, initiating the genetic response. Therefore, to examine the effects of glucocorticoids on muscle cell protein degradation, we first examined the expression and localization of the glucocorticoid receptor in C2C12 cells. GR is abundantly expressed during C2C12 myogenic differentiation (Fig. 1A). Subcellular localization of GR in control C2C12 myoblasts is primarily in the cytoplasm. Upon dexamethasone exposure, GR is activated and translocates to the nucleus in C2C12 cells (Fig. 1B). Although prolonged exposure of muscle cells to pharmacological doses of glucocorticoids is associated with activation of the ubiquitin system and loss of muscle protein (17), it remains unknown as to whether glucocorticoid affects the protein expression and the ubiquitin-dependent degradation of muscle regulatory factors such as MyoD and Id1. As shown in Fig. 2A, dexamethasone exposure did not significantly alter the protein expression level of either MyoD or Id1 in myoblasts. In myotubes, however, MyoD protein expression was markedly reduced upon dexamethasone exposure. No significant change was observed for the Id1 protein level. In addition, the reduction in MyoD protein expression upon dexamethasone exposure was abrogated by RU486, an antagonist of GR. This indicates that the observed decrease in MyoD in myotubes is a specific 3340 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0800165105
MG132 + CHX
8.0 h
CHX
1.0 h dexamethasone
MG132 + CHX
10 h
CHX
0.4 h T0
T0.5
T1
T2
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Fig. 2. MyoD and Id1 protein expression (A) and MyoD protein degradation half-lives (t1/2 h) (B) in C2C12 myoblasts (A) and myotubes (B) with or without dexamethasone treatment. 1, control; 2, dexamethasone at 1 M for 16 h; 3, mifepristone (RU486) at 1 M for 16 h; and 4, dexamethasone and RU486 at 1 M each for 16 h. For half-life in myotubes, C2C12 myoblasts were transiently transfected with MyoD encoding plasmid DNA. Upon confluence, transfected C2C12 cells were switched to differentiation medium and maintained for 4 days with medium changed every 24 h. Protein half-life analysis was then performed by using cycloheximide (CHX) and proteasome inhibitor MG132 as described in Materials and Methods. The lower MyoD band represents the hypophosphorylated form (18).
response to glucocorticoid and depends on the glucocorticoid receptor. The reduction in MyoD observed in myotubes exposed to glucocorticoid could be the result of decreased protein synthesis or of accelerated protein degradation or both. To determine whether this reduction is associated with a change in MyoD degradation via the ubiquitin system, we compared endogenous MyoD protein-degradation half-lives in C2C12 myoblasts and myotubes with or without dexamethasone treatment. As shown in Fig. 2B, MyoD was rapidly degraded (t1/2 ⬇ 1.0 h) in myoblasts independent of dexamethasone exposure. However, in myotubes, exposure to dexamethasone significantly accelerated MyoD degradation, shortening the MyoD protein half-life from ⬇1 h in control myotubes to ⬇0.4 h in myotubes treated with dexamethasone. MG132, a potent proteasome inhibitor, greatly stabilized MyoD protein in control and in dexamethasonetreated myotubes, indicating that the ubiquitin–proteasome proteolytic pathway is responsible for the accelerated MyoD protein degradation in glucocorticoid treated myotubes. To further clarify the mechanism involved, we determined the protein-degradation half-lives of two MyoD mutants (19), one unable to undergo N terminus-dependent ubiquitination, the other unable to undergo internal lysine-dependent ubiquitination, in muscle cells with or without glucocorticoid administration. In myoblasts (Fig. 3A), exogenous wild-type MyoD is rapidly degraded with a t1/2 ⬇ 1 h. Lysineless MyoD Sun et al.
A
myoblasts
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0.4 ± 0.1
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10 ± 2.7
1.6 ± 0.2
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1.4 ± 0.3
3.8 ± 0.6
Fig. 3. Half-lives of wild-type MyoD (anti-MyoD), MyoD-LL-HA (anti-HA), and 6⫻Myc-MyoD (anti-Myc) in myoblasts (A) and in myotubes (B) with or without dexamethasone treatment. Protein half-lives were determined in a similar fashion as described in the Fig. 2 legend.
(MyoD-LL-HA), which is unable to undergo internal lysinedependent ubiquitination, is stable, with a t1/2 ⬇ 10 h, whereas MyoD with an N terminus 6⫻ Myc tag (6⫻Myc-MyoD), which is unable to undergo N-terminus-dependent ubiquitination, is rapidly degraded, with a t1/2 ⬇ 1.4 h. These results indicate that the lysine-dependent ubiquitination pathway plays the primary role in MyoD protein degradation in myoblasts. In control myotubes (Fig. 3B), differentiation did not alter the protein half-lives of MyoD, MyoD-LL-HA, and 6⫻Myc-MyoD as expected based on our previous studies (18). Dexamethasone administration, however, induced accelerated protein degradation of exogenous wild-type MyoD in C2C12 myotubes (t1/2 from ⬇1 h to ⬇0.4 h) similar to that observed for endogenous MyoD (Fig. 2B). MyoD-LL-HA degradation is significantly accelerated by glucocorticoids (t1/2 ⬇ 1–2 h), whereas 6⫻MycMyoD appears to be stabilized (t1/2 ⬇ 4 h). Thus, in myotubes, glucocorticoid appears to activate the N terminus-dependent ubiquitination pathway. Previous studies suggested that the two ubiquitination pathways were differentially regulated in the cell nucleus versus the cytoplasm (19). Although MyoD is a nuclear protein, it can be retained in the cytoplasm by mutation of its nuclear localization signal (NLS). In myoblasts, MyoD-LL-NLS-HA is localized to the cytoplasm (Fig. 4A) and rapidly degraded (t1/2 ⬇ 1.6 h) (Fig. 4B), in contrast to that seen for MyoD-LL-HA that is localized to the nucleus (Fig. 4 A), yet slowly degraded (t1/2 ⬇ 10 h) (Fig. 3). The 6⫻Myc-MyoD-NLS is also localized to the myoblast cytoplasm (Fig. 4 A) yet is slowly degraded (t1/2 ⬇ 8 h) (Fig. 4B) in contrast to that seen for 6⫻Myc-MyoD, which is localized to the nucleus (Fig. 4 A) yet rapidly degraded (t1/2 ⬇ 1.4 h) (Fig. 3). Sun et al.
The rates of degradation of MyoD-LL-NLS-HA and 6⫻Myc-MyoD-NLS were unaltered in myotubes compared with that seen in myoblasts (Fig. 4B). Furthermore, there was no effect of dexamethasone on the degradation rates of these cytoplasmically localized proteins in myotubes (Fig. 4B). This is in marked contrast to the effect of dexamethasone on these proteins in the nucleus (Fig. 3B). Taken together, these results reaffirm differences in MyoD degradation in the nucleus versus cytoplasm and demonstrate a specific effect of glucocorticoids on the degradation of nuclear N terminusdependent MyoD-LL (Table 1). Because Id proteins (Id1/Id2) are also degraded via the N terminus-dependent pathway, we examined the effect of glucocorticoid on the degradation of wild-type Id1, Id1-LL, and 6⫻Myc-Id1 in C2C12 cells. In myoblasts, in contrast to that seen for MyoD, Id1-LL is rapidly degraded (t1/2 ⬇ 0.7 h), whereas 6⫻Myc-Id1 is quite stable (t1/2 ⬇ 10 h) (Fig. 5A). Similar rates of degradation for Id1-LL and 6⫻Myc-Id1 were observed in myotubes (Fig. 5B). Furthermore, the presence of dexamethasone did not alter the degradation rates for wildtype or mutated Id1 proteins (Fig. 5). Discussion Although it is well established that activation of the ubiquitin– proteasome pathway is the principle pathway underlying skeletal muscle wasting, the molecular mechanisms responsible are poorly understood. Most studies to date have focused on evaluation of degradation of total muscle protein or that of myofibrillar proteins. In the present study, we examined the degradation of MyoD and Id1 in C2C12 cells exposed to glucocorticoid at levels in the high physiologic to pharmarcologic range. This catabolic hormone activates the ubiquitin PNAS 兩 March 4, 2008 兩 vol. 105 兩 no. 9 兩 3341
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Time (h)
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t1/2 (h)
Fig. 5. Half-lives of wild-type Id1(anti-Id1), Id1-LL (anti-Id1) and 6⫻Myc-Id1 (anti-Myc) in C2C12 myoblasts (A) and myotubes (B) with or without dexamethasone treatment. Id1 half-lives were determined in a similar fashion to that described in the Fig. 2 legend.
dexamethasone MyoD-LL-NLS-HA
T1.5
Degradation rates (t, h) are taken from Figs. 3 and 4.
chexia, was shown to reduce the MyoD protein abundance in C2C12 myotubes (20). Taken together, these results suggest that, in muscle cells, the reduction in MyoD protein expression may be a common catabolic consequence. Using two MyoD mutants that are deficient in either lysine-dependent ubiquitination or in N-terminus-dependent ubiquitination, we further demonstrate that glucocorticoid activates the N-terminal ubiquitination pathway of MyoD degradation. Previous studies in nonmuscle cells have shown that MyoD can be degraded via the recently described Nterminal ubiquitination pathway (11, 19). In muscle cells, lysineless MyoD (MyoD-LL-HA) was very stable (t1/2 ⬇ 10 h), whereas N terminus-blocked MyoD (6⫻Myc-MyoD) was rapidly degraded (t1/2 ⬇ 1.4 h). Thus, MyoD is primarily degraded via the lysine-dependent ubiquitination pathway in normal muscle cells. Upon glucocorticoid treatment, degradation of lysineless MyoD was significantly accelerated (i.e., 6-fold) via activation of the N terminus ubiquitination pathway. We note that the N-terminus-blocked MyoD appeared to be stabilized upon glucocorticoid treatment, suggesting a reduction in activity of the lysine-dependent ubiquitination pathway. Exogenous wild-type MyoD degradation, which occurs via both ubiquitination pathways, was significantly accelerated in glucocorticoid-treated myotubes (t1/2 ⬇ 1 h to t1/2 ⬇ 0.4 h). Thus, activation of the N terminus-dependent ubiquitination pathway appears to be the dominant event induced by glucocorticoid. Our results clearly demonstrate the differential roles of the two ubiquitination pathways in MyoD degradation. The subcellular locus of the ubiquitin system (e.g., the nucleus and the cytoplasm) is also of importance, because distinct ubiquitination pathways appear to be regulated differently within different subcellular compartments. Wild-type MyoD is a nuclear protein, as is lysineless MyoD. Their rapid degradation indicates the presence of a ubiquitin system capable of N terminus-dependent ubiquitination in the cell nucleus (19). Using MyoD nuclear localization signal mutants, which localize to the cytoplasm, we further show that MyoD-
3342 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0800165105
Sun et al.
T0
T1.5
T3
t1/2 (h)
Fig. 4. Subcelluar localization (A) of MyoD-LL-NLS-HA and 6⫻Myc-MyoDNLS in C2C12 myoblasts and protein degradation half-lives (B) of MyoD-LLNLS-HA and 6⫻Myc-MyoD-NLS in C2C12 myoblasts and myotubes with or without dexamethasone treatment. (A) Localization of various MyoD protein forms was determined with immunofluorescent staining and fluorescence microscopy. (B) MyoD half-lives were determined in a similar fashion as described in the Fig. 2 legend. MG132 stabilized the degradation of these MyoD mutants (data not shown).
system and induces muscle wasting in vivo. Our underlying hypothesis is that the ubiquitin–proteasome-mediated degradation of muscle regulatory factors, such as MyoD and Id1, is precisely regulated to maintain their cellular abundance during normal muscle development and regeneration. Furthermore, under catabolic conditions, degradation and cellular abundance of these factors is modulated via activation of the ubiquitin system. Glucocorticoid selectively decreased MyoD expression level in differentiated C2C12 myotubes, because of accelerated MyoD degradation by the ubiquitin system. Tumor necrosis factor, another mediator of skeletal muscle wasting in caTable 1. Summary of MyoD species degradation rates Myoblasts MyoD species MyoD-WT MyoD-LL-HA MyoD-LL-NLS-HA 6xMyc-MyoD 6xMyc-MyoD-NLS
Myotubes
Control
Control
Dexamethasone
0.9 ⫾ 0.1 10 ⫾ 2.9 1.6 ⫾ 0.4 1.4 ⫾ 0.2 7.7 ⫾ 0.9
0.9 ⫾ 0.1 10 ⫾ 2.7 1.5 ⫾ 0.4 1.4 ⫾ 0.3 7.0 ⫾ 0.8
0.4 ⫾ 0.1 1.6 ⫾ 0.2 1.4 ⫾ 0.4 3.8 ⫾ 0.6 7.4 ⫾ 1.9
Sun et al.
N-terminal
Lysine
Id1 degradation ± GC
myoblasts
Muscle differentiation
myotubes + GC
N-terminal
cytoplasm
Lysine
N-terminal
Lysine
nucleus
accelerated MyoD degradation via activation of N-terminal ubiquitination in the nucleus
Fig. 6. A proposed model for the ubiquitin–proteasome-mediated degradation of MyoD and Id1 in muscle cells. For Id1 degradation, the Nterminal ubiquitination pathway is dominantly active in both myoblasts and myotubes with or without glucocorticoid administration. For MyoD degradation in myoblasts and myotubes without exposure to excess glucocorticoids, the internal lysine-dependent ubiquitination pathway is much more active in the nucleus, whereas, in the cytoplasm, the N-terminal ubiquitination pathway is more active. In myotubes upon glucocorticoid administration, the N-terminal ubiquitination pathway is activated and becomes dominant in MyoD degradation in the nucleus, whereas no significant change is induced regarding the activities of the two ubiquitination pathways within the cytoplasm. The thickness of the arrows indicates relative pathway activity.
to be only activated in muscle cells under catabolic conditions, elicited by glucocorticoid increases. Taken together, these results strongly support a significant role for the N terminusdependent ubiquitination pathway in the physiology of muscle protein degradation. Materials and Methods Materials. The C2C12 mouse myoblast cell line was obtained from the American Type Culture Collection. For cells seeded for protein degradation assays where glucocorticoid treatment was performed, charcoal/dextrantreated FBS (HyClone) is used in lieu of regular serum. Transient transfections were performed by using the Fugene 6 reagent (Roche Molecular Biochemicals) (18). Dexamethasone (Sigma) and Mifepristone (RU486) (Cayman) were freshly prepared as a stock solution in ethanol and methanol, respectively, before the addition to cell culture medium at a final concentration of 1 M. Vectors encoding various forms of MyoD and Id1 were either reported previously or constructed with insertion of the specified tags or by site-directed mutagenesis (19, 21). Determination of Protein Degradation Half-Life. Protein degradation half-lives were determined as described (18) by using cycloheximide (CHX, 100 g/ml; Sigma) and/or proteasome inhibitor MG132 (N-benzyloxycarbonayl-leu-leuleucinal, 20 M; Peptides International). The half-life data reported were evaluated by three to six independent determinations and are expressed as mean ⫾ SD. More detailed information is available in supporting information (SI) Text and SI Figs. 7 and 8. ACKNOWLEDGMENTS. This work was supported by the National Institutes of Health. PNAS 兩 March 4, 2008 兩 vol. 105 兩 no. 9 兩 3343
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MyoD degradation
N-terminal
cytoplasm
Lysine
N-terminal
nucleus
Lysine
LL-NLS-HA can be degraded via the N terminus-dependent pathway in the cytoplasm. However, in muscle cells, the regulation of the two ubiquitination pathways in the cytoplasm appears to be markedly different from that in the nucleus. In the nucleus, MyoD-LL-HA is stable (t1/2 ⬇ 10 h) and 6⫻MycMyoD is rapidly degraded (t1/2 ⬇ 1.4 h). In the cytoplasm MyoD-LL-NLS-HA was rapidly degraded (t1/2 ⬇ 1 h), whereas 6⫻Myc-MyoD-NLS is stabilized (t1/2 ⬇ 10 h). Glucocorticoid treatment did not significantly alter the degradation rate of either MyoD-LL-NLS-HA or 6⫻Myc-MyoD-NLS, both cytoplasmically localized. Glucocorticoids did, however, markedly accelerate N terminus-dependent degradation of MyoD-LL localized to the nucleus. These results suggest that, for MyoD degradation, the N terminus-dependent pathway is far more active than the lysine-dependent pathway in the cytoplasm of muscle cells. Furthermore, glucocorticoid activates only the N terminus-dependent pathway of MyoD degradation in the nucleus. Although the major effect of glucocorticoids on muscle catabolism is not likely via MyoD, the degradation of MyoD via the N terminus-dependent pathway represents one important mechanism for muscle protein catabolism. At least three potential mechanisms may account for the activation of the N terminus-dependent ubiquitination pathway of MyoD degradation. First, the two ubiquitination pathways (N terminus-dependent and lysine-dependent) are likely catalyzed by distinct ubiquitin E3 ligases or E2/E3 complexes. In this scenario, activation and regulation of E3 or E2/E3 expression would determine which pathway is more active. Second, the two pathways may be catalyzed by the same ubiquitin E3 ligase, but the ligase may be directed to different pathways upon modification (e.g., phosphor ylation) or through interaction with other regulatory molecules. Third, it is possible that the protein substrate itself undergoes modification, such that its accessibility to different ubiquitination pathway machinery is affected. In addition, it is possible that glucocorticoid treatment promotes MyoD export to the cytoplasm, where it is rapidly degraded. The precise role of glucocorticoid with respect to these alternatives remains to be elucidated. The N terminus-dependent pathway appears to be the predominant pathway for Id1 degradation in C2C12 cells. Replacement of all lysine residues did not alter the degradation rate; lysineless Id1 is as rapidly degraded as is wild-type Id1, whereas N-terminus-blocked Id1 is stable. Glucocorticoid did not alter the degradation rate of either lysineless or N-terminus-blocked Id1. Id2 is also degraded primarily via the N terminus-dependent pathway, because lysineless Id2 is degraded as efficiently as the wild type (15). Id1 protein level is markedly down-regulated in differentiated muscle cells (18). Id1 colocalizes to the nucleus with MyoD in myoblasts. However, in myotubes, Id1 localizes to the cytoplasm (18). Thus, there may be little benefit in regulating Id1 expression in muscle cells under catabolic conditions, where MyoD transcription activity appears to be downregulated independent of its interaction with Id1. The effects of glucocorticoid on MyoD and Id1 protein degradation also likely depend on the various cellular compartments in which their degradation occurs. We have shown that MyoD and Id1 reside within different compartments in C2C12 myotubes, with MyoD in the nucleus and Id1 in the cytoplasm (18). Therefore, it is possible that glucocorticoid affects only the ubiquitin– proteasome system in the nucleus. Consistent with this notion, glucocorticoid did not affect cytoplasmic MyoD degradation. In summary, we present a scheme for MyoD and Id1 degradation by the ubiquitin proteasome system in Fig. 6. MyoD and Id1 are degraded via the N terminus-dependent pathway, although they are physiologically regulated differently. For MyoD, the N terminus-dependent pathway appears
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