Doucet C et al. (2005), Multiple phosphorylation events control mitotic...

XB-ART-1015

BMC Biochem 2005 Sep 28;6:27. doi: 10.1186/1471-2091-6-27.

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Multiple phosphorylation events control mitotic degradation of the muscle transcription factor Myf5.

Doucet C , Gutierrez GJ , Lindon C , Lorca T , Lledo G , Pinset C , Coux O .

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The two myogenic regulatory factors Myf5 and MyoD are basic helix-loop-helix muscle transcription factors undergoing differential cell cycle dependent proteolysis in proliferating myoblasts. This regulated degradation results in the striking expression of these two factors at distinct phases of the cell cycle, and suggests that their precise and alternated disappearance is an important feature of myoblasts, maybe connected to the maintenance of the proliferative status and/or commitment to the myogenic lineage of these cells. One way to understand the biological function(s) of the cyclic expression of these proteins is to specifically alter their degradation, and to analyze the effects of their stabilization on cells. To this aim, we undertook the biochemical analysis of the mechanisms governing Myf5 mitotic degradation, using heterologous systems. We show here that mitotic degradation of Myf5 is conserved in non-myogenic cells, and is thus strictly under the control of the cell cycle apparatus. Using Xenopus egg extracts as an in vitro system to dissect the main steps of Myf5 mitotic proteolysis, we show that (1) Myf5 stability is regulated by a complex interplay of phosphorylation/dephosphorylation, probably involving various kinases and phosphatases, (2) Myf5 is ubiquitylated in mitotic extracts, and this is a prerequisite to its degradation by the proteasome and (3) at least in the Xenopus system, the E3 responsible for its mitotic degradation is not the APC/C (the major E3 during mitosis). Altogether, our data strongly suggest that the mitotic degradation of Myf5 by the ubiquitin-proteasome system is precisely controlled by multiple phosphorylation of the protein, and that the APC/C is not involved in this process.

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Species referenced: Xenopus
Genes referenced: ccnb1.2 cdc27 cdh1 cdk1 kif22 mapk1 myf5 myod1 rpsa

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	Figure 1. Myf5 mitotic degradation is conserved in non-muscle cells. HeLa-S3 cells expressing Myf5 under an inducible promotor (tetoff) were treated (lanes 2, 3, 4) or not (AS, lane 1) with nocodazole (200 ng/ml) for 16 h, as in [20]. In lanes 3 and 4, cells were treated with a proteasome inhibitor (MG132, 50 Î¼M) for 2 h prior to lysis (as described in Methods). In lane 4, extract was incubated with Lambda phosphatase (New England Biolabs) for 30 min at 30Â°C. 25 Î¼g of each extract were resolved by 10% SDS-PAGE and immunoblotted with anti-Myf5 antibodies. AS: asynchronous cells; * indicates a non specific band recognized by the antibodies that can be used as a loading control.
	Figure 2. Myf5 is degraded in a cell-cycle dependent manner in Xenopus egg extracts, and its degradation is correlated with changes in its phosphorylation status. (A) Myf5 is phosphorylated and degraded in mitotic (CSF) extracts, but dephosphorylated and stable in interphase extracts. Degradation assays were performed as described in Methods; 2 Î¼l samples were taken at the indicated times and resolved by 10% SDS-PAGE. (B) Degradation of Myf5 was quantified and normalized (start = 100%) : black squares represent the average degradation of Myf5 in interphase extracts (5 independent experiments), open circles the average degradation of Myf5 in CSF extracts (7 independent experiments), and black circles the average degradation of dephosphorylated (deP) Myf5 (see Methods section) in CSF extracts (4 independent experiments). Bars represent standard deviation. For each lane, a region corresponding to all non-phosphorylated and phosphorylated forms of Myf5 was quantified using the ImageQuant software (Molecular Dynamics). The apparent increase of Myf5 level in interphase extracts is probably due to the accumulation of all the initially phosphorylated forms of Myf5 (some of them being close to background) into the fastest migrating form of Myf5. (C) All the slower migrating forms of Myf5 are phosphorylated: lane 1: in vitro translated Myf5; lane 2: in vitro translated Myf5 after 1 h incubation with CSF extract in the presence of 1 Î¼M microcystin LR (this treatment leads to hyperphosphorylation of Myf5 (Myf5PPP), see text and figure 3 for details); lanes 3â6: in vitro translated Myf5 (lanes 3 and 4), or Myf5PPP (lanes 5 and 6) were immunoprecipitated with anti-Myf5 antibodies and incubated in the absence or presence of Lambda phosphatase (Î» PPase), as indicated. Samples were resolved by 10% SDS-PAGE and analyzed using a PhosphoImager. (D) A mitotic extract was first incubated with 200 Î¼M MG132, or the same volume of pure DMSO as a control, for 10 min at 25Â°C; dephosphorylated (deP) Myf5 (see method) was then added to the treated extract. At each time point, 2 Î¼l samples were resolved by 10% SDS-PAGE. (E) Relative amounts of Myf5 in the gel shown in D were quantified as above and reported on the graph.
	Figure 3. Hyperphosphorylated Myf5 is stable. An equal volume of reticulocyte lysate containing in vitro translated Myf5 and of Xenopus mitotic egg extract (CSF) were incubated together for 60 min in the presence of 1 Î¼M microcystin LR. At each time point, 0.5 Î¼l samples were analyzed by 10% SDS-PAGE (panel A). After this initial incubation, 2 Î¼l of the reaction mixture containing hyperphosphorylated Myf5 (Myf5PPP) were further incubated in 18 Î¼l of fresh mitotic extract, either in the presence of 1 Î¼M microcystin (panel B) or without inhibitor addition (panel C), or without microcystin and in the presence of the proteasome inhibitor MG132 (200 Î¼M, panel D). At each time point, 2 Î¼l samples were analyzed by 10% SDS-PAGE.
	Figure 4. Several signaling pathways are likely to control Myf5 mitotic degradation (A) 1 Î¼l of untreated (upper panel) or dephosphorylated (deP, lower panel) Myf5 was incubated with buffer (lane 2), recombinant CDK1/cyclin B (New England Biolabs, 40U, lane 3), recombinant Erk2 (New England Biolabs, 200U, lane 4) or both kinases (lane 5) for 30 min at 30Â°C. The reaction mix was resolved by 10% SDS-PAGE. "Start" is a non-incubated control. (B) Myf5 degradation is sensitive to very low concentrations of calcium: a mitotic extract was prepared using XB without CaCl2 but supplemented with 6 mM EGTA (see Methods), then incubated with 0.1 mM CaCl2 and 6 mM EGTA, or an equivalent volume of water as a control, for 10 min at 25Â°C. 2 Î¼l of Myf5 were incubated for 1 hour with 18 Î¼l of treated extract. 2 Î¼l samples were resolved by 10% SDS-PAGE at the beginning and the end of the reaction. Myf5 degradation rate was quantified as previously described; the graph represents mean values of 3 experiments. Bars represent standard deviations.
	Figure 5. Polyubiquitylation of Myf5 is required for its degradation. (A) Myf5 is polyubiquitylated in mitotic extracts: Myf5 was incubated in a mitotic extract in the presence of ubiquitin (1 mg/ml), MG132 (200 Î¼M) and ubiquitin aldehyde (5 Î¼M). At the times indicated, 2 Î¼l samples were analyzed by 10% SDS-PAGE to visualize the formation of high-MW adducts ((Ub)n-Myf5, poly-ubiquitylated Myf5 molecules). (B) Lysine-less (K0) ubiquitin inhibits Myf5 polyubiquitylation: Myf5 ubiquitylation was performed in a mitotic extract in the presence of MG132 (200 Î¼M), ubiquitin aldehyde (1 Î¼M), ubiquitin (Ub) and lysine-less-ubiquitin (UbK0) at the indicated concentrations, for 30 min at 25Â°C. (C) Lysine-less ubiquitin stabilizes Myf5: Degradation assays were performed in 20 Î¼l reaction mixtures containing 16 Î¼l of mitotic extract, 2 Î¼l of radiolabeled in vitro translated Myf5 and a mixture of wild type and K0 ubiquitin as indicated. The reaction was performed at 25Â°C for 40 min, 2 Î¼l samples were resolved by 10% SDS-PAGE. "START" corresponds to a 2 Î¼l sample taken at time 0 from the reaction containing 1 mg/ml Ub. Radioactivity was quantified as previously described (see legend of figure 2) and percentages of degradation were calculated for each Ub/UbK0 ratio from the difference between the times 0 (not shown) and 40 minutes.
	Figure 6. The E3 responsible for Myf5 ubiquitylation is not the APC/C. (A) In vitro translated Xkid (upper panel) or Myf5 (lower panel) were incubated in 15 Î¼l Xenopus interphase egg extract in which Cdh1 has been translated [39]. After the times indicated, 2.5 Î¼l were analyzed by SDS-PAGE and fluorography. (B) 1 ml of mitotic extract was activated with 1 Î¼M microcystin LR (in order to fully activate APC/C), then fractionated on a DEAE column (see Methods). Bound proteins were eluted in two steps with buffer A containing 0.25 M and 0.5 M NaCl, respectively. Each fraction (lanes 2 & 3) was compared to a control reaction containing buffer (lane 1) for its ability to mediate ubiquitylation of either cyclin B (upper panel) or Myf5 (lower panel). Lanes 4â7: the 0.25 M NaCl eluate that mediates both cyclin B and Myf5 ubiquitylation was then subjected to immunoprecipitations using either anti-Cdc27 or control antibodies. For each immunoprecipitation, both the material bound to the beads or the supernatant were analyzed for ubiquitylation activity using cyclin B (upper panel) or Myf5 (lower panel) as a substrate. (Ub)n-cycB and (Ub)n-Myf5 indicate poly-ubiquitylated forms of cyclin B and Myf5, respectively.
	Figure 7. Myf5 ubiquitylation is controlled by phosphorylation. (A) Scheme of Myf5 E3 purification : for details see Methods section. (B) 2 ml of mitotic extract were fractionated as described in A; the fractions obtained from the UnoQ column were screened for their ability to ubiquitylate radiolabeled Myf5 in the presence of a ubiquitylation mix containing GST-Ub (see Methods). The reactions were resolved by 10% SDS-PAGE. (Ub)n-Myf5 indicates poly-ubiquitylated forms of Myf5. (C) The fraction containing the E3 was concentrated about 10-fold using a Centricon 10 K (Millipore). Untreated Myf5 (lanes 1 to 3) or dephosphorylated (deP) Myf5 (lanes 4 to 6) was incubated with the ubiquitylation mix alone (lanes 2, 5) or together with 6 Î¼l of concentrated fraction containing the E3 (lanes 3, 6) for 30 minutes at 25Â°C. Non incubated Myf5 was loaded in lanes 1 and 4 (START) as control. Similar amounts of radioactive Myf5 and dephosphorylated Myf5 were used.
	Figure 8. A model for Myf5 mitotic degradation pathway. Myf5 is subjected to multiple phosphorylations in Xenopus mitotic egg extracts, that tightly control its stability: phosphorylation of the protein leads to its ubiquitylation by an E3 distinct from APC/C, and its subsequent degradation by the proteasome. However, a hyperphosphorylated form of Myf5 remains stable (see text for details).

References [+] :

Abu Hatoum, Degradation of myogenic transcription factor MyoD by the ubiquitin pathway in vivo and in vitro: regulation by specific DNA binding. 1998, Pubmed