Figure 2
From: Multiple phosphorylation events control mitotic degradation of the muscle transcription factor Myf5
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 μl 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 μM microcystin LR (this treatment leads to hyperphosphorylation of Myf5 (Myf5PPP), see text and figure 3 for details); lanes 3–6: 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 (λ PPase), as indicated. Samples were resolved by 10% SDS-PAGE and analyzed using a PhosphoImager. (D) A mitotic extract was first incubated with 200 μM MG132, or the same volume of pure DMSO as a control, for 10 min at 25°C; dephosphorylated (deP) Myf5 (see method) was then added to the treated extract. At each time point, 2 μl 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.