Figure 5.

Involvement of PDE4D3 phosphorylation in the myogenic response induced by overexpression of the enzyme. L6-C5 myoblasts were transiently transfected either with 1 μg per dish of pCMV5 containing wild-type PDE4D3 cDNA (Sette et al., 1994) or various amounts of pCMV5 containing mutant ser⁵⁴ala-PDE4D3 cDNA (Sette and Conti, 1996) or with the empty vector (mock), and plated in 10% FBS-containing medium. (A) After 24 h, the medium was replaced by medium containing 1% BSA with or without the addition of 1 nM IGF-I. Myogenin was detected by immunofluorescence, by using the antimyogenin antibody F5D. The results are expressed as percentages of myogenin-positive nuclei (the total number of nuclei was evaluated by UV microscopy using Hoechst 33342 nuclear dye). Normalization of the efficiency of transfection was obtained by cotransfecting a GFP-carrying plasmid. Results are the mean ± SEM of four independent experiments in which 10 fields were counted. ⁺⁺p < 0.02 compared with Mock-transfected cells −IGF; *p < 0.05 compared with wt-PDE4D3–transfected cells − IGF; **p < 0.02 compared with wt-PDE4D3–transfected cells −IGF; ###p < 0.0001 compared with Mock-transfected cells +IGF; ^∧∧∧p < 0.0001 compared with wt-PDE4D3–transfected cells +IGF. The specific activities of PDE in the homogenates of cells transfected with empty, 1 μg wild-type-PDE4D3-, 1 μg, 0.3 μg, 0.1 μg ser⁵⁴ala mutant-containing plasmid DNA were, respectively, of 25.2 ± 0.6, 114.8 ± 4.6, 141.4 ± 6.2, 35.4 ± 4.6, 16.6 ± 1.6 pmol·min⁻¹·mg proteins⁻¹. (B) Twenty-four hours after plating in 10% FBS medium, the transfected cells were shifted to 1% FBS medium. Myogenin nuclear accumulation was quantified as above. Results are the mean ±. SEM of three independent experiments in which 10 fields were counted. *p < 0.05 compared with Mock-transfected cells.