PMC

Schematic diagram of the MyoD protein indicating functional domains and putative CDK phosphorylation sites. NLS, nuclear localization signal.

Increased stability of MyoD S200A. The half-lives of nuclear wild-type MyoD and MyoD S200A were determined as described in Materials and Methods. C3H10T1/2 fibroblasts transiently transfected with either wild-type MyoD (WT-MyoD) (A) or MyoD S200A (B) were treated with cycloheximide for the times indicated; nuclear extracts were prepared and subjected to Western analysis. Identical blots were subjected to Western analysis with anti-Cdc2 antibodies to verify sample loading.

Expression of a dnCDC34 stabilizes MyoD. C3H10T1/2 fibroblasts were transiently transfected with MyoD, CDC34, or both; nuclear extracts were prepared and subjected to Western analysis as described for Fig. . (A) Western analysis of nuclear extracts prepared from cells transfected with the indicated plasmids and incubated in the presence of anti-MyoD. Lanes: 1, pCB6+; 2, pCB6+ and wild-type MyoD; 3, wild-type MyoD and wild-type CDC34; 4, wild-type MyoD and dnCDC34. (B) Western analysis of the samples described for panel A, incubated with anti-Cdc34.

The proteasome inhibitor LLnL stabilizes the hyperphosphorylated form of MyoD. (A and B) C3H10T1/2 fibroblasts transiently transfected with wild-type MyoD were treated with cycloheximide for the times indicated in the absence (A) or presence (B) of LLnL; nuclear extracts were prepared and subjected to Western analysis. The same blots were subjected to Western analysis with anti-Cdc2 antibodies to verify sample loading. (C) C3H10T1/2 fibroblasts transiently transfected with wild-type MyoD were not treated (lane 1) or treated with LLnL for 1 h (lane 2) or 2 h (lane 3) prior to harvest; nuclear extracts were prepared and subjected to Western analysis.

Increased 4TRTK-CAT activity in C3H10T1/2 fibroblasts expressing MyoD proteins containing mutations of CDK consensus phosphorylation sites. Wild-type (WT) or mutant MyoD expression vectors, 4TRTK-CAT, and a TK-luciferase vector were cotransfected into C3H10T1/2 fibroblasts. Cultures were harvested 36 h posttransfection, and luciferase and CAT activities were determined. The results are presented as percentages of the transactivating ability of wild-type MyoD, which was arbitrarily set to 100%. Each experiment was done in duplicate, and each CAT assay contained equivalent amounts of luciferase activity. The results presented are means ± standard deviations of three separate experiments.

Hyperphosphorylation of MyoD requires S200. Thirty micrograms of pCB6+ vector or pCB6+ containing wild-type or mutated MyoD was transfected into C3H10T1/2 fibroblasts as described in Materials and Methods. At 36 h after transfection, cells were harvested; nuclear extracts were prepared and separated by SDS-PAGE. Separated proteins were subjected to Western analysis using anti-MyoD antibodies. (A) Lanes (oligonucleotide sequences in brackets): 1, vector alone; 2, MyoD (wild-type MyoD); 3, SP1-MyoD (MyoD S5A [ELLSPPLR ELLAPPLR]); 4, SP2-MyoD (MyoD S37A [CFDSPDLR CFDAPDLR]); 5, SP3-MyoD (MyoD S200A [DASSPRSN DASAPRSN]); 6, SP4-MyoD (MyoD S262A [STDSPAAP STDAPAAP]); 7, SP5-MyoD (MyoD S277A [PPESPPGP PPEAPPGP]); 8, SP6-MyoD (MyoD T296A/S298A [GTQTPSPDA GTQA PAPDA]). (B) Lanes: 1, wild-type MyoD-containing nuclear extracts; 2, wild-type MyoD-containing nuclear extracts pretreated with calf intestine alkaline phosphatase; 3, MyoD S200A-containing nuclear extracts; 4, MyoD S200A nuclear extracts pretreated with calf intestine alkaline phosphatase.