B-Myb repressor function is regulated by cyclin A phosphorylation and sequences within the C-terminal domain

Area of Science:

• Molecular Biology
• Gene Regulation
• Oncogene Research

Background:

• B-Myb, a member of the myb oncogene family, regulates gene transcription.
• B-Myb can act as either a transcriptional activator or repressor.
• In aortic smooth muscle cells, B-Myb represses alpha2(V) collagen gene transcription.

Purpose of the Study:

• To investigate the effect of cyclin A on B-Myb's repressive function.
• To determine if cyclin A's modulation of B-Myb repression is linked to its known effects on B-Myb transactivation.
• To identify regions of B-Myb involved in cyclin A-mediated repression.

Main Methods:

• Coexpression of B-Myb with cyclin A in relevant cell models.
• Use of dominant-negative cyclin-dependent kinase 2 (cdk2) to modulate kinase activity.
• Site-directed mutagenesis of putative cyclin A phosphorylation sites on B-Myb.
• Analysis of alpha2(V) collagen promoter activity.
• Assessment of B-Myb protein stability and decay rates.

Main Results:

• Cyclin A coexpression abolished B-Myb-mediated repression of the alpha2(V) collagen promoter.
• Dominant-negative cdk2 enhanced B-Myb repression.
• Mutations in cyclin A sites did not affect cyclin A's ability to alleviate repression.
• Cyclin A accelerated the decay of wild-type B-Myb but not mutant forms.
• Specific B-Myb mutants (Mut3, 491) showed positive regulation and were unaffected by cyclin A.

Conclusions:

• Cyclin A abolishes B-Myb's repressive function on the alpha2(V) collagen promoter.
• The mechanisms of B-Myb activation and repression by cyclin A are distinct.
• Negative regulation by B-Myb is mapped to amino acids 491-582.