Integration of Rac-dependent regulation of cyclin D1 transcription through a nuclear factor-kappa B-dependent pathway

The small GTP-binding protein Rad, a member of the Ras superfamily, plays a fundamental role in cytoskeleton reorganization, cellular transformation, the induction of DNA synthesis, and superoxide production. Cyclin D1 abundance is rate-limiting in normal G(1), phase progression, and the abundance of cyclin D1 is induced by activating mutations of both Res and Rad, Nuclear factor-KB (NF-kappa B) proteins consist of cytoplasmic hetero- or homodimeric Rel-related proteins complexed to a member of the I kappa B family of inhibitor proteins. In the current studies, activating mutants of Rac1 (Rac(Leu-61), Rac(Val-12)) induced cyclin D1 expression and the cyclin D1 promoter in NIH 3T3 cells. Induction of cyclin D1 by Rad required both an NF-kappa B and an ATF-2 binding site. Inhibiting NF-kappa B by overexpression of an NF-kappa B transdominant inhibitor (nonphosphorylatable IKBa) reduced cyclin D1 promoter activation by the Rad mutants, placing NF-kappa B in a pathway of Rad activation of cyclin D1, Specific amino acid mutations in the aminoterminal effector domain of Rac(Leu-61) had comparable effects on NF-kappa B transcriptional activity and activation of the cyclin D1 promoter. The NF-kappa B factors Rel A (p65) and NF-kappa B-1 (p50) induced the cyclin D1 promoter, requiring both the NF-kappa B binding site and the ATF-8 site. Stable overexpression of Rae(Leu-61) increased binding of Rel A and NF-kappa B, to the cyclin D1 promoter NF-kappa B site. Activation of Rad in NIH 3T3 cells induces both NF-kappa B binding and activity and enhances expression of cyclin D1 through an NF-kappa B and ATF-2 site in the proximal promoter, suggesting a critical role for NP-kappa B in cell cycle regulation through cyclin D1 and Rad.