INHIBITION OF CYCLIN D1 KINASE-ACTIVITY IS ASSOCIATED WITH E2F-MEDIATED INHIBITION OF CYCLIN D1 PROMOTER ACTIVITY THROUGH E2F AND SP1

Coordinated interactions between cyclin-dependent kinases (Cdks), thei r target ''pocket proteins'' (the retinoblastoma protein [pRB], p107, and p130), the pocket protein binding E2F-DP complexes, and the Cdk in hibitors regulate orderly cell cycle progression. The cyclin D1 gene e ncodes a regulatory subunit of the Cdk holoenzymes, which phosphorylat e the tumor suppressor pRB, leading to the release of free E2F-1. Over expression of E2F-1 can induce apoptosis and may either promote or inh ibit cellular proliferation, depending upon the cell type. In these st udies overexpression of E2F-1 inhibited cyclin D1-dependent kinase act ivity, cyclin D1 protein levels, and promoter activity. The DNA bindin g domain, the PRE pocket binding region, and the amino-terminal Sp1 bi nding domain of E2F-1 were required for full repression of cyclin D1, Overexpression of pRB activated the cyclin D1 promoter, and a dominant interfering pRB mutant was defective in cyclin D1 promoter activation . Two regions of the cyclin D1 promoter were required for full E2F-1-d ependent repression. The region proximal to the transcription initiati on site at -127 bound Sp1, Sp3, and Sp4, and the distal region at -143 bound E2F-4-DP-1-p107. In contrast with E2F-1, E2F-4 induced cyclin D 1 promoter activity. Differential regulation of the cyclin D1 promoter by E2F-1 and E2F-4 suggests that E2Fs may serve distinguishable funct ions during cell cycle progression. Inhibition of cyclin D1 abundance by E2F-1 may contribute to an autoregulatory feedback loop to reduce p RB phosphorylation and E2F-1 levels in the cell.