Transduced p16 super(INK4a) Peptides Inhibit Hypophosphorylation of the Retinoblastoma Protein and Cell Cycle Progression Prior to Activation of Cdk2 Complexes in Late G sub(1)

Progression of cells through the G sub(1) phase of the cell cycle requires cyclin D:Cdk4/6 and cyclin E:Cdk2 complexes; however, the duration and ordering of these complexes remain unclear. To address this, we synthesized a peptidyl mimetic of the Cdk4/6 inhibitor, p16 super(INK4a) that contained an NH sub(2)-terminal TAT protein transduction domain. Transduction of TAT-p16 wild-type peptides into cells resulted in the loss of active, hypophosphorylated pRb and elicited an early G sub(1) cell cycle arrest, provided cyclin E:Cdk2 complexes were inactive. We conclude that cyclin D:Cdk4/6 activity is required for early G sub(1) phase cell cycle progression up to, but not beyond, activation of cyclin E:Cdk2 complexes at the restriction point and is thus nonredundant with cyclin E:Cdk2 in late G sub(1).