Transduced p16(INK4a) peptides inhibit hypophosphorylation of the retinoblastoma protein and cell cycle progression prior to activation of Cdk2 complexes in late G(1)

Progression of cells through the G(1) phase of the cell cycle requires cycl in D:Cdk4/6 and cyclin E:Cdk2 complexes; however, the duration and ordering of these complexes remain unclear. To address this, we synthesized a pepti dyl mimetic of the Cdk4/6 inhibitor, p16(INK4a) that contained an NH2-termi nal TAT protein transduction domain. Transduction of TAT-p16 wild-type pept ides into cells resulted in the loss of active, hypophosphorylated pRb and elicited an early G(1) cell cycle arrest, provided cyclin E:Cdk2 complexes mere inactive. We conclude that cyclin D:Cdk4/6 activity is required for ea rly G(1) phase cell cycle progression up to, but not beyond, activation of cyclin E:Cdk2 complexes at the restriction point and is thus nonredundant w ith cyclin E:Cdk2 in late G(1).