The retinoblastoma tumor suppressor protein (pRB) negatively regulates early-G 1 cell cycle progression, in part, by sequestering E2F transcription factors and repressing E2F-responsive genes. Although pRB is phosphorylated on up to 16 cyclin-dependent kinase (Cdk) sites by multiple G 1 cyclin-Cdk complexes, the active form(s) of pRB in vivo remains unknown. pRB is present as an unphosphorylated protein in G 0 quiescent cells and becomes hypophosphorylated (ϳ2 mol of PO 4 to 1 mol of pRB) in early G 1 and hyperphosphorylated (ϳ10 mol of PO 4 to 1 mol of pRB) in late G 1 phase. Here, we report that hypophosphorylated pRB, present in early G 1 , represents the biologically active form of pRB in vivo that is assembled with E2Fs and E1A but that both unphosphorylated pRB in G 0 and hyperphosphorylated pRB in late G 1 fail to become assembled with E2Fs and E1A. Furthermore, using transducible dominant-negative TAT fusion proteins that differentially target cyclin D-Cdk4 or cyclin D-Cdk6 (cyclin D-Cdk4/6) and cyclin E-Cdk2 complexes, namely, TAT-p16 and TATdominant-negative Cdk2, respectively, we found that, in vivo, cyclin D-Cdk4/6 complexes hypophosphorylate pRB in early G 1 and that cyclin E-Cdk2 complexes inactivate pRB by hyperphosphorylation in late G 1 .
Moreover, we found that cycling human tumor cells expressing deregulated cyclin D-Cdk4/6 complexes, due to deletion of the p16INK4a gene, contained hypophosphorylated pRB that was bound to E2Fs in early G 1 and that E2F-responsive genes, including those for dihydrofolate reductase and cyclin E, were transcriptionally repressed. Thus, we conclude that, physiologically, pRB is differentially regulated by G 1 cyclin-Cdk complexes.Stimulation by growth factors of resting G 0 quiescent cells to enter the early-G 1 phase of the cell cycle and to transit across the restriction point into late G 1 phase requires the concerted activities of multiple cyclin-dependent kinases (Cdks) that phosphorylate substrates in a cell cycle-specific fashion (for reviews, see references 13, 42, 56, and 64). Activation of cyclin E-Cdk2 at the late G 1 restriction point and activation of cyclin A-Cdk2 at the transition from late G 1 to S phase suggest the involvement of these cyclin-Cdk complexes at specific cell cycle regulatory checkpoints (42,56,57). In contrast, cyclin D-Cdk4 or cyclin D-Cdk6 (cyclin D-Cdk4/6) complexes are inactive in G 0 quiescent cells but become activated by growth factor addition in early G 1 phase (36, 38). In addition, whereas cyclin Eand A-associated kinase activities remain cell cycle regulated in cycling cells, cyclin D-Cdk4/6 activity is constitutive in cycling cells (5,17,35,44). Importantly, these observations indicate a distinct role for cyclin D-Cdk4/6 complexes in regulating G 1 cell cycle progression from that of cyclin E-Cdk2 and cyclin A-Cdk2. Indeed, Datar et al. (7) and Meyer et al. (37) using genetic models of Drosophila melanogaster have recently demonstrated that cyclin D-Cdk4 complexes regulate cellular growth (accumulation of mass) and not c...