Differential Regulation of Retinoblastoma Tumor Suppressor Protein by G₁ Cyclin-Dependent Kinase Complexes In Vivo

Abstract

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...

RB and cyclin dependent kinase pathways: defining a distinction between RB and p16 loss in lung cancer

“…Since inhibition of cyclin E activity, but not cyclin D, can induce G1 arrest of proliferating cells, the indirect suppression of CDK2:cyclin E by p27Kip1 may be the primary growth suppressor function mediated by p16 induction (Jiang et al, 1998). In fact, the specific role for cyclin D during the G1 phase of the cell cycle is still controversial (Dowdy et al, 1993;Ezhevsky et al, 2001;Geng et al, 1999;Ho and Dowdy, 2002). In addition, although RB is maximally phosphorylated by sequential cyclin D and cyclin E phosphorylation (Adams, 2001) and RB status has been inversely correlated only with p16 protein expression in lung cancer, the possibility still exists that the deregulated phosphorylation of another unknown cyclin E substrate(s) may also be critical for p16 tumor suppressor activity during lung tumorigenesis.…”

Eps8 decreases chemosensitivity and affects survival of cervical cancer patients

“…Rb is differentially phosphorylated by cyclindependent kinases (cdk) in G 1 phase (12,13). In the presence of growth factor stimulation, quiescent cells reenter the cell cycle and Rb is hypophosphorylated by cyclin D-cdk4/6 complexes in early G 1 phase (12).…”

“…Rb is differentially phosphorylated by cyclindependent kinases (cdk) in G 1 phase (12,13). In the presence of growth factor stimulation, quiescent cells reenter the cell cycle and Rb is hypophosphorylated by cyclin D-cdk4/6 complexes in early G 1 phase (12). It is the hypophosphorylated Rb that associates with E2F transcription factors (E2F) and represses the transcription of their target genes required for DNA replication and the progression of cells from G 1 to S phase (12,14).…”

“…It is the hypophosphorylated Rb that associates with E2F transcription factors (E2F) and represses the transcription of their target genes required for DNA replication and the progression of cells from G 1 to S phase (12,14). When cyclin E-cdk2 further phosphorylates Rb into the hyperphosphorylayted form in late G 1 phase (12), E2F are released and turn on their downstream target genes. As a pivotal transcription factor for cell cycle arrest and apoptosis, the expression level of p53 can be modulated by Mdm2-mediated ubiquitination and proteosome degradation in normal cells (15,16).…”

“…In the presence of growth factor stimulation, quiescent cells reenter the cell cycle and Rb is hypophosphorylated by cyclin D-cdk4/6 complexes in early G 1 phase (12). It is the hypophosphorylated Rb that associates with E2F transcription factors (E2F) and represses the transcription of their target genes required for DNA replication and the progression of cells from G 1 to S phase (12,14). When cyclin E-cdk2 further phosphorylates Rb into the hyperphosphorylayted form in late G 1 phase (12), E2F are released and turn on their downstream target genes.…”