pRB phosphorylation mutants reveal role of pRB in regulating S phase completion by a mechanism independent of E2F

Abstract

Progression of cells into S phase is controlled by the retinoblastoma protein (pRB) and relies on the functional inactivation of this tumour suppressor in late G1 via protein phosphorylation. We provide evidence here that, besides controlling entry of cells into S phase, pRB can operate to inhibit S phase completion. Dierential arrays of phosphorylation appear to regulate these dierent events, suggesting that cycle progression at these two stages of the cell cycle may be achieved via activation of distinct downstream pRB eector pathways. In agreement with this hypothesis, pRB's ability to prevent S phase entry, but not its ability to inhibit S phase completion, correlates with repression of E2F-regulated promoters. Furthermore, ectopic expression of E2F or the E2F-regulated cyclin E gene promote S phase entry in cells expressing phosphorylation-defective pRB but neither is sucient to trigger completion of S phase. Our ®ndings raise the possibility that pRB, in addition to its well-established role in controlling a checkpoint in late G1, could be involved in the control of a further checkpoint operating during S phase and that implementation of this checkpoint relies on an as yet unidenti®ed pRB eector distinct from E2F.Keywords: retinoblastoma protein; pRB; E2F; phosphorylation; checkpoint control; cell cycle IntroductionThe growth-controlling activity of the tumour suppressor, retinoblastoma protein (pRB), relies on its capacity to reversibly associate with E2F and other cellular transcription factors (Herwig and Strauss, 1997;Weinberg, 1995). The phosphorylation state of pRB determines its ability to bind to these factors (reviewed in Bandara et al., 1993;Taya, 1997). Evidence to this eect was ®rst provided through analysis of pRB's association with SV40 T antigen (Sterner et al., 1998;Templeton et al., 1991;Whyte et al., 1989). Only un-or poorly phosphorylated pRB associated with this viral oncoprotein and this has since been similarly demonstrated for many of the cellular pRB ligands.Phosphorylation of pRB, leading to its inactivation, is executed by members of the cyclin-dependent family of protein kinases (ConnellCrowley et al., 1997;Johnson et al., 1995;Lees et al., 1991;Lin et al., 1991;Mittnacht et al., 1994;Sterner et al., 1998;Templeton et al., 1991;Whyte et al., 1989;. In growing cells, the phosphorylation state of pRB changes periodically and in accordance with the cellular division cycle. Un-or poorly phosphorylated pRB, distinguished from its highly phosphorylated variant by a faster migration in SDS gels and its tight association with the cell nucleus, exists in cells only during the early phases of G1 (Buchkovich et al., 1989;Chen et al., 1989;DeCaprio et al., 1989;Mittnacht et al., 1991Mittnacht et al., , 1994. This form of pRB is no longer detectable upon passage of cells into S phase. Instead, highly phosphorylated pRB accumulates and prevails throughout the remainder of the cell cycle until the cells enter the next G1 phase, when pRB is converted into an un-or poorly phosphorylated form by d...

Conserved region 2 of adenovirus E1A has a function distinct from pRb binding required to prevent cell cycle arrest by p16INK4a or p27Kip1

“…However, E2F is not su cient to prevent growth inhibition by p27, implying that cyclin E/CDK2 complexes have an additional role(s) at the G1-S transition, distinct from pRb phosphorylation (Alevizopoulos et al, 1997;Mann and Jones, 1996). Similar conclusions were reached by other groups (Chew et al, 1998;Hofmann and Livingston, 1996;Knudsen et al, 1998;Leng et al, 1997;Lukas et al, 1997;Ohtsubo et al, 1995;Resnitzky and Reed, 1995). In summary, the ability of the CDK4 inhibitor p16 to arrest the cell cycle in G1 seems to depend essentially on activation of pRb and sequestration of E2F, whereas p27 not only activates pRb, but also impinges on other CDK2-regulated events (discussed in Dyson, 1998).…”

Overexpressed BCL6 (LAZ3) oncoprotein triggers apoptosis, delays S phase progression and associates with replication foci

“…Note that the di use nuclear BCL6fg staining (visible on Figures 1c and Figure 8a), but not the nuclear dots, largely disappears upon the`pre-permeabilization' procedure (before formalin ®xation) used in this experiment (see Materials and methods) Growth suppression and apoptosis by BCL6 (LAZ3) O Albagli et al Albagli et al, 1998;Foley et al, 1998 and references therein). Of particular interest is the case of RB, which inhibits both the entry and the progression through S phase (Brehm et al, 1998;Chew et al, 1998;Knudsen et al, 1998;Luo et al, 1998;MagnaghiJaulin et al, 1998 and references therein). On the contrary, certain histone acetyltransferases may facilitate the progression in the cell cycle, as P300 undergoes an activating phosphorylation at the G1/S boundary (Ait-Si-Ali et al, 1998) while mice lacking P300 have defects in cell proliferation (Yao et al, 1998).…”

Retinoblastoma protein and the leukemia-associated PLZF transcription factor interact to repress target gene promoters

“…Mammalian cDNA expression vectors for PML, PLZF, 5 0 PLZF (encoding amino acids 1-455 of PLZF, but lacking the last seven C-terminal zinc-fingers), poxvirus and zinc-finger (POZ) domain (encoding the N-terminal amino acids 1-120 of PLZF) and the GST-PLZF plasmid have been previously described (Chen et al, 1994;Dong et al, 1996;Guidez et al, 1998). Expression vectors for E2F1 (Hsieh et al, 1997), Rb (Chew et al, 1998) and GST-Rb (full-length and deletions) (Zarkowska and Mittnacht, 1997) have also been previously described. (Figure 1c).…”

“…This is facilitated through selective partnering with other transcription factors, which in turn bring promoters with combinatorial binding sites under E2F and, hence, Rb control (Schlisio et al, 2002; Figure 4 Co-expression of retinoblastoma (Rb) and promyelocytic leukemia zinc-finger (PLZF) enhances transcriptional repression of PLZF and E2F1/Rb target genes. C33A cells were transiently transfected (Lipofectamine 2000, Qiagen) with luciferase reporters containing (a) three copies of an E2F consensus binding sequence along with mammalian expression plasmids encoding PLZF, phosphorylation-deficient Rb protein (Rb NPC ) or E2F1 and DP1 as indicated (Chen et al, 1994;Chew et al, 1998) or (b) a PLZFresponsive Hoxb2 r3/r5 enhancer luciferase reporter (Ivins et al, 2003) along with PLZF and Rb NPC as indicated. Cells were harvested 36 h after transfection and luciferase assays performed as previously described (Petrie et al, 2003).…”

“…Rb and PLZF co-localize. (b) C33A cells were transiently transfected (Lipofectamine 2000; Qiagen, Valencia, CA, USA) with PLZF and Rb (Chen et al, 1994;Chew et al, 1998). After fixation with 4% paraformaldehyde, cells were stained with To-pro-3 iodide (Invitrogen, Carlsbad, CA, USA, stained blue) and rabbit polyclonal anti-PLZF (H300; Santa Cruz Biotechnology, shown in green) and mouse monoclonal anti-Rb (G3-245; BD Biosciences; shown in red) antibodies.…”