Evidence of p53-dependent cross-talk between ribosome biogenesis and the cell cycle: effects of nucleolar protein Bop1 on G(1)/S transition

Abstract

Bop1 is a novel nucleolar protein involved in rRNA processing and ribosome assembly. We have previously shown that expression of Bop1Delta, an amino-terminally truncated Bop1 that acts as a dominant negative mutant in mouse cells, results in inhibition of 28S and 5.8S rRNA formation and deficiency of newly synthesized 60S ribosomal subunits (Z. Strezoska, D. G. Pestov, and L. F. Lau, Mol. Cell. Biol. 20:5516-5528, 2000). Perturbation of Bop1 activities by Bop1Delta also induces a powerful yet reversible cell cycle arrest in 3T3 fibroblasts. In the present study, we show that asynchronously growing cells are arrested by Bop1Delta in a highly concerted fashion in the G(1) phase. Kinase activities of the G(1)-specific Cdk2 and Cdk4 complexes were downregulated in cells expressing Bop1Delta, whereas levels of the Cdk inhibitors p21 and p27 were concomitantly increased. The cells also displayed lack of hyperphosphorylation of retinoblastoma protein (pRb) and decreased expression of cyclin A, indicating their inability to progress through the restriction point. Inactivation of functional p53 abrogated this Bop1Delta-induced cell cycle arrest but did not restore normal rRNA processing. These findings show that deficiencies in ribosome synthesis can be uncoupled from cell cycle arrest and reveal a new role for the p53 pathway as a mediator of the signaling link between ribosome biogenesis and the cell cycle. We propose that aberrant rRNA processing and/or ribosome biogenesis may cause "nucleolar stress," leading to cell cycle arrest in a p53-dependent manner.

FIG. 1

Expression of Bop1Δ in asynchronously growing cells induces reversible G₁ growth arrest. (A) LAP3 cells were cotransfected with pPGK-puro, which confers resistance to puromycin, and either the empty IPTG-inducible vector (pX11) or constructs for inducible expression of the Cdk inhibitor p21 (pX11-p21) or Bop1Δ (pX11-Bop1Δ). Equal numbers of stably transfected, puromycin-resistant cells were treated in parallel with IPTG for 24 h to induce expression and then with IPTG and BrdU for 48 h to selectively kill proliferating cells, as previously described (46). Cells that did not replicate DNA during this period and therefore survived the treatment were rescued by removal of the BrdU and IPTG, grown for 8 days, and stained with crystal violet. The number of colonies thus reflects the number of cells that were reversibly cell cycle arrested while under IPTG induction of the transfected gene. (B) Bop1Δ causes accumulation of cells in G₁. Parallel cultures of asynchronously growing cells were left untreated (−IPTG) or treated with inducer for 24 h (+IPTG) and subjected to flow cytometry analysis. Two independent clonal lines that inducibly express Bop1Δ (Bop1Δ/2 and Bop1Δ/6) and a control clonal line (LAP3/1) transfected with the empty vector were analyzed. Histograms of the cellular DNA content and the calculated distributions of cell populations in different phases of the cell cycle are shown.

FIG. 2

Bop1Δ inhibits Cdk4-associated activity but not the abundance of Cdk4 or cyclin D1. (A) pRb kinase activity in Cdk4 immune complexes. Cdk4-specific phosphorylation of recombinant pRb as a substrate (see Materials and Methods) was determined by immunoblotting of reaction products with antibodies specific to phosphorylated Ser780 of pRb [anti-pRb(P-Ser780)] (top), and then the same blot was reprobed with anti-pRb antibody to show equal amounts of the substrate in each lane (bottom). Assays were performed with immunoprecipitates from Bop1Δ/6 and parental LAP3 cells that were untreated (−) or treated with IPTG for 24 h (+). Lane 0, LAP3 cells serum starved for 48 h to determine basal kinase activity; lane c, no-cell-lysate control. (B) Cdk4 and cyclin D1 levels in the cell lysates shown in panel A were determined by immunoblotting of cell lysates with the indicated antibodies.

FIG. 3

Effects of Bop1Δ on cyclin E/A-Cdk2, CKIs, and pRb phosphorylation. (A) Histone H1 kinase activity of Cdk2 immunoprecipitates (top; duplicate assays shown) and cyclin E and cyclin A immunoprecipitates (bottom) were determined in the presence of [γ-³²P]ATP. Reaction products were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and detected by autoradiography. (B) Lysates from cells that were untreated (−) or treated with IPTG for 24 h (+) were resolved by SDS-PAGE and immunoblotted with antibodies against the indicated proteins. (C) At the indicated times after IPTG addition to growing cultures of Bop1Δ/6 and control LAP3/1 cells, pRb was immunoprecipitated from cell lysates, separated on a 7.5% polyacrylamide gel, and detected by immunoblotting with anti-pRb antibody. pRb∗, hyperphosphorylated pRb.

FIG. 4

Time course of Bop1Δ induction and its effects on DNA synthesis and rRNA processing. (A) The Bop1Δ and LAP3 cell lines were treated with IPTG for the indicated times, and their rates of DNA synthesis (top) were determined by measuring incorporation of [³H]thymidine. Histograms show the average counts per minute in quadruplicate samples, expressed as a percentage of that in untreated cultures; error bars indicate standard deviation. For analysis of rRNA processing (bottom), RNA from cells undergoing parallel treatment and labeled with [³H]uridine was separated on an agarose gel, transferred to a nylon membrane, and visualized by fluorography. Positions of mature 28S and 18S rRNAs and major precursors are marked. (B) Bop1Δ detected by immunoblotting with anti-Bop1 antibodies at various times after IPTG induction in Bop1Δ/6 cells. Parental LAP3 cells were used as a control.

FIG. 5

Rate of global protein synthesis is unaffected by Bop1Δ at the time of cell cycle arrest. [³⁵S]methionine incorporation was measured in triplicate cultures of Bop1Δ/6 or LAP3 cells that were either untreated (−) or treated with IPTG for 24 h (+). Histograms show average incorporation normalized to cell number, and error bars show standard deviation. The background incorporation was determined in cells treated with cycloheximide (CH) (10 μg/ml) for 30 min to block protein synthesis.

FIG. 6

Expression of human papillomavirus type 16 E6 protein alleviates Bop1Δ-induced cell cycle arrest. LAP3 cells were cotransfected with pX11-Bop1Δ, the selection marker pPGK-puro, and either pJ4Ω16E6 or pJ4Ω16E6Δ111–115, which drive expression of wild-type E6 and mutant E6 defective in p53 binding, respectively (11), or vector DNA. Pools of stably transfected clones were obtained by puromycin selection, and equal numbers of cells from each pool were subjected to BrdU and light treatment as described in the legend to Fig. 1.

FIG. 7

Bop1Δ-induced cell cycle arrest is p53 dependent. (A) Bop1Δ/6 cells were infected with pBabe-puro-GSE56, which antagonizes p53 function (43), or pBabe-puro vector. BrdU-light treatment with puromycin-selected pools was performed as described in the legend to Fig. 1 except that BrdU was added at 15 h after IPTG induction. Parental LAP3 cells infected with the same viruses and treated in parallel are shown for comparison. (B) [³H]thymidine incorporation was measured in Bop1Δ/6 and control LAP3 cells carrying retrovirus transduced GSE56 or empty pBabe-puro vector that were either untreated or treated with IPTG for 20 h. Histograms show average label incorporation normalized to cell number in triplicate cultures, and error bars indicate standard deviation. (C) Induction of Bop1Δ is unaffected by GSE56. Whole-cell lysates prepared from Bop1Δ/6 cells used for the above experiments and normalized to protein content were analyzed by immunoblotting with antibodies against Bop1.

FIG. 8

Functional inactivation of p53 in Bop1Δ-expressing cells does not affect rRNA processing block but decreases p21 induction. (A) Synthesis of 28S rRNA is impaired in Bop1Δ/6 cells expressing GSE56. rRNA processing was analyzed by [³H]uridine labeling as in Fig. 4; note the lack of 28S rRNA labeling and aberrant accumulation of the 36S precursor. (B) Expression of Bop1Δ was induced in puromycin-selected Bop1Δ/6 cell populations infected with pBabe-puro or pBabe-puro-GSE56. Cell lysates were prepared at different times after induction, normalized to protein content, and analyzed by immunoblotting with antibodies against p21.

FIG. 9

Nucleolar stress model of cell cycle arrest due to perturbation in ribosome biogenesis. Expression of mutant proteins such as Bop1Δ, exposure to chemical inhibitors of synthesis, and maturation of rRNA and other ribosome components in mammalian cells induce nucleolar stress, causing cell cycle arrest by triggering activation of the p53 pathway. p53 is required for sensing cellular stress caused by a variety of conditions, including DNA damage, replication defects, and gratuitous oncogene expression.