p21Waf1/Cip1/Sdi1 mediates retinoblastoma protein degradation

Abstract

Damage-induced G1 checkpoint in mammalian cells involves upregulation of p53, which activates transcription of p21Waf1 (CDKN1A). Inhibition of cyclin-dependent kinase (CDK)2 and CDK4/6 by p21 leads to dephosphorylation and activation of Rb. We now show that ectopic p21 expression in human HT1080 fibrosarcoma cells causes not only dephosphorylation but also depletion of Rb; this effect was p53-independent and susceptible to a proteasome inhibitor. CDK inhibitor p27 (CDKN1B) also caused Rb dephosphorylation and depletion, but another CDK inhibitor p16 (CDKN2A) induced only dephosphorylation but not depletion of Rb. Rb depletion was observed in both HT1080 and HCT116 colon carcinoma cells, where p21 was induced by DNA-damaging agents. Rb depletion after DNA damage did not occur in the absence of p21, and it was reduced when p21 induction was inhibited by p21-targeting short hairpin RNA or by a transdominant inhibitor of p53. These results indicate that p21 both activates Rb through dephosphorylation and inactivates it through degradation, suggesting negative feedback regulation of damage-induced cell-cycle checkpoint arrest.Oncogene ( Keywords: p21; Rb; p27; damage response p53-inducible cyclin-dependent kinase (CDK) inhibitor p21 (CDKN1A) is the key mediator of damage-induced cell-cycle arrest. p21 interacts with different cyclin/CDK complexes and other regulators of transcription and signal transduction, exerting broad effects on cell survival, gene expression and morphology (Roninson, 2002). p21 effects are partially mediated by Rb, which is inactivated in proliferating cells through phosphorylation by CDK2 and CDK4/6, both of which are inhibited by p21. As a result, p21 induction leads to Rb dephosphorylation and activation, with ensuing G1 arrest.Whereas p21 activates Rb by dephosphorylation, several oncoproteins inactivate Rb by degradation via the proteasome. Proteasome-mediated Rb degradation is promoted by Mdm2 (Sdek et al., 2005) and gankyrin (Higashitsuji et al., 2000), E7 of papilloma virus (Boyer et al., 1996) and Tax of HTLV1 (Kehn et al., 2005). Oncoprotein-induced proteasomal degradation of Rb is one of the mechanisms for Rb inactivation in carcinogenesis (Ying and Xiao, 2006), but Rb degradation has not been described in DNA damage response.Changes in Rb phosphorylation are most commonly detected by immunoblotting through changes in the protein's electrophoretic mobility. Examination of numerous Rb immunoblots published by different groups showed that in many (but not all) cases Rb dephosphorylation, which results from drug treatment, cell senescence or ectopic p21 expression, is associated with a reduction in the Rb protein signal. In the present study, we have asked (i) whether a decrease in the Rb signal in response to p21 reflects protein degradation or merely altered immunoreactivity of dephosphorylated Rb, (ii) if p53 plays a p21-independent role in the decrease in Rb, (iii) whether such decrease can be induced by other CDK inhibitors that induce Rb dephosphorylation and (iv) if the decrease...

The centrosomal protein TACC3 controls paclitaxel sensitivity by modulating a premature senescence program

“…Moreover, the induction of senescence in TACC3-depleted cells was associated with a rapid decrease of the retinoblastoma protein (pRb) (Supplementary Figure 3b). Inhibition of pRb is a typical event during progression of senescence and has been reported to be mediated by elevated levels of p21 WAF that promote pRb degradation in response to DNA damage (Broude et al, 2007). Another noticeable feature of TACC3-depleted MCF-7 cells was the strong increase of HP1g (phospho-Ser83)-positive SAHF in addition to an abundant expression of nuclear p21 WAF (Supplementary Figure 4).…”

Hypoxia suppresses conversion from proliferative arrest to cellular senescence

“…S1A). Next, we determined whether the effect of hypoxia requires the p53 function by using parental HT-p21-9 and HT-p21-9-GSE56 cells, which lack functional p53 (21,43,44). As expected, in HT-p21-9-GSE56 cells, IPTG-induced senescence was preventable by rapamycin but not by nutlin-3a (21) (Fig.…”

“…HT-p21-9 cells, derived from HT1080 human fibrosarcoma cells (American Type Culture Collection), provided by Igor Roninson (University of South Carolina, Charleston, SC), were previously described (1,2,7,8). HT-p21-9-GSE56 cells, which lack transcriptional function of p53, were described previously (21,43,44). HT-p21-9 cells were cultured in highglucose DMEM without pyruvate supplemented with FC2 serum (HyClone FetalClone II; Thermo Scientific).…”

MEK drives cyclin D1 hyperelevation during geroconversion

“…Next, we investigated the effect of U0126 on cyclin D1 in p16-induced senescence in the HT-p16 cell line, in which IPTG stimulated irreversible senescence by inducing p16. 50 We have previously observed that IPTG-induced cyclins D1 and E in HT-p16 cells.…”