Interaction with cyclin-dependent kinases and PCNA modulates proteasome-dependent degradation of p21

Oncogene (1998) 17, 2437 ± 2444 ã 1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00 http://www.stockton-press.co.uk/onc Interaction with cyclin-dependent kinases and PCNA modulates proteasome-dependent degradation of p21 Corinne Cayrol and Bernard Ducommun I.P.B.S - C.N.R.S, Universite Paul Sabatier, 205 route de Narbonne, 31077 Toulouse cedex, France The cyclin-dependent kinase (CDK) inhibitor p21Cip1/Waf1 plays an essential role in the control of cell proliferation by modulating the activity of cyclin/CDK complexes in response to various intracellular or extracellular signals. Small variations in p21 expression levels may determine whether it acts as an inhibitor or an assembly factor for cyclin/CDK complexes. It is therefore critical to better characterize the mechanisms regulating p21 abundance. Here, we show, using a tetracycline-regulated system in p53-de®cient DLD-1 human colon cancer cells, that p21 protein levels and stability are regulated by the proteasome-dependent degradation pathway and by association with its partners, CDKs and PCNA. A p21 mutant de®cient for interaction with CDKs, p21CDK7, displayed an enhanced stability and greatly reduced sensitivity to proteasome-mediated proteolysis, indicating that association with cyclin/CDK complexes may trigger p21 degradation. In contrast, a p21 mutant impaired in the interaction with PCNA, p21PCNA7, exhibited a decreased stability, suggesting that association with PCNA protects p21 from proteasome-dependent degradation. Furthermore, the abundance of p21 itself, in addition to protein-protein interactions, may also modulate p21 stability since we found that high levels of p21 expression overcome proteasome-dependent regulation of p21 accumulation. Keywords: cyclin-dependent kinases; p21Cip1; PCNA; degradation Introduction Cell cycle progression is driven by the sequential activation and inactivation of cyclin-dependent kinases (CDKs). CDK activity is regulated at di€erent levels (Morgan, 1995), including association with a cyclin regulatory subunit, phosphorylation/dephosphorylation cycles and association with a number of negative regulatory proteins known as CKI (for CDK inhibitors) (Elledge et al., 1996; Sherr and Roberts, 1995). The CIP/KIP family of mammalian CKIs consists of three members, p21CIP1/WAF1/SDI1 (hereafter referred to as p21), p27KIP1 and p57KIP2, which contain discrete CDK and cyclin-binding domains (Chen et al., 1996; Goubin and Ducommun, 1995; Lin et al., 1996; Russo et al., 1996). CIP/KIP proteins regulate cell proliferation through the modulation of cyclin/CDK Correspondence: B Ducommun Received 26 March 1998; revised 2 June 1998; accepted 2 June 1998 complexes activity in response to various intracellular or extracellular signals (Elledge et al., 1996; Sherr and Roberts, 1995). p21, for instance, is a key mediator of the G1 growth arrest induced by the tumor suppressor protein p53 in response to DNA damage (Brugarolas et al., 1995; Deng et al., 1995; Waldman et al., 1995). A unique feature of p21 that distinguishes it from the other CKIs is its capacity to bind to the Proliferating Cell Nuclear Antigen (PCNA), a processivity factor for DNA polymerase d and e, that is essential for both DNA replication and DNA repair (Gulbis et al., 1996; Kelman, 1997). We have recently shown that p21 binding to PCNA is sucient to inhibit cell cycle progression at the G1/S and G2/M transitions in human cancer cells (Cayrol et al., 1998). p21 may therefore inhibit cell cycle progression by two independent mechanisms, inhibition of cyclin/CDK complexes and/or inhibition of PCNA function. The e€ectiveness of these two mechanisms is likely to be determined by the relative abundance of p21, CDKs and PCNA. Furthermore, the abundance of the p21 protein may also determine whether p21 acts as an inhibitor or as an assembly factor of cyclin/CDK complexes, since it has recently been found that at low concentration p21 promotes the assembly of active kinase complexes, whereas at higher concentrations it inhibits activity (LaBaer et al., 1997). It is therefore critical to better understand the molecular mechanisms underlying the regulation of p21 levels. Although most studies have focused on the mechanisms of transcriptional regulation of p21, it has recently been shown that p21 expression can also be regulated at the level of protein stability. Overexpression of the transcription factors C/EBPa (Timchenko et al., 1996) or p120E4F (Fernandes et al., 1998) was shown to result in a signi®cant increase of p21 half-life. In addition, p21 has been found to be ubiquitinated in vivo (Maki and Howley, 1997) suggesting that p21 levels may be regulated by the ubiquitin-proteasome pathway. This system plays a key role in cell cycle control by regulating the abundance of a number of regulatory proteins, including the human CKI p27 (Pagano et al., 1995) and the yeast CKIs Sic1p (Verma et al., 1997) and Far1p (Henchoz et al., 1997). Although p21 was initially thought not to be regulated by the proteasome-dependent pathway (Pagano et al., 1995), more recent studies indicate that proteasome mediated proteolysis may play a role in the regulation of p21 abundance (Blagosklonny et al., 1996; Maki and Howley, 1997). Here, we report that p21 expression levels and stability in p53-de®cient cells are regulated by the proteasome-dependent proteolytic pathway and that this regulation is abrogated when p21 is expressed to CDK and PCNA binding modulates p21 stability C Cayrol and B Ducommun 2438 very high levels. Futhermore, we show that p21 mutants de®cient for interaction with either CKDs or PCNA exhibit respectively, decreased and enhanced sensitivity to proteasome-mediated degradation. Association with cyclin/CDKs complexes may therefore facilitate p21 degradation while binding to PCNA may protect p21 from proteasome-dependent proteolysis. These later results suggest that association of p21 with its di€erent partners regulates p21 expression levels and stability in vivo. p21 were clearly detected in cells treated with LLnL (Figure 1b). In contrast, no change in electrophoretic mobility of p21 was observed in cells treated with LLM Results Proteasome-dependent degradation modulates p21 protein levels and stability in vivo Proteasome inhibitors have previously been shown to upregulate p21 expression levels in human cancer cells. This e€ect was most signi®cant in cell lines expressing wild-type p53 and appeared to be due in part to p53dependent increase in p21 mRNA levels (Blagosklonny et al., 1996; Maki and Howley, 1997). To further characterize the role of the proteasome in the regulation of p21 stability in vivo, and to overcome the limitations due to p53-dependent transactivation of the p21 promoter, we expressed wild-type and mutant HA-tagged p21 proteins, under the control of a tetracycline-regulated promoter, in the p53-de®cient human colon cancer cell line DLD1, which constitutively expresses very low levels of (...truncated)