14-3-3τ Regulates Ubiquitin-Independent Proteasomal Degradation of p21, a Novel Mechanism of p21 Downregulation in Breast Cancer

14-3-3 proteins are a family of about 30-kDa dimeric well-conserved α-helical phosphoserine/threonine binding proteins. They contain seven mammalian isoforms (β, ɛ, γ, η, σ, τ, ζ) and are able to bind to multiple protein ligands. The 14-3-3 binding proteins are very diverse; therefore, 14-3-3 is involved in many different cellular processes, including mitogenesis, DNA damage checkpoint, cell cycle control, and apoptosis (12). Most 14-3-3 ligands require phosphorylation to bind to 14-3-3; and their consensus motifs are R(S/Ar)XpSXP (mode 1), RX(Ar/S)XpSXP (mode 2) (12, 46), and (pS/pT)X1-2-COOH (mode 3) (13). However, this consensus is not absolutely required, since a few 14-3-3 binding ligands have sequences significantly different from the sequences of these motifs or do not even require phosphorylation for binding (12, 46). In general, 14-3-3 proteins play a role in promoting survival and repressing apoptosis (33). However, each isoform may have unique functions in certain physiological contexts. For example, 14-3-3τ binds to ATM-phosphorylated E2F1 during DNA damage and promotes E2F1 stability, leading to the induction of E2F1 proapoptotic target genes such as p73, Apaf1, and caspases (44). Like other 14-3-3 isoforms, however, there appears to be a role for 14-3-3τ in cell survival as well. The deletion of 14-3-3τ in mice leads to embryonic lethality, probably due to developmental arrest (25). Examination of 14-3-3τ+/− mice reveals a role for 14-3-3τ in cardiomyocyte survival (25). This is probably due to its activity that antagonizes ASK1 and sequesters BAD and FOXO family members. However, whether and how 14-3-3τ is involved in cell cycle progression remain poorly understood. In the study described here, we investigated the role of 14-3-3τ in cell cycle control and uncovered its involvement in the regulation of the cyclin-dependent kinase inhibitor p21(Waf1/Cip1). p21 is a p53 target gene and a major regulator that mediates p53-dependent G1 arrest and senescence. The turnover of the p21 protein is under very tight control. p21 can be degraded through both ubiquitin-dependent and ubiquitin-independent mechanisms. In the ubiquitin-dependent pathway, Skp2 and CRL4(Cdt2) are responsible for p21 degradation in S phase (1, 5, 23, 30, 48), whereas APC/CCdc20 controls the degradation of p21 in prometaphase (3). There are also data demonstrating that Skp2 is not required for basal p21 ubiquitylation and degradation (8). p21 can also be directly targeted to the proteasome for degradation without ubiquitylation (38). This process is mediated by an interaction between p21 and the C8 α subunit of the 20S proteasome (40) and can be promoted by MDM2 and MDMX (20, 21, 49). The MDM2/MDMX-regulated degradation of p21 occurs at the G1 and early S phases (21). The stability of the p21 protein is also regulated by heat shock proteins. An Hsp90 binding protein, WISp39, recruits Hsp90 to p21 and protects p21 from degradation during DNA damage (19). Therefore, it appears that several different regulators control the stability of the p21 protein, probably depending on the phases of the cell cycle and cellular contexts. Given the evidence of both ubiquitin-dependent and -independent degradation of p21, Pagano and colleagues proposed that both mechanisms of degradation of p21 in different protein complexes may occur in cells (3). It has been shown that free p21, but not cyclin E/cdk2-bound p21, can be degraded by the proteasome in vitro without ubiquitylation (4, 26, 27). Thus, when p21 is complexed with cdk2, it may be degraded by the ubiquitin-dependent pathway, while the ubiquitin-independent mechanism may target free p21 for degradation. It has been shown that this process involves the REGγ proteasome complex (7, 26), which forms the 11S lid and activates the 20S catalytic core proteasome. However, the regulatory mechanisms for the ubiquitin-independent degradation of p21 remain unclear. In the present study, we identify 14-3-3τ as the protein that regulates the ubiquitin-independent proteasomal degradation of p21 in G1 phase. We demonstrate a direct role for 14-3-3τ in the 20S-mediated p21 degradation via facilitation of an interaction between p21, MDM2, and C8 in vitro. This new role of 14-3-3τ might have an important clinical implication. The extracellular matrix tenascin-C induces 14-3-3τ and degrades p21 through the induction of 14-3-3τ and ameliorates adriamycin-induced cell cycle arrest. 14-3-3τ is often overexpressed in breast cancer, and its overexpression is associated with the downregulation of p21 and shorter patient survival. Through the downregulation of p21, 14-3-3τ overexpression also leads to tamoxifen resistance in MCF7 breast cancer cells.