The tumor suppressor programmed cell death protein 4 (PDCD4) inhibits the translation initiation factor eIF4A, an RNA helicase that catalyzes the unwinding of secondary structure at the 5' untranslated region (5'UTR) of messenger RNAs (mRNAs). In response to mitogens, PDCD4 was rapidly phosphorylated on Ser67 by the protein kinase S6K1 and subsequently degraded via the ubiquitin ligase SCF(betaTRCP). Expression in cultured cells of a stable PDCD4 mutant that is unable to bind betaTRCP inhibited translation of an mRNA with a structured 5'UTR, resulted in smaller cell size, and slowed down cell cycle progression. We propose that regulated degradation of PDCD4 in response to mitogens allows efficient protein synthesis and consequently cell growth.

Skp2 and its cofactor Cks1 are the substrate-targeting subunits of the SCF(Skp2-Cks1) (Skp1/Cul1/F-box protein) ubiquitin ligase complex that regulates entry into S phase by inducing the degradation of the cyclin-dependent kinase inhibitors p21 and p27 (ref. 1). Skp2 is an oncoprotein that often shows increased expression in human cancers; however, the mechanism that regulates its cellular abundance is not well understood. Here we show that both Skp2 and Cks1 proteins are unstable in G1 and that their degradation is mediated by the ubiquitin ligase APC/C(Cdh1) (anaphase-promoting complex/cyclosome and its activator Cdh1). Silencing of Cdh1 by RNA interference in G1 cells stabilizes Skp2 and Cks1, with a consequent increase in p21 and p27 proteolysis. Depletion of Cdh1 also increases the percentage of cells in S phase, whereas concomitant downregulation of Skp2 reverses this effect, showing that Skp2 is an essential target of APC/C(Cdh1). Expression of a stable Skp2 mutant that cannot bind APC/C(Cdh1) induces premature entry into S phase. Thus, the induction of Skp2 and Cks1 degradation in G1 represents a principal mechanism by which APC/C(Cdh1) prevents the unscheduled degradation of SCF(Skp2-Cks1) substrates and maintains the G1 state.

The Cdc25A phosphatase is essential for cell-cycle progression because of its function in dephosphorylating cyclin-dependent kinases. In response to DNA damage or stalled replication, the ATM and ATR protein kinases activate the checkpoint kinases Chk1 and Chk2, which leads to hyperphosphorylation of Cdc25A. These events stimulate the ubiquitin-mediated proteolysis of Cdc25A and contribute to delaying cell-cycle progression, thereby preventing genomic instability. Here we report that beta-TrCP is the F-box protein that targets phosphorylated Cdc25A for degradation by the Skp1/Cul1/F-box protein complex. Downregulation of beta-TrCP1 and beta-TrCP2 expression by short interfering RNAs causes an accumulation of Cdc25A in cells progressing through S phase and prevents the degradation of Cdc25A induced by ionizing radiation, indicating that beta-TrCP may function in the intra-S-phase checkpoint. Consistent with this hypothesis, suppression of beta-TrCP expression results in radioresistant DNA synthesis in response to DNA damage--a phenotype indicative of a defect in the intra-S-phase checkpoint that is associated with an inability to regulate Cdc25A properly. Our results show that beta-TrCP has a crucial role in mediating the response to DNA damage through Cdc25A degradation.