Cullins assemble a potentially large number of ubiquitin ligases by binding to the RING protein ROC1 to catalyse polyubiquitination, as well as binding to various specificity factors to recruit substrates. The Cul4A gene is amplified in human breast and liver cancers, and loss-of-function of Cul4 results in the accumulation of the replication licensing factor CDT1 in Caenorhabditis elegans embryos and ultraviolet (UV)-irradiated human cells. Here, we report that human UV-damaged DNA-binding protein DDB1 associates stoichiometrically with CUL4A in vivo, and binds to an amino-terminal region in CUL4A in a manner analogous to SKP1, SOCS and BTB binding to CUL1, CUL2 and CUL3, respectively. As with SKP1-CUL1, the DDB1-CUL4A association is negatively regulated by the cullin-associated and neddylation-dissociated protein, CAND1. Recombinant DDB1 and CDT1 bind directly to each other in vitro, and ectopically expressed DDB1 bridges CDT1 to CUL4A in vivo. Silencing DDB1 prevented UV-induced rapid CDT1 degradation in vivo and CUL4A-mediated CDT1 ubiquitination in vitro. We suggest that DDB1 targets CDT1 for ubiquitination by a CUL4A-dependent ubiquitin ligase, CDL4A(DDB1), in response to UV irradiation.

Cullins assemble the largest family of ubiquitin ligases by binding with ROC1 and various substrate receptors. CUL4 function is linked with many cellular processes, but its substrate-recruiting mechanism remains elusive. We identified a protein motif, the DWD box (DDB1-binding WD40 protein), and demonstrated the binding of 15 DWD proteins with DDB1-CUL4A. We provide evidence supporting the critical function of the DWD box and DDB1's role as the linker mediating DWD protein association with CUL4A. A database search predicts that about one-third of WD40 proteins, 90 in humans, contain the DWD box, suggesting a potentially large number of DWD-DDB1-CUL4-ROC1 E3 ligases. The ubiquitin-proteasome pathway regulates the concentration and conformation of many cellular proteins in response to changes in physiological conditions. This pathway consists of a cascade of three activities performed by E1 (ubiquitin-activating), E2 (ubiquitin-conjugating), and E3 (ubiquitin ligase) enzymes (Hochstrasser 1996;King et al. 1996;Hershko and Ciechanover 1998). A critical step in this process is how individual protein substrates are recruited to specific E3 ligases. The RING family represents the major family of E3 ligases. Members either contain an intrinsic RING finger domain (as in MDM2 and BRCA1) or bind in trans with a small RING finger protein, such as ROC1 (also known as Rbx1 and Hrt1) by the cullins, to recruit and activate an E2 (Jackson et al. 2000;Petroski and Deshaies 2005).A remarkable aspect of cullin E3 ligases is that each cullin can assemble into many distinct cullin-RING-dependent ligases (CRLs) by interacting with a conserved motif present in multiple proteins (Petroski and Deshaies 2005). To recruit specific substrates, CUL1 utilizes an N-terminal domain to bind with a linker protein, SKP1 (Feldman et al. 1997;Skowyra et al. 1997;, which does not interact with other cullins (Michel and Xiong 1998). SKP1 uses a separate domain to bind with a conserved protein motif, the F box, which, via its additional protein-protein interaction modules, recruits various substrates, often phosphorylated, to the CUL1-ROC1 catalytic core. To bring specific substrates to CUL2-and CUL5-dependent ligases, a heterodimeric linker complex containing elongins B and C binds simultaneously to an analogous N-terminal domain in CUL2 and CUL5 and to two similar protein motifs, the VHL box and SOCS box. VHL and SOCS proteins, via their additional protein-protein interaction modules, target various substrates differentially to the CUL2-ROC1 or CUL5-ROC2 catalytic cores (Kamura et al. 1998(Kamura et al. , 2001(Kamura et al. , 2004Stebbins et al. 1999;Zhang et al. 1999). Omitting a linker, CUL3 utilizes its N-terminal domain to bind to proteins with a conserved 100-residue protein motif known as a BTB domain, which, via additional protein-protein interaction domains, then target various substrates to the CUL3-ROC1 catalytic core (Furukawa et al. 2003;Geyer et al. 2003;Pintard et al. 2003;Xu et al. 2003). The presence of multiple substrate receptors-...

Tuberous sclerosis (TSC) is an autosomal dominant disease characterized by hamartoma formation in various organs and is caused by mutations targeting either theThe tuberous sclerosis 1 (TSC1) locus encodes a 130-kDa protein termed "hamartin," and the TSC2 locus encodes a 180-kDa protein termed "tuberin" that contains a coiled-coil domain and a GAP (GTPase-activating protein) domain. Genetic studies in Drosophila melanogaster and biochemical analyses in mammalian cells have established the TSC1-TSC2 heterodimeric complex as a critical regulator in coupling various cellular conditions to cell growth through stimulation of GTP hydrolysis of RheB to antagonize the mTOR signaling pathway (Inoki et al. 2005). These studies have identified both TSC subunits as substrates of various kinases that mediate different cellular conditions such as nutrient availability, energy, hormones, and growth factor stimulation. TSC2 is a short-lived protein, and it is actively ubiquitinated (Chong-Kopera et al. 2006). Many disease-associated mutations in TSC1 and TSC2 result in a substantial decrease in the level of hamartin and tuberin, respectively (Inoki et al. 2002;Nellist et al. 2005), suggesting that protein turnover plays a critical role in TSC regulation. Identification of the ubiquitin ligase for TSC2 would shed light on the understanding of the regulation of TSC and cell growth pathways. Results and Discussion TSC2 binds to WD40 protein FBW5, DDB1, and CUL4We used a yeast two-hybrid screen and coupled immunoprecipitation and immunoblotting (IP-Western) analyses to search for TSC2-interacting protein with potential ubiquitin ligase activity and identified two F-box proteins-FBW5 (Fig. 1A) and FBL6 (data not shown)-that bind with TSC2. Three additional control F-box proteins-SKP2, ␤-TrCP, and FBX5-did not bind with TSC2 in the same assay (data not shown), indicating specificity of the FBL6-TSC2 and FBW5-TSC2 interactions and also suggesting that the F-box motif is not sufficient for binding to TSC2. Consistent with TSC1-TSC2 dimerization, FBW5 also associates with TSC1 (Fig. 1A). Our subsequent genetic study in Drosophila (see below) led us to focus on determining the function and mechanism of the FBW5-TSC interaction. The functional significance of the FBL6-TSC interaction remains to be determined.Human FBW5 is a 60-kDa (566 residues) protein containing two recognizable domains; an N-terminally located F-box motif and seven WD40 repeats occupying most of the rest of the sequence. The F-box motif functions to bridge a substrate protein to the CUL1-ROC1 E3 ligase via the SKP1 linker (Bai et al. 1996;Feldman et al. 1997;Skowyra et al. 1997). Recently, we and others found that DDB1 binds to a subset of WD40 proteins referred to as DWD proteins (also known as DCAF, for Ddb1-and Cul4-associated factors; or CDW, for CUL4-and DDB1-associated WD40 repeat proteins), and bridges them to CUL4-ROC1 to constitute a potentially large and distinct family of DWD-DDB1-CUL4-ROC1 E3 ubiquitin ligases (Angers et al. 2006;He et al. 2006;Higa et al. ...

The DNA replication licensing factor Cdt1 is degraded by the ubiquitinproteasome pathway during S phase of the cell cycle, to ensure one round of DNA replication during each cell division and in response to DNA damage to halt DNA replication. Constitutive expression of Cdt1 causes DNA re-replication and is associated with the development of a subset of human non-small cell-lung carcinomas. In mammalian cells, DNA damage-induced Cdt1 degradation is catalyzed by the Cul4-Ddb1-Roc1 E3 ubiquitin ligase. We report here that overexpression of the proliferating cell nuclear antigen (PCNA) inhibitory domain from the CDK inhibitors p21 and p57, but not the CDK-cyclin inhibitory domain, blocked Cdt1 degradation in cultured mammalian cells after UV irradiation. In vivo soluble Cdt1 and PCNA co-elute by gel filtration and associate with each other physically. Silencing PCNA in cultured mammalian cells or repression of pcn1 expression in fission yeast blocked Cdt1 degradation in response to DNA damage. Unexpectedly, deletion of Ddb1 in fission yeast cells also accumulated Cdt1 in the absence of DNA damage. We suggest that the Cul4-Ddb1 ligase evolved to ubiquitinate Cdt1 during normal cell growth as well as in response to DNA damage and a separate E3 ligase, possibly SCF Skp2 , evolved to either share or take over the function of Cdt1 ubiquitination during normal cell growth and that PCNA is involved in mediating Cdt1 degradation by the Cul4-Ddb1 ligase in response to DNA damage.Cdt1, first identified in the fission yeast Schizosaccharomyces pombe as a G 1 START component Cdc10-dependent transcript whose loss-of-function prevents DNA replication (1), binds to the origin recognition complex with Cdc6 at the origin of replication and together with Cdc6 and origin recognition complex recruits the minichromosome maintenance 2-7 (MCM2-7) to assemble the prereplication complexes during G 1 , thereby controlling the initiation of DNA replication (2). Constitutive expression of Cdt1 alone in Caenorhabditis elegans, with Cdc6 in S. pombe or with Cdc6 and cyclin A-cdk2 in p53-deficient mammalian cells causes DNA re-replication (3-5). Constitutive expression of Cdt1 is also associated with the development of a subset of human non-small lung carcinomas (6), indicating the critical importance of regulating Cdt1 level for both initiating DNA replication and maintaining genome integrity.In addition to Cdc10-dependent transcriptional regulation, at least four mechanisms have been proposed for controlling Cdt1 function at the protein level; Cdt1 is exported from the nucleus in S phase in the budding yeast Saccharomyces cerevisiae (7), is inhibited by the binding of geminin from S to M phase in metazoans (8), is degraded in human cell lines, possibly by the SCF Skp2 E3 2 ubiquitin ligase (9, 10), and is degraded in response to DNA damage and during normal C. elegans embryogenesis by the Cul4-Ddb1-Roc1 ubiquitin E3 ligase (3,11,12). Evolving multiple distinct mechanisms to negatively regulate the level of Cdt1 protein presumably functions to m...

Cullin 3 (Cul3)-family ubiquitin ligases use the BTB-domain-containing proteins for the recruitment of substrates, but the regulation of this family of ubiquitin ligases has not been completely understood. KLHL20 is a BTB-family protein and targets tumor suppressor promyelocytic leukemia protein (PML) and death-associated protein kinase (DAPK) to its kelch-repeat domain for ubiquitination and degradation. Here, we show that another BTB-kelch protein KLHL39 is recruited to the substrate-binding domain of KLHL20 but is not a substrate of Cul3-KLHL20 complex. Interestingly, KLHL39 does not bind Cul3 because of the absence of certain conserved residues in the BTB domain. Instead, KLHL39 blocks KLHL20-mediated ubiquitination of PML and DAPK by disrupting the binding of these substrates to KLHL20 as well as the binding of KLHL20 to Cul3. Through the two mechanisms, KLHL39 increases the stability of PML and DAPK. In human colon cancers, downregulations of KLHL39, PML and DAPK are associated with metastatic progression. Furthermore, preclinical data indicate that KLHL39 promotes colon cancer migration, invasion and survival in vitro and metastasis in vivo through a PML- and DAPK-dependent mechanism. Our study identifies KLHL39 as a negative regulator of Cul3-KLHL20 ubiquitin ligase and reveals a role of KLHL39-mediated PML and DAPK stabilization in colon cancer metastasis.