The ECS (Elongin B/C-Cul2/Cul5-SOCS-box protein) complex is a member of a family of ubiquitin ligases that share a Cullin-Rbx module. SOCS-box proteins recruit substrates to the ECS complex and are linked to Cullin-Rbx via Elongin B/C. VHL has been implicated as a SOCS-box protein, but lacks a C-terminal sequence (downstream of the BC box) of the SOCS box. We now show that VHL specifically interacts with endogenous Cul2-Rbx1 in mammalian cells, whereas SOCS-box proteins associate with Cul5-Rbx2. We also identify LRR-1 and FEM1B as proteins that share a region of homology with VHL (the VHL box, including the BC box and downstream residues) and associate with Cul2-Rbx1. ECS complexes can thus be classified into two distinct protein assemblies, that is, those that contain a subunit with a VHL box (composed of the BC box and a downstream Cul2 box) that interacts with Cul2-Rbx1, and those that contain a subunit with a SOCS box (BC box and downstream Cul5 box) that interacts with Cul5-Rbx2. Domain-swapping analyses showed that the specificity of interaction of VHL-box and SOCS-box proteins with Cullin-Rbx modules is determined by the Cul2 and Cul5 boxes, respectively. Finally, RNAi-mediated knockdown of the Cul2-Rbx1 inhibited the VHL-mediated degradation of HIF-2␣, whereas knockdown of Cul5-Rbx2 did not affect it. These data suggest that the functions of the Cul2-Rbx1 and Cul5-Rbx2 modules are distinct.

The abundance of the cyclin-dependent kinase (CDK) inhibitor p57 Kip2 , an important regulator of cell cycle progression, is thought to be controlled by the ubiquitin-proteasome pathway. The Skp1͞ Cul1͞F-box (SCF)-type E3 ubiquitin ligase complex SCF Skp2 has now been shown to be responsible for regulating the cellular level of p57 Kip2 by targeting it for ubiquitylation and proteolysis. The elimination of p57 Kip2 was impaired in Skp2 ؊/؊ cells, resulting in abnormal accumulation of the protein. Coimmunoprecipitation analysis also revealed that Skp2 interacts with p57 Kip2 in vivo. Overexpression of WT Skp2 promoted degradation of p57 Kip2 , whereas expression of a dominant negative mutant of Skp2 prolonged the half-life of p57 Kip2 . Mutation of the threonine residue (Thr-310) of human p57 Kip2 that is conserved between the COOH-terminal QT domains of p57 Kip2 and p27 Kip1 prevented the effect of Skp2 on the stability of p57 Kip2 , suggesting that phosphorylation at this site is required for SCF Skp2 -mediated ubiquitylation. Finally, the purified recombinant SCF Skp2 complex mediated p57 Kip2 ubiquitylation in vitro in a manner dependent on the presence of the cyclin E-CDK2 complex. These observations thus demonstrate that the SCF Skp2 complex plays an important role in cell-cycle progression by determining the abundance of p57 Kip2 and that of the related CDK inhibitor p27 Kip1 .

The cyclin-dependent kinase (CDK) inhibitor p27 is degraded at the G 0 -G 1 transition of the cell cycle by the ubiquitin-proteasome pathway in a Skp2-independent manner. We recently identified a novel ubiquitin ligase, KPC (Kip1 ubiquitylation-promoting complex), consisting of KPC1 and KPC2, which regulates the ubiquitindependent degradation of p27 at G 1 phase. We have now investigated the structural requirements for the interactions of KPC1 with KPC2 and p27. The NH 2 -terminal region of KPC1 was found to be responsible for binding to KPC2 and to p27. KPC1 mutants that lack this region failed to mediate polyubiquitylation of p27 in vitro and expression of one such mutant delayed p27 degradation in vivo. We also generated a series of deletion mutants of p27 and found that KPC failed to polyubiquitylate a p27 mutant that lacks the CDK inhibitory domain. Interestingly, the cyclin E⅐CDK2 complex prevented both the interaction of KPC with p27 as well as KPC-mediated polyubiquitylation of p27. A complex of cyclin E with a kinase-negative mutant of CDK2 also exhibited these inhibitory effects, suggesting that cyclin E⅐CDK2 competes with KPC1 for access to the CDK inhibitory domain of p27. These results suggest that free p27 is recognized by the NH 2 -terminal region of KPC1, which also associates with KPC2, and that p27 is then polyubiquitylated by the COOH-terminal RING-finger domain of KPC1.

KPC2 (Kip1 ubiquitylation-promoting complex 2) together with KPC1 forms the ubiquitin ligase KPC, which regulates degradation of the cyclin-dependent kinase inhibitor p27 at the G1 phase of the cell cycle. KPC2 contains a ubiquitin-like (UBL) domain, two ubiquitin-associated (UBA) domains, and a heat shock chaperonin-binding (STI1) domain. We now show that KPC2 interacts with KPC1 through its UBL domain, with the 26S proteasome through its UBL and NH2-terminal UBA domains, and with polyubiquitylated proteins through its UBA domains. The association of KPC2 with KPC1 was found to stabilize KPC1 in a manner dependent on the STI1 domain of KPC2. KPC2 mutants that lacked either the NH2-terminal or the COOH-terminal UBA domain supported the polyubiquitylation of p27 in vitro, whereas a KPC2 derivative lacking the STI1 domain was greatly impaired in this regard. Depletion of KPC2 by RNA interference resulted in inhibition of p27 degradation at the G1 phase, and introduction of KPC2 derivatives into the KPC2-depleted cells revealed that the NH2-terminal UBA domain of KPC2 is essential for p27 degradation. These observations suggest that KPC2 cooperatively regulates p27 degradation with KPC1 and that the STI1 domain as well as the UBL and UBA domains of KPC2 are indispensable for its function.

Cullin-based ubiquitin ligases (E3s) constitute one of the largest E3 families. Fbxw8 (also known as Fbw6 or Fbx29) is an F-box protein that is assembled with Cul7 in an SCF-like E3 complex. Here we show that Cul7 forms a heterodimeric complex with Cul1 in a manner dependent on Fbxw8. We generated mice deficient in Fbxw8 and found that Cul7 did not associate with Cul1 in cells of these mice. Two-thirds of Fbxw8 ؊/؊ embryos die in utero, whereas the remaining one-third are born alive and grow to adulthood. Fbxw8 ؊/؊ embryos show intrauterine growth retardation and abnormal development of the placenta, characterized by both a reduced thickness of the spongiotrophoblast layer and abnormal vessel structure in the labyrinth layer. Although the placental phenotype of Fbxw8 ؊/؊ mice resembles that of Cul7 ؊/؊ mice, other abnormalities of Cul7 ؊/؊ mice are not apparent in Fbxw8 ؊/؊ mice. These results suggest that the Cul7-based SCF-like E3 complex has both Fbxw8-dependent and Fbxw8-independent functions.Ubiquitin-dependent proteolysis plays indispensable roles in various biological processes (6,17,38). Protein ubiquitylation is mediated by several enzymes that act in concert. A ubiquitinactivating enzyme (E1), with ATP as a substrate, catalyzes the formation of a thioester bond between itself and ubiquitin, and it then transfers the activated ubiquitin to a ubiquitin-conjugating enzyme (E2). Certain E2 enzymes transfer ubiquitin directly to the protein substrate, whereas others require the participation of a third component, a ubiquitin ligase (E3), to achieve this effect (16). Hundreds of E3s have been identified in eukaryotes and classified into three groups: HECT-type, U-box-type, and RING-type E3s. Cullin-based E3s, a subtype of RING-type E3s, are multisubunit complexes (13,30). To date, seven mammalian proteins, Cul1, Cul2, Cul3, Cul4A, Cul4B, Cul5, and Cul7, have been identified as members of the cullin family. These proteins interact via their COOH-terminal cullin homology domain with Rbx1 or Rbx2, each of which contains a RING finger domain that interacts with E2. They also interact via their NH 2 -terminal cullin repeat (with or without the participation of an adapter molecule such as Skp1 or Elongin C) with substrate recognition molecules, such as F-box proteins in the Cul1-based SCF complex (7,20,23,28,39), VHL-box proteins in the Cul2-based ECV complex (22), BTB domain proteins in the 31,40), and SOCS-box proteins in the Cul5-based ECS complex (22). At least two other molecules, the APC2 subunit of the anaphase-promoting complex/cyclosome and the Cul7-related protein Parc, also possess a cullin homology domain (30, 34).Cul7 was originally identified as p185, a protein that binds the large T antigen of simian virus 40 (24). The interaction of p185 with large T antigen is important for the cellular transformation activity of the latter (2), and the BH3 domain identified in the COOH-terminal region of p185 is thought to function in the promotion of apoptosis (36).

To overcome the sensitivity limit in immunoassays for small molecules (haptens), we established a noncompetitive immunoenzymometric assay (IEMA) format that can detect attomole-range hapten molecules. We selected 11-deoxycortisol (11-DC; Mr 346.5), a corticosteroid serving a diagnostic index for pituitary-adrenal function, as a model target hapten. A fusion of a single-chain Fv fragment (scFv) specific for 11-DC and alkaline phosphatase (ALP) was generated for use as an enzyme-labeled antibody, instead of the conventional chemically linked enzyme-antibody conjugates. After binding reaction of 11-DC and fixed amounts of the fusion protein (scFv-ALP), the unbound fusion protein was removed by incubation with a mouse beta-type anti-idiotype antibody recognizing the scFv paratope. These complexes were captured by magnetic separation using anti-mouse IgG antibody-coated magnetic beads. Following magnetic sedimentation of the beads, immune complexes of scFv-ALP and 11-DC remained in the supernatant were further purified by capture on microtiter plates with immobilized alpha-type anti-idiotype antibody. As measured fluorometrically, ALP activity from bound immune complexes on the plates increased with increasing 11-DC, which is characteristic of a noncompetitive relationship. This IEMA afforded an extremely low detection limit (20 amol/assay), a very wide measurable range, and practical specificity. The plasma 11-DC levels determined for healthy subjects were validated as reliable.

The G1/S-phase transition is a well-toned switch in the mammalian cell cycle. Cdk2, Cdk4, and the rate-limiting tumor suppressor retinoblastoma protein (Rb) have been studied in separate animal models, but interactions between the kinases and Rb in vivo have yet to be investigated. To further dissect the regulation of the G 1 to S-phase progression, we generated Cdk2 ؊/؊ Cdk4 ؊/؊ Rb ؊/؊ (TKO) mutant mice. TKO mice died at midgestation with major defects in the circulatory systems and displayed combined phenotypes of Rb ؊/؊ and Cdk2 ؊/؊ Cdk4 ؊/؊ mutants. However, TKO mouse embryonic fibroblasts were not only resistant to senescence and became immortal but displayed enhanced S-phase entry and proliferation rates similar to wild type. These effects were more remarkable in hypoxic compared with normoxic conditions. Interestingly, depletion of the pocket proteins by HPV-E7 or p107/p130 shRNA in the absence of Cdk2/Cdk4 elicited a mechanism for the G 1/S regulation with increased levels of p27 Kip1 binding to Cdk1/ cyclin E complexes. Our work indicates that the G 1/S transition can be controlled in different ways depending on the situation, resembling a regulatory network.cell cycle ͉ p27 ͉ retinoblastoma protein ͉ Skp2

Cyclin-dependent kinase 2 (Cdk2) is dispensable for mitotic cell cycle progression and Cdk2 knockout mice are viable due to the compensatory functions of other Cdks. In order to assess the role of Cdk2 under limiting conditions, we used Skp2 knockout mice that exhibit increased levels of Cdk inhibitor, p27Kip1, which is able to inhibit Cdk2 and Cdk1. Knockdown of Cdk2 abrogated proliferation of Skp2−/− mouse embryonic fibroblasts, encouraging us to generate Cdk2−/−Skp2−/− double knockout mice. Cdk2−/−Skp2−/− double knockout mice are viable and display similar phenotypes as Cdk2−/− and Skp2−/− mice. Unexpectedly, fibroblasts generated from Cdk2−/−Skp2−/− double knockout mice proliferated at normal rates. The increased stability of p27 observed in Skp2−/− MEFs was not observed in Cdk2−/−Skp2−/− double knockout fibroblasts indicating that in the absence of Cdk2, p27 is regulated by Skp2-independent mechanisms. Ablation of other ubiquitin ligases for p27 such as KPC1, DDB1, and Pirh2 did not restore stability of p27 in Cdk2−/−Skp2−/− MEFs. Our findings point towards novel and alternate pathways for p27 regulation.