p27 is regulated independently of Skp2 in the absence of Cdk2.

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, p27(Kip1), 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.

Genetic mouse models to investigate cell cycle regulation.

Early studies on cell cycle regulation were based on experiments in model systems (Yeast, Xenopus, Starfish, Drosophila) and have shaped the way we understand many events that control the cell cycle. Although these model systems are of great value, the last decade was highlighted by studies done in human cells and using in vivo mouse models. Mouse models are irreplaceable tools for understanding the genetics, development, and survival strategies of mammals. New developments in generating targeting vectors and mutant mice have improved our approaches to study cell cycle regulation and cancer. Here we summarize the most recent advances of mouse model approaches in dissecting the mechanisms of cell cycle regulation and the relevance to human disease.

Rb/Cdk2/Cdk4 triple mutant mice elicit an alternative mechanism for regulation of the G1/S transition.

The G(1)/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.

Fbxw8 is essential for Cul1-Cul7 complex formation and for placental development.

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.

Immunoenzymometric assay for a small molecule,11-deoxycortisol, with attomole-range sensitivity employing an scFv-enzyme fusion protein and anti-idiotype antibodies.

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 degradation of p27 and cancer].

The cell cycle of eukaryotic cells is regulated by a series of protein complexes composed of cyclins and cyclin-dependent kinases (CDKs), the activity of which is suppressed by a group of CDK inhibitors (CKIs). Among the CKIs, p27 plays a pivotal role in the control of cell proliferation. Degradation of p27 is a critical event for reentry of cells into the cell cycle from G0 phase and occurs through ubiquitination by two ubiquitin ligase complexes (KPC and SCFSkP2) and subsequent degradation by the 26S-proteasome. A tumor suppressing function of p27 has been demonstrated in mouse models and studies of human tumors. This review will focus on the regulation of p27 proteolysis and its consequences for tumorigenesis.

Role of the UBL-UBA protein KPC2 in degradation of p27 at G1 phase of the cell cycle.

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 G(1) 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 NH(2)-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 NH(2)-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 G(1) phase, and introduction of KPC2 derivatives into the KPC2-depleted cells revealed that the NH(2)-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.

Molecular dissection of the interaction between p27 and Kip1 ubiquitylation-promoting complex, the ubiquitin ligase that regulates proteolysis of p27 in G1 phase.

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 ubiquitin-dependent 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 E.CDK2 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 E.CDK2 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.

VHL-box and SOCS-box domains determine binding specificity for Cul2-Rbx1 and Cul5-Rbx2 modules of ubiquitin ligases.

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-2alpha, 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.

Degradation of p57Kip2 mediated by SCFSkp2-dependent ubiquitylation.

The abundance of the cyclin-dependent kinase (CDK) inhibitor p57Kip2, an important regulator of cell cycle progression, is thought to be controlled by the ubiquitin-proteasome pathway. The Skp1/Cul1/F-box (SCF)-type E3 ubiquitin ligase complex SCFSkp2 has now been shown to be responsible for regulating the cellular level of p57Kip2 by targeting it for ubiquitylation and proteolysis. The elimination of p57Kip2 was impaired in Skp2-/- cells, resulting in abnormal accumulation of the protein. Coimmunoprecipitation analysis also revealed that Skp2 interacts with p57Kip2 in vivo. Overexpression of WT Skp2 promoted degradation of p57Kip2, whereas expression of a dominant negative mutant of Skp2 prolonged the half-life of p57Kip2. Mutation of the threonine residue (Thr-310) of human p57Kip2 that is conserved between the COOH-terminal QT domains of p57Kip2 and p27Kip1 prevented the effect of Skp2 on the stability of p57Kip2, suggesting that phosphorylation at this site is required for SCFSkp2-mediated ubiquitylation. Finally, the purified recombinant SCFSkp2 complex mediated p57Kip2 ubiquitylation in vitro in a manner dependent on the presence of the cyclin E-CDK2 complex. These observations thus demonstrate that the SCFSkp2 complex plays an important role in cell-cycle progression by determining the abundance of p57Kip2 and that of the related CDK inhibitor p27Kip1.