Pivotal role for the ubiquitin Y59-E51 loop in lysine 48 polyubiquitination

Lysine 48 (K48)-polyubiquitination is the predominant mechanism for mediating selective protein degradation, but the underlying molecular basis of selecting ubiquitin (Ub) K48 for linkage-specific chain synthesis remains elusive. Here, we present biochemical, structural, and cell-based evidence demonstrating a pivotal role for the Ub Y59-E51 loop in supporting K48-polyubiquitination. This loop is established by a hydrogen bond between Ub Y59’s hydroxyl group and the backbone amide of Ub E51, as substantiated by NMR spectroscopic analysis. Loop residues Y59 and R54 are specifically required for the receptor activity enabling K48 to attack the donor Ub-E2 thiol ester in reconstituted ubiquitination catalyzed by Skp1-Cullin1-F-box (SCF)^βTrCP E3 ligase and Cdc34 E2-conjugating enzyme. When introduced into mammalian cells, loop-disruptive mutant Ub^R54A/Y59A diminished the production of K48-polyubiquitin chains. Importantly, conditional replacement of human endogenous Ub by Ub^R54A/Y59A or Ub^K48R yielded profound apoptosis at a similar extent, underscoring the global impact of the Ub Y59-E51 loop in cellular K48-polyubiquitination. Finally, disulfide cross-linking revealed interactions between the donor Ub-bound Cdc34 acidic loop and the Ub K48 site, as well as residues within the Y59-E51 loop, suggesting a mechanism in which the Ub Y59-E51 loop helps recruit the E2 acidic loop that aligns the receptor Ub K48 to the donor Ub for catalysis.Central to selective protein turnover by the 26S proteasome is the formation of homotypic lysine 48 (K48)-linked ubiquitin (Ub) chains that tag substrate proteins for degradation (1). Among the most extensively studied systems that produce K48-linked Ub chains is the SCF (Skp1-Cullin1-F-box) E3-directed ubiquitination. SCF is a member of the multisubunit Cullin-RING E3 Ub ligase (CRL) family, the largest of all E3s (2). CRL contains a tandem of a large scaffold protein [Cullin (CUL)] and a RING domain-containing protein (ROC1/Rbx1) that typically associates with an adaptor protein (such as Skp1) in complex with a substrate recognition protein (such as F-box protein). As such, the organization of CRL subunits positions the substrate receptor (such as the F-box protein) within the proximity of ROC1, which recruits an E2-conjugating enzyme that catalyzes the transfer of Ub to a bound substrate. In the SCF reconstitution system, K48-linked polyubiquitin chains on a substrate such as IκBα and β-catenin are produced in a two-step reaction. The E2 UbcH5c deposits the first Ub moiety, forming a substrate–Ub linkage, which is followed by repeated discharge of subsequent Ubs by E2 Cdc34 to form K48-specific Ub chains (3). Human Cdc34 contains a highly conserved charged acidic loop (residues 102–113) that participates in the elongation of K48 chains (4, 5). The current work addresses whether there are determinants on the Ub itself that dictate K48 linkage specificity and, moreover, how Cdc34 might recognize Ub K48.     

Poxvirus ankyrin repeat proteins are a unique class of F-box proteins that associate with cellular SCF1 ubiquitin ligase complexes

F-box proteins direct the degradation of an extensive range of proteins via the ubiquitin-proteasome system. Members of this large family of proteins are typically bipartite. They recruit specific substrates through a substrate-binding domain and, via the F-box, link these to core components of a major class of ubiquitin ligases (SCF1). F-box proteins thus determine the specificity of SCF1-mediated ubiquitination. F-box-like motifs were recently detected in poxvirus ankyrin repeat (ANK) proteins but clear compositional differences to typical F-box proteins raise questions regarding the classification and function of the motif. Here we show that all five ANK proteins of a representative poxvirus, Orf virus, interact in vivo with core components of the SCF1 ubiquitin ligase complex. Interaction is dependent on the poxviral F-box-like motif and the adaptor subunit of the complex (SKP1). The viral protein does not block enzymatic activity of the complex. These observations identify the poxviral motif as a functional F-box. They also identify a new class of F-box that in contrast to cellular counterparts is truncated, has an extreme C-terminal location and is paired with an ANK protein-binding domain. ANK proteins constitute the largest family of poxviral proteins but their function and the significance of their abundance have remained an enigma. We propose that poxviruses use these unique ANK/F-box proteins to dictate target specificity to SCF1 ubiquitin ligases and thereby exploit the cell's ubiquitin-proteasome machinery.     

Down-regulation of NKG2D and NKp80 ligands by Kaposi's sarcoma-associated herpesvirus K5 protects against NK cell cytotoxicity

Natural killer (NK) cells are important early mediators of host immunity to viral infections. The NK activatory receptors NKG2D and NKp80, both C-type lectin-like homodimeric receptors, stimulate NK cell cytotoxicity toward target cells. Like other herpesviruses, Kaposi's sarcoma-associated herpesvirus (KSHV) down-regulates MHC class I molecules to avoid detection by cytotoxic T lymphocytes but renders cells susceptible to NK cell cytotoxicity. We now show that the KSHV immune evasion gene, K5, reduces cell surface expression of the NKG2D ligands MHC class I-related chain A (MICA), MICB, and the newly defined ligand for NKp80, activation-induced C-type lectin (AICL). Down-regulation of both MICA and AICL requires the ubiquitin E3 ligase activity of K5 to target substrate cytoplasmic tail lysine residues. The common MICA *008 allele has a frameshift mutation leading to a premature stop codon and is resistant to down-regulation because of the loss of lysine residues. K5-mediated ubiquitylation signals internalization but not degradation of MICA and causes a potent reduction in NK cell-mediated cytotoxicity. The down-regulation of ligands for both the NKG2D and NKp80 activation pathways provides KSHV with a powerful mechanism for evasion of NK cell antiviral functions.     

The ubiquitin ligase Nedd4-1 is dispensable for the regulation of PTEN stability and localization

PTEN is a tumor suppressor frequently mutated in cancer. Recent reports implicated Nedd4-1 as the E3 ubiquitin ligase for PTEN that regulates its stability and nuclear localization. We tested the physiological role of Nedd4-1 as a PTEN regulator by using cells and tissues derived from two independently generated strains of mice with their Nedd4-1 gene disrupted. PTEN stability and ubiquitination were indistinguishable between the wild-type and Nedd4-1-deficient cells, and an interaction between the two proteins could not be detected. Moreover, PTEN subcellular distribution, showing prominent cytoplasmic and nuclear staining, was independent of Nedd4-1 presence. Finally, activation of PKB/Akt, a major downstream target of cytoplasmic PTEN activity, and the ability of PTEN to transactivate the Rad51 promoter, a measure of its nuclear function, were unaffected by the loss of Nedd4-1. Taken together, our results fail to support a role for Nedd4-1 as the E3 ligase regulating PTEN stability and subcellular localization.     

Spectrin's E2/E3 ubiquitin conjugating/ligating activity is diminished in sickle cells

Erythrocyte spectrin contains E2/E3 ubiquitin conjugating/ligating activity in its alpha subunit. Ankyrin is a target of spectrin's E2/E3 ubiquitin conjugating/ligating activity in vitro and in vivo. We compare the ubiquitination levels of ankyrin mediated by control and sickle cell spectrin using a biotinylated ubiquitin cell-free assay. Sickle cell spectrin has diminished ability to transfer ubiquitin from an intermediate spectrin-ubiquitin thioester adduct (alpha' spectrin) to ankyrin, which may be due to glutathiolation of spectrin's E2 and/or E3 active site cysteines. There is also a diminished ability of the sickle cell ankyrin to serve as target of spectrin's E2/E3 activity, probably due to oxidative damage to ankyrin. A direct correlation exists between the alpha'/alpha spectrin ratio and spectrin's ability to ubiquitinate ankyrin. There is also an inverse correlation between severity of the disease and the alpha'/alpha spectrin ratio in SS erythrocytes. These results suggest that reduced spectrin E2/E3 activity is an important determinant of sickle cell severity.     

Mode of interaction of TRIP13 AAA-ATPase with the Mad2-binding protein p31comet and with mitotic checkpoint complexes

The AAA-ATPase thyroid hormone receptor interacting protein 13 (TRIP13), jointly with the Mad2-binding protein p31^comet, promotes the inactivation of the mitotic (spindle assembly) checkpoint by disassembling the mitotic checkpoint complex (MCC). This checkpoint system ensures the accuracy of chromosome segregation by delaying anaphase until correct bipolar attachment of chromatids to the mitotic spindle is achieved. MCC inhibits the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that targets for degradation securin, an inhibitor of anaphase initiation. MCC is composed of the checkpoint proteins Mad2, BubR1, and Bub3, in association with the APC/C activator Cdc20. The assembly of MCC in active checkpoint is initiated by the conversion of Mad2 from an open (O-Mad2) to a closed (C-Mad2) conformation, which then binds tightly to Cdc20. Conversely, the disassembly of MCC that takes place when the checkpoint is turned off involves the conversion of C-Mad2 back to O-Mad2. Previously, we found that the latter process is mediated by TRIP13 together with p31^comet, but the mode of their interaction remained unknown. Here, we report that the oligomeric form of TRIP13 binds both p31^comet and MCC. Furthermore, p31^comet and checkpoint complexes mutually promote the binding of each other to oligomeric TRIP13. We propose that p31^comet bound to C-Mad2–containing checkpoint complex is the substrate for the ATPase and that the substrate-binding site of TRIP13 is composed of subsites specific for p31^comet and C-Mad2–containing complex. The simultaneous occupancy of both subsites is required for high-affinity binding to TRIP13.Thyroid hormone receptor interacting protein 13 (TRIP13 ) is an AAA-ATPase that is required for the inactivation of the mitotic (spindle assembly) checkpoint (1, 2). This checkpoint system delays anaphase until correct bipolar attachment of sister chromatids to the mitotic spindle is achieved and thus ensures accuracy of chromosome segregation in mitosis (3–5). When the mitotic checkpoint system is on, it inhibits the action of the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that targets for degradation specific cell cycle regulatory proteins, such as securin, an inhibitor of anaphase initiation (6). APC/C is inhibited by the mitotic checkpoint complex (MCC), which is composed of the checkpoint proteins Mad2, BubR1, and Bub3, in association with the APC/C activator Cdc20. The active checkpoint converts Mad2 from an open (O-Mad2) to a closed (C-Mad2) conformation, and the latter associates with Cdc20 in a very tight complex. It is thought that the C-Mad2–Cdc20 (MC) subcomplex associates with BubR1-Bub3 to form the MCC (4, 5).In studying the mechanisms of the disassembly of MCC, we found that ATP hydrolysis is required for this process (7). ATP was also required for the action of p31^comet, a Mad2-binding protein involved in the exit from the mitotic checkpoint (8) and in MCC dissociation (9). Subsequently, we purified a factor that promotes ATP- and p31^comet-dependent release of Mad2 from MC and MCC and identified it as the TRIP13 ATPase (1). The role of TRIP13 in checkpoint inactivation was corroborated by in vivo results of other investigators indicating that TRIP13 knockdown delays metaphase–anaphase transition (2). We proposed that the energy of ATP hydrolysis is used by the TRIP13 ATPase to promote conformational transition of C-Mad2 to O-Mad2, thus leading to its release from MCC or MC (1). The action of TRIP13 to convert C-Mad2 to O-Mad2 was recently demonstrated by direct methods (10).The question arose concerning what is the role of p31^comet in the action of the TRIP13 ATPase. Because it had been suggested by a proteomic data-mining study that TRIP13 interacts with p31^comet (11) and because p31^comet specifically binds to the closed conformation of Mad2 (12), it seemed reasonable to assume that p31^comet serves as an adaptor protein that targets the TRIP13 AAA-ATPase to C-Mad2–containing checkpoint complexes. However, in our previous experiments on immunodepletion of TRIP13 or p31^comet from checkpoint extracts, using antibodies directed against either protein, we could not detect coimmunodepletion of either protein with its presumed partner (1). The present investigation was initiated to solve this problem and to gain insight into the role of p31^comet in TRIP13 action. We find that p31^comet and checkpoint complexes mutually stimulate the binding of each other to the oligomeric form of the TRIP13 ATPase. We propose that p31^comet bound to C-Mad2–containing checkpoint complex is the substrate for the ATPase and that the substrate-binding site of TRIP13 is composed of subsites specific for p31^comet and the C-Mad2 moiety of the checkpoint complex. The simultaneous binding of p31^comet and C-Mad2 to these subsites is required for their high-affinity interaction with TRIP13.     

Distinct activation of an E2 ubiquitin-conjugating enzyme by its cognate E3 ligases

A significant portion of ubiquitin (Ub)-dependent cellular protein quality control takes place at the endoplasmic reticulum (ER) in a process termed “ER-associated degradation” (ERAD). Yeast ERAD employs two integral ER membrane E3 Ub ligases: Hrd1 (also termed “Der3”) and Doa10, which recognize a distinct set of substrates. However, both E3s bind to and activate a common E2-conjugating enzyme, Ubc7. Here we describe a novel feature of the ERAD system that entails differential activation of Ubc7 by its cognate E3s. We found that residues within helix α2 of Ubc7 that interact with donor Ub were essential for polyUb conjugation. Mutagenesis of these residues inhibited the in vitro activity of Ubc7 by preventing the conjugation of donor Ub to the acceptor. Unexpectedly, Ub chain formation by mutant Ubc7 was restored selectively by the Hrd1 RING domain but not by the Doa10 RING domain. In agreement with the in vitro data, Ubc7 α2 helix mutations selectively impaired the in vivo degradation of Doa10 substrates but had no apparent effect on the degradation of Hrd1 substrates. To our knowledge, this is the first example of distinct activation requirements of a single E2 by two E3s. We propose a model in which the RING domain activates Ub transfer by stabilizing a transition state determined by noncovalent interactions between the α2 helix of Ubc7 and Ub and that this transition state may be stabilized further by some E3 ligases, such as Hrd1, through additional interactions outside the RING domain.The ubiquitin (Ub) conjugation machinery employs three basic enzymatic activities, E1, E2, and E3, that work in concert to transfer Ub to client substrates and to form polyUb chains (1). Initially, an E1 Ub-activating enzyme forms a high-energy thioester bond with the C terminus of Ub, after which the Ub molecule is transferred to the active-site Cys of an E2 Ub-conjugating (Ubc) enzyme. The Ub-charged E2 binds to an E3 ligase and catalyzes the transfer of Ub to the ε-amino group of a Lys side chain within the substrate. Additional Ubs then can be ligated to the initial Ub molecule through sequential ubiquitylation cycles, ultimately forming a polyUb chain. Ub can be conjugated to itself via specific Lys residues, resulting in diverse types of chain linkages. Linkage through Lys48 is linked primarily to substrate degradation. Consequently, protein substrates carrying Lys48-linked polyUb chains bind to and are degraded by 26S proteasome.Although it is well established that E3 ligases activate Ub ligation by E2s via their RING domains, very little is actually known about the underlying regulatory mechanism. Several recent studies determined the structure of RING domain complexes with Ub-charged UbcH5 (2–4). In one of these studies, the structure in solution of Ub-charged UbcH5c together with the mouse E3 ligase E4B U-box domain revealed that Ub can adopt an array of “open” and “closed” conformations (2). The productive closed conformation promotes a nucleophilic attack on the Ub∼E2 thioester by an incoming Lys (acceptor) residue (2). A similar closed conformation was identified in the structures of UbcH5a and UbcH5b, together with their cognate RING domains (3, 4). Taken together, these structural studies suggest that RING domains can catalyze Ub transfer by stabilizing a transition state of a closed conformation of the E2-bound (donor) Ub (5).Among the fundamental intracellular functions of the Ub–proteasome system is maintenance of cellular protein quality control (PQC) by targeting a diverse array of transiently or permanently misfolded substrates for proteolysis. A central branch of PQC degradation takes place in the endoplasmic reticulum (ER) in a process termed “ER-associated degradation” (ERAD) (6). Despite the multitude of misfolded substrates, ERAD employs only a few E3–ligase complexes (7). In fact, the bakers'' yeast S. cerevisiae ERAD system employs only two Ub-ligation complexes, specified by their E3 ligase components, Hrd1 and Doa10 (8–12). Importantly, each of the two E3 ligase complexes recognizes a distinct set of substrates, with minor overlaps (13).Degradation by the yeast ERAD Ub-ligation system entails the combined activity of two E2 enzymes: Ubc6 and Ubc7 for the Doa10 pathway and Ubc1 and Ubc7 for the Hrd1 pathway (14, 15). The shared E2 enzyme, Ubc7, is a soluble cytosolic protein whose binding to either of the E3–ligase complexes at the ER membrane is mediated by the auxiliary ER membrane protein Cue1. Binding to Cue1 not only mediates the interaction with the E3–ligase complex but also protects Ubc7 from degradation and stimulates its Ub-transfer activity (16–20). Ubc7 is highly conserved in evolution, as evident from substantial sequence and structure similarities with its orthologs from other species (21). The human Ubc7 ortholog, Ube2g2 (21), functions together with several ER membrane-embedded E3 ligases, the best characterized of which is the tumor autocrine motility factor receptor, gp78 (22). Ubc7 and Ube2g2 are subjected to similar regulatory mechanisms: They bind to and are activated by the RING domains of their cognate E3s as well as by the E2-binding regions and CUE domains within Cue1 and gp78 (19, 20, 23–26). The evolutionarily conserved sequence, structure, and regulatory mechanisms of the Ubc7 E2s imply an essential physiological function.In this study we explored the role of helix α2 of Ubc7 in enzyme activation. Based on our in vivo and in vitro observations and on the available structural information, we propose a mechanism whereby activation of Ubc7, mediated by noncovalent interaction with Ub at helix α2, is differentially affected by the RING domains of its cognate E3 ligases Hrd1 and Doa10.     

Identification of an unconventional E3 binding surface on the UbcH5?～?Ub conjugate recognized by a pathogenic bacterial E3 ligase.

Gram-negative bacteria deliver a cadre of virulence factors directly into the cytoplasm of eukaryotic host cells to promote pathogenesis and/or commensalism. Recently, families of virulence proteins have been recognized that function as E3 Ubiquitin-ligases. How these bacterial ligases integrate into the ubiquitin (Ub) signaling pathways of the host and how they differ functionally from endogenous eukaryotic E3s is not known. Here we show that the bacterial E3 SspH2 from S. typhimurium selectively binds the human UbcH5 ∼ Ub conjugate recognizing regions of both UbcH5 and Ub subunits. The surface of the E2 UbcH5 involved in this interaction differs substantially from that defined for other E2/E3 complexes involving eukaryotic E3-ligases. In vitro, SspH2 directs the synthesis of K48-linked poly-Ub chains, suggesting that cellular protein targets of SspH2-catalyzed Ub transfer are destined for proteasomal destruction. Unexpectedly, we found that intermediates in SspH2-directed reactions are activated poly-Ub chains directly tethered to the UbcH5 active site (UbcH5 ∼ Ub_n). Rapid generation of UbcH5 ∼ Ub_n may allow for bacterially directed modification of eukaryotic target proteins with a completed poly-Ub chain, efficiently tagging host targets for destruction.     

Genetic study of Saudi diabetes (GSSD): significant association of the KCNJ11 E23K polymorphism with type 2 diabetes

BACKGROUND: The E23K variant of KCNJ11 has been associated with type 2 diabetes (T2D) in several but not all populations studied. Thus far, despite a high incidence of T2D, the role of this variant in Arabs has not been established. METHODS: We performed a case-control association study using 550 T2D Saudi patients (WHO criteria), and 335 controls (age>or=60; fasting plasma glucose<7 mmol/L). E23K genotyping was performed by using molecular beacon-based real time PCR assays. RESULTS: The difference in K or risk allele frequency of cases and controls was significant with an OR of 1.7 (p=0.0001). The K allele is more common among T2D patients (21%) than in the age and sex matched controls (13.6%). This was consistent with a likely eventual conversion to T2D of younger normoglycemic individuals as they grow older. CONCLUSIONS: Our results report for the first time a positive association of the E23K variant with T2D in an Arab population. Confirmation by a larger study is indicated.     

截断逆挽方对慢加急性肝衰竭大鼠肝细胞进入S期关键蛋白的影响

目的:观察截断逆挽方对慢加急性肝衰竭(ACLF)大鼠肝脏细胞进入S期关键蛋白的影响。方法:将150只Wistar大鼠随机分成3组,正常组(NC)、模型组(MC)及截断逆挽方组(JD)。采用人血清白蛋白联合D-氨基半乳糖和脂多糖急性攻击制作ACLF模型。JD组大鼠在急性攻击24 h后连续灌胃,在第5、10、15天平行取材。运用HE染色法观察大鼠肝脏组织结构,Western blot法检测转录因子E2F1的表达量,RT-PCR检测CyclinA、E和Cdc25 mRNA表达量。结果:与模型组相比,JD组大鼠E2F1的表达量明显增高,并在第10天时差异最为显著(P<0.05),CyclinA、E、Cdc25表达量均有不同程度升高;与正常组相比,JD组大鼠10 d、15 d时CyclinA mRNA表达量差异显著,JD组大鼠在10 d、15 d时Cdc25和CyclinE mRNA表达量均差异显著(P<0.05)。结论:截断逆挽方可以通过调节E2F1介导的细胞再生信号通路上的关键蛋白E2F1、CyclinA、CyclinE、Cdc25的表达,从而促使细胞进入S期,加快DNA复制进程,提高肝细胞的增殖率,发挥治疗ACLF的作用。     

Prognostic significance of the F-box protein Skp2 expression in diffuse large B-cell lymphoma

The F-box protein Skp2 positively regulates the G1-S transition by promoting degradation of the cyclin-dependent kinase inhibitor p27(kip1) (p27). Recent evidence has suggested an oncogenic role of Skp2 in not only carcinogenesis but also lymphomagenesis. In this study, we performed immunohistochemical analysis on the cell-cycle-associated proteins, Skp2, p27, and Ki-67, in 27 patients with de novo diffuse large B-cell lymphoma (DLBCL), evaluating the correlation between the clinical characteristics and expression levels of these proteins. The patients were classified into two groups according to the positivity for Skp2 expression: a high Skp2 expression group (>60% positive for Skp2 in lymphoma cells) and a low Skp2 expression group (< or = 60%). A high level of Skp2 expression significantly correlated with advanced clinical stage (P = 0.029), although the increase did not correlate with age, gender, LDH levels, presence of extranodal disease, or performance status and resulted in no correlation with the International Prognostic Index-based risk grading. However, it was noteworthy that the high Skp2 expression group demonstrated a significantly worse prognosis than the low Skp2 expression group (P = 0.0007). The expression level of Skp2 correlated with that of Ki-67 but not necessarily with that of p27. The p27 expression level did not correlate patients' prognosis. Taken together, it was suggested that Skp2 was a valuable and independent marker predicting the outcome in DLBCL.     

Composite low affinity interactions dictate recognition of the cyclin-dependent kinase inhibitor Sic1 by the SCFCdc4 ubiquitin ligase

The ubiquitin ligase SCF(Cdc4) (Skp1/Cul1/F-box protein) recognizes its substrate, the cyclin-dependent kinase inhibitor Sic1, in a multisite phosphorylation-dependent manner. Although short diphosphorylated peptides derived from Sic1 can bind to Cdc4 with high affinity, through systematic mutagenesis and quantitative biophysical analysis we show that individually weak, dispersed Sic1 phospho sites engage Cdc4 in a dynamic equilibrium. The affinities of individual phosphoepitopes serve to tune the overall phosphorylation site threshold needed for efficient recognition. Notably, phosphoepitope affinity for Cdc4 is dramatically weakened in the context of full-length Sic1, demonstrating the importance of regional environment on binding interactions. The multisite nature of the Sic1-Cdc4 interaction confers cooperative dependence on kinase activity for Sic1 recognition and ubiquitination under equilibrium reaction conditions. Composite dynamic interactions of low affinity sites may be a general mechanism to establish phosphorylation thresholds in biological responses.     

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

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 G₁ 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 G₁/S regulation with increased levels of p27^Kip1 binding to Cdk1/cyclin E complexes. Our work indicates that the G₁/S transition can be controlled in different ways depending on the situation, resembling a regulatory network.