Interaction between Mnk2 and CBCVHL ubiquitin ligase E3 complex

MAP kinase-interacting kinase-2 (Mnk2) is one of the downstream kinases activated by MAP kinases. It phosphorylates the eukaryotic initiation factor 4E (elF4E), although the role of elF4E phosphorylation and the role of Mnk2 in the process of protein translation are not well understood. Except for elF4E, other physiological substrates of Mnk2 are still unidentified. To look for these unidentified substrates and to reveal the physiological function of Mnk2, we performed a yeast two-hybrid screening with Mnk2 as the bait. The results demonstrated Mnk2 could interact with VHL (von Hippel-Lindau tumor suppressor), Rbx1 (ring-box 1) and Cul2 (Cullin2) proteins in yeast cells. Furthermore, we validated the interaction between Mnk2 and VHL proteins in mammalian cells by co-immunoprecipitation analysis. Because the three proteins VHL, Rbx1 and Cul2 are all components of the CBC^VHL ubiquitin ligase E3 complex, it has been shown that Mnk2 can interact with CBC^VHL complex, and is probably one of the new substrates of the CBC^VHL complex. Furthermore, during the interaction of Mnk2 with von Hippel-Lindau (VHL) tumor suppressor-binding protein 1 (VBP1), it appears that Mnk2 also joins to modulate cell shape as VBP1 plays an important role in the process of the maturation of the cytoskeleton and in the process of morphogenesis.     

Interaction between Mnk2 and CBCVHL ubiquitin ligase E3 complex

MAP kinase-interacting kinase-2 (Mnk2) is one of the downstream kinases activated by MAP kinases. It phosphorylates the eukaryotic initiation factor 4E (elF4E), although the role of elF4E phosphorylation and the role of Mnk2 in the process of protein translation are not well understood. Except for elF4E, other physiological substrates of Mnk2 are still unidentified. To look for these unidentified substrates and to reveal the physiological function of Mnk2, we performed a yeast two-hybrid screening with Mnk2 as the bait. The results demonstrated Mnk2 could interact with VHL (von Hippel-Lindau tumor suppressor), Rbx1 (ring-box 1) and Cul2 (Cullin2) proteins in yeast cells. Furthermore, we validated the interaction between Mnk2 and VHL proteins in mammalian cells by co-immunoprecipitation analysis. Because the three proteins VHL, Rbx1 and Cul2 are all components of the CBC^VHL ubiquitin ligase E3 complex, it has been shown that Mnk2 can interact with CBC^VHL complex, and is probably one of the new substrates of the CBC^VHL complex. Furthermore, during the interaction of Mnk2 with von Hippel-Lindau (VHL) tumor suppressor-binding protein 1 (VBP1), it appears that Mnk2 also joins to modulate cell shape as VBP1 plays an important role in the process of the maturation of the cytoskeleton and in the process of morphogenesis.     

The ubiquitin proteasome system — Implications for cell cycle control and the targeted treatment of cancer

Two families of E3 ubiquitin ligases are prominent in cell cycle regulation and mediate the timely and precise ubiquitin–proteasome-dependent degradation of key cell cycle proteins: the SCF (Skp1/Cul1/F-box protein) complex and the APC/C (anaphase promoting complex or cyclosome). While certain SCF ligases drive cell cycle progression throughout the cell cycle, APC/C (in complex with either of two substrate recruiting proteins: Cdc20 and Cdh1) orchestrates exit from mitosis (APC/C^Cdc20) and establishes a stable G1 phase (APC/C^Cdh1). Upon DNA damage or perturbation of the normal cell cycle, both ligases are involved in checkpoint activation. Mechanistic insight into these processes has significantly improved over the last ten years, largely due to a better understanding of APC/C and the functional characterization of multiple F-box proteins, the variable substrate recruiting components of SCF ligases. Here, we review the role of SCF- and APC/C-mediated ubiquitylation in the normal and perturbed cell cycle and discuss potential clinical implications of SCF and APC/C functions. This article is part of a Special Issue entitled: Ubiquitin–Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf.     

PAR-6 levels are regulated by NOS-3 in a CUL-2 dependent manner in Caenorhabditiselegans

The PAR proteins have an essential and conserved function in establishing polarity in many cell types and organisms. However, their key upstream regulators remain to be identified. In C. elegans, regulators of the PAR proteins can be identified by their ability to suppress the lethality of par-2 mutant embryos. Here we show that a nos-3 loss of function mutant suppresses the lethality of par-2 mutants by regulating PAR-6 protein levels. The suppression requires the activity of the sex determination genes fem-1/2/3 and of the cullin cul-2. FEM-1 is a substrate-specific adaptor for a CUL-2-based ubiquitin ligase (CBC^FEM-1). Interestingly, we find that CUL-2 is required for the regulation of PAR-6 levels and that PAR-6 physically interacts with FEM-1. Our data strongly suggest that PAR-6 levels are regulated by the CBC^FEM-1 ubiquitin ligase thereby uncovering a novel role for the FEM proteins and cullin-dependent degradation in regulating PAR proteins and polarity processes.     

Characterization of the DOC1/APC10 subunit of the yeast and the human anaphase-promoting complex.

The anaphase-promoting complex/cyclosome (APC) is a ubiquitin-protein ligase whose activity is essential for progression through mitosis. The vertebrate APC is thought to be composed of 8 subunits, whereas in budding yeast several additional APC-associated proteins have been identified, including a 33-kDa protein called Doc1 or Apc10. Here, we show that Doc1/Apc10 is a subunit of the yeast APC throughout the cell cycle. Mutation of Doc1/Apc10 inactivates the APC without destabilizing the complex. An ortholog of Doc1/Apc10, which we call APC10, is associated with the APC in different vertebrates, including humans and frogs. Biochemical fractionation experiments and mass spectrometric analysis of a component of the purified human APC show that APC10 is a genuine APC subunit whose cellular levels or association with the APC are not cell cycle-regulated. We have further identified an APC10 homology region, which we propose to call the DOC domain, in several protein sequences that also contain either cullin or HECT domains. Cullins are present in several ubiquitination complexes including the APC, whereas HECT domains represent the catalytic core of a different type of ubiquitin-protein ligase. DOC domains may therefore be important for reactions catalyzed by several types of ubiquitin-protein ligases.     

An orc1 allele with a mutated APC motif is female sterile with amplification defects

The origin recognition complex 1 (ORC1) is the largest subunit of the ORC, the heteromeric hexamer. ORC1 is an essential component of the pre-replicative complex (pre-RC) that licenses eukaryote DNA replication origins. The levels of ORC1 fluctuate during the mitotic cell cycle in Drosophila as well as in some human cells. Proteolysis of ORC1 occurs at the end of M phase in Drosophila, which is mediated by the anaphase-promoting complex (APC), and in late S phase in human cells by Skip-Cullin-F box (SCF). Previously we showed that proteolysis of ORC1 by APC is mediated by the ORC1 destruction box (the O-box), an APC motif conserved among species yet distinct from the D-box or KEN-box. Recently we showed that replacing the O-box with the D-box (ORC1^O→D) changes the degradation profile of ORC1 during a canonical cell cycle. Here we report further characterization of the ORC1^O→D allele that turned out to be a useful tool to examine the function of ORC1 in other modes of DNA replication during oogenesis. In endoreplication stages ORC1^O→D does not change any DNA content profiles, consistent with our previous finding that ORC is dispensable for endoreplication. However, in amplification stage replication efficiency of ORC1^O→D is drastically reduced, which resulted in amplification defects that led to thin egg shell phenotype. Taken together, our analyses show that orc1 allele newly identified is female sterile and possesses a unique feature of phenotypes that are distinct in different modes of DNA replication.     

The Arabidopsis APC4 subunit of the anaphase-promoting complex/cyclosome (APC/C) is critical for both female gametogenesis and embryogenesis

The anaphase‐promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase that is involved in regulating cell‐cycle progression. It has been widely studied in yeast and animal cells, but the function and regulation of the APC/C in plant cells are largely unknown. The Arabidopsis APC/C comprises at least 11 subunits, only a few of which have been studied in detail. APC4 is proposed to be a connector in the APC/C in yeast and animals. Here, we report the functional characterization of the Arabidopsis APC4 protein. We examined three heterozygous plant lines carrying apc4 alleles. These plants showed pleiotropic developmental defects in reproductive processes, including abnormal nuclear behavior in the developing embryo sac and aberrant cell division in embryos; these phenotypes differ from those reported for mutants of other subunits. Some ovules and embryos of apc4/+ plants also accumulated cyclin B protein, a known substrate of APC/C, suggesting a compromised function of APC/C. Arabidopsis APC4 was expressed in meristematic cells of seedlings, ovules in pistils and embryos in siliques, and was mainly localized in the nucleus. Additionally, the distribution of auxin was distorted in some embryos of apc4/+ plants. Our results indicate that Arabidopsis APC4 plays critical roles in female gametogenesis and embryogenesis, possibly as a connector in APC/C, and that regulation of auxin distribution may be involved in these processes.     

Structure, function and mechanism of the anaphase promoting complex (APC/C)

The complex molecular events responsible for coordinating chromosome replication and segregation with cell division and growth are collectively known as the cell cycle. Progression through the cell cycle is orchestrated by the interplay between controlled protein synthesis and degradation and protein phosphorylation. Protein degradation is primarily regulated through the ubiquitin proteasome system, mediated by two related E3 protein ubiquitin ligases, the Skp1 cullin F-box (SCF) and the anaphase promoting complex (also known as the cyclosome) (APC/C). The APC/C is a multi-subunit cullin-RING E3 ubiquitin ligase that regulates progression through the mitotic phase of the cell cycle and controls entry into S phase by catalysing the ubiquitylation of cyclins and other cell cycle regulatory proteins. Selection of APC/C targets is controlled through recognition of short destruction motifs, predominantly the D-box and KEN-box. APC/C-mediated coordination of cell cycle progression is achieved through the temporal regulation of APC/C activity and substrate specificity, exerted through a combination of co-activator subunits, reversible phosphorylation and inhibitory proteins and complexes. The aim of this article is to discuss the APC/C from a structural and mechanistic perspective. Although an atomic structure of the APC/C is still lacking, a combination of genetic, biochemical, electron microscopy studies of intact APC/C and crystallographic analysis of individual subunits, together with analogies to evolutionarily related E3 ligases of the RING family, has provided deep insights into the molecular mechanisms of catalysis and substrate recognition, and structural organisation of the APC/C.     

Proteolysis and the cell cycle: with this RING I do thee destroy

The ubiquitin system drives the cell division cycle by the timely destruction of numerous regulatory proteins. Remarkably, the two main activities that catalyze substrate ubiquitination in the cell cycle, the Skp1-Cdc53/cullin-F-box protein (SCF) complexes and the anaphase-promoting complex/cyclosome (APC/C), define a new superfamily of E3 ubiquitin ligases, all based on related cullin and RING-H2 finger protein subunits. The circuits that interconnect the SCF, APC/C and cyclin-dependent kinase activities form a master oscillator that coordinates the replication and segregation of the genome.     

The COP9 signalosome regulates cell proliferation of Dictyostelium discoideum

Regulated protein destruction involving SCF (Skp1/Cullin/F-box, E3 ubiquitin ligase) complexes is required for multicellular development of Dictyostelium discoideum. Dynamic modification of cullin by nedd8 is required for the proper action of SCF. The COP9 signalosome (CSN), first identified in a signaling pathway for light response in plants, functions as a large multi-protein complex that regulates cullin neddylation in eukaryotes. Still, there is extreme sequence divergence of CSN subunits of the yeasts in comparison to the multicellular plants and animals. Using the yeast two-hybrid system, we have identified the CSN5 subunit as a potential interacting partner of a cell surface receptor of Dictyostelium. We further identified and characterized all 8 CSN subunits in Dictyostelium discoideum. Remarkably, despite the ancient origin of Dictyostelium, its CSN proteins cluster very closely with their plant and animal counterparts. We additionally show that the Dictyostelium subunits, like those of other systems are capable of multi-protein interactions within the CSN complex. Our data also indicate that CSN5 (and CSN2) are essential for cell proliferation in Dictyostelium, a phenotype similar to that of multicellular organisms, but distinct from that of the yeasts. Finally, we speculate on a potential role of CSN in cullin function and regulated protein destruction during multicellular development of Dictyostelium.     

Proteomics analysis identifies new components of the fission and budding yeast anaphase-promoting complexes

The anaphase-promoting complex (APC) is a conserved multisubunit ubiquitin ligase required for the degradation of key cell cycle regulators. Components of the APC have been identified through genetic screens in both Schizosaccharomyces pombe and Saccharomyces cerevisiae as well as through biochemical purification coupled with mass spectrometric protein identification. With these approaches, 11 subunits of the core S. cerevisiae APC have been identified. Here, we have applied a tandem affinity purification approach coupled with direct analysis of the purified complexes by mass spectrometry (DALPC) to reveal additional subunits of both the S. pombe and S. cerevisiae APCs. Our data increase the total number of identified APC subunits to 13 in both yeasts and indicate that previous approaches were biased against the identification of small subunits. These results underscore the power of direct analysis of protein complexes by mass spectrometry and set the foundation for further functional and structural studies of the APC.     

Neddylation and deneddylation of CUL-3 is required to target MEI-1/Katanin for degradation at the meiosis-to-mitosis transition in C. elegans

BACKGROUND: SCF (Skp1-Cullin-F-box) complexes are a major class of E3 ligases that are required to selectively target substrates for ubiquitin-dependent degradation by the 26S proteasome. Conjugation of the ubiquitin-like protein Nedd8 to the cullin subunit (neddylation) positively regulates activity of SCF complexes, most likely by increasing their affinity for the E2 conjugated to ubiquitin. The Nedd8 conjugation pathway is required in C. elegans embryos for the ubiquitin-mediated degradation of the microtubule-severing protein MEI-1/Katanin at the meiosis-to-mitosis transition. Genetic experiments suggest that this pathway controls the activity of a CUL-3-based E3 ligase. Counteracting the Nedd8 pathway, the COP9/signalosome has been shown to promote deneddylation of the cullin subunit. However, little is known about the role of neddylation and deneddylation for E3 ligase activity in vivo. RESULTS: Here, we identified and characterized the COP9/signalosome in C. elegans and showed that it promotes deneddylation of CUL-3, a critical target of the Nedd8 conjugation pathway. As in other species, the C. elegans signalosome is a macromolecular complex containing at least six subunits that localizes in the nucleus and the cytoplasm. Reducing COP9/signalosome function by RNAi results in a failure to degrade MEI-1, leading to severe defects in microtubule-dependent processes during the first mitotic division. Intriguingly, reducing COP9/signalosome function suppresses a partial defect in the neddylation pathway; this suppression suggests that deneddylation and neddylation antagonize each other. CONCLUSIONS: We conclude that both neddylation and deneddylation of CUL-3 is required for MEI-1 degradation and propose that cycles of CUL-3 neddylation and deneddylation are necessary for its ligase activity in vivo.     

The Caenorhabditis elegans Skp1-related gene family: diverse functions in cell proliferation, morphogenesis, and meiosis

BACKGROUND: The SCF ubiquitin-ligase complex targets the ubiquitin-mediated degradation of proteins in multiple dynamic cellular processes. A key SCF component is the Skp1 protein that functions within the complex to link the substrate-recognition subunit to a cullin that in turn binds the ubiquitin-conjugating enzyme. In contrast to yeast and humans, Caenorhabditis elegans contains multiple expressed Skp1-related (skr) genes. RESULTS: The 21 Skp1-related (skr) genes in C. elegans form one phylogenetic clade, suggesting that a single ancestral Skp1 gene underwent independent expansion in C. elegans. The cellular and developmental functions of the 21 C. elegans skr genes were probed by dsRNA-mediated gene inactivation (RNAi). The RNAi phenotypes of the skr genes fall into two classes. First, the highly similar skr-7, -8, -9, and -10 genes are required for posterior body morphogenesis, embryonic and larval development, and cell proliferation. Second, the related skr-1 and -2 genes are required for the restraint of cell proliferation, progression through the pachytene stage of meiosis, and the formation of bivalent chromosomes at diakinesis. CUL-1 was found to interact with SKR-1, -2, -3, -7, -8, and -10 in the yeast two-hybrid system. Interestingly, SKR-3 could interact with both CUL-1 and its close paralog CUL-6. CONCLUSIONS: Members of the expanded skr gene family in C. elegans perform critical functions in regulating cell proliferation, meiosis, and morphogenesis. The finding that multiple SKRs are able to bind cullins suggests an extensive set of combinatorial SCF complexes.     

A Minimal Anaphase Promoting Complex/Cyclosome (APC/C) in Trypanosoma brucei

The anaphase-promoting complex/cyclosome (APC/C) is a multi-subunit E3 ubiquitin ligase that initiates chromosome segregation and mitotic exit by targeting critical cell-cycle regulators for proteolytic destruction. Previously, seven APC/C subunit homologues were identified in the genome of Trypanosoma brucei. In the present study, we tested five of them in yeast complementation studies and found none of them capable of complementing the yeast mutants lacking the corresponding subunits, suggesting significant discrepancies between the two APC/C’s. Subunit homologues of mitotic checkpoint complex (MCC) have not yet been identified in T. brucei, raising the possibility that a MCC-APC/C complex equivalent may not exist in T. brucei. We performed tandem affinity purification of the protein complex containing a APC1 fusion protein expressed in the cells enriched in different phases of the cell cycle of procyclic form T. brucei, and compared their protein profiles using LC-MS/MS analyses. The seven putative APC/C subunits were identified in the protein complex throughout the cell cycle together with three additional proteins designated the associated proteins (AP) AP1, AP2 and AP3. Abundance of the 10 proteins remained relatively unchanged throughout the cell cycle, suggesting that they are the core subunits of APC/C. AP1 turned out to be a homologue of APC4. An RNAi knockdown of APC4 and AP3 showed no detectable cellular phenotype, whereas an AP2 knockdown enriched the cells in G2/M phase. The AP2-depleted cells showed stabilized mitotic cyclin B. An accumulation of poly-ubiquitinated cyclin B was indicated in the cells treated with the proteasome inhibitor MG132, demonstrating the involvement of proteasome in degrading poly-ubiquitinated cyclin B. In all, a 10-subunit APC/C machinery with a conserved function is identified in T. brucei without linking to a MCC-like complex, thus indicating a unique T. brucei APC/C.     

SCF and APC: the Yin and Yang of cell cycle regulated proteolysis

Progression through the cell cycle requires the activity of two ubiquitination complexes, the Skp1—cullin—F-box-protein complex (SCF) and the anaphase-promoting complex/cyclosome (APC). Observations in the past year have revealed unexpected similarities between the SCF and the APC and have allowed detailed insight into the regulation of their activities. Both complexes are now known to exist in different forms that target different substrates for ubiquitin-dependent proteolysis.     

APC-targeted RAAI degradation mediates the cell cycle and root development in plants

Protein degradation by the ubiquitin-proteasome system is necessary for a normal cell cycle. As compared with knowledge of the mechanism in animals and yeast, that in plants is less known. Here we summarize research into the regulatory mechanism of protein degradation in the cell cycle in plants. Anaphase-promoting complex/cyclosome (APC), in the E3 family of enzymes, plays an important role in maintaining normal mitosis. APC activation and substrate specificity is determined by its activators, which can recognize the destruction box (D-box) in APC target proteins. Oryza sativa root architecture-associated I (OsRAA1) with GTP-binding activity was originally cloned from rice. Overexpression of of OsRAA1 inhibits the growth of primary roots in rice. Knockdown lines showed reduced height of seedlings because of abnormal cell division. OsRAA1 transgenic rice and fission yeast show a higher proportion of metaphase cells than that of controls, which suggests a blocked transition from metaphase to anaphase during mitosis. OsRAA1 co-localizes with spindle tubulin. It contains the D-box motif and interacts with OsRPT4 of the regulatory particle of 26S proteasome. OsRAA1 may be a cell cycle inhibitor that can be degraded by the ubiquitin-proteasome system, and its disruption is necessary for the transition from metaphase to anaphase during root growth in rice.Key words: cell cycle, APC, RAA1, rice, protein degradationProtein degradation by the ubiquitin-proteasome system is necessary for the normal cell cycle. The activation of 3 enzymes, E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme) and E3 (ubiquitin ligase), are required for the addition of ubiquitin molecules to the target protein. E1 catalyzes the formation of the thiol-ester bond between C-terminal glycine in ubiquitin and cysteine in E1, and activated ubiquitin is transferred to a cysteine in E2. With the help of an E3, ubiquitin is linked to the lysine in the target protein. Subsequent ubiquitins can be attached to the previously bound ubiquitin because of the seven lysine residues in the ubiquitin molecule. Finally, the ubiquitinated substrates are degraded by the 26S proteasome.E3 confers substrate specificity. E3 ubiquitin ligases comprise a large and diverse family of proteins or protein complexes. E3s are of two classes: homology to E6-AP carboxy terminus-containing proteins, and RING-finger domain-containing proteins. The RING-finger E3s have 4 subgroups: single subunit RING E3, VCB-Cul2 complex (VBC), Skp1/Cullin/F-box protein (SCF) and anaphase-promoting complex/cyclosome (APC/C).¹ The SCF ligases regulate the transition from G₁/S and G₂/M, and APC is required for mitosis. Many APC substrates have been identified in animals.² The polyubiquitinated substrates can be recognized by different ubiquitin receptors and degraded via 26S proteasome.^3,4 However, little is known about APC substrates in plants.     

The Caenorhabditis elegans cell-cycle regulator ZYG-11 defines a conserved family of CUL-2 complex components

The cullin CUL-2 is a crucial component of a subclass of multisubunit cullin-RING ubiquitin-ligases. The specificity of CUL-2-based complexes is provided by variable substrate-recognition subunits that bind to specific substrates. In Caenorhabditis elegans, CUL-2 regulates several key processes in cell division and embryonic development, including meiotic progression, anterior-posterior polarity and mitotic chromatin condensation. However, the substrate recognition subunits that work in these CUL-2-dependent processes were unknown. Here, we present evidence that ZYG-11 is the substrate-recognition subunit for a CUL-2-based complex that regulates these functions. We show that ZYG-11 interacts with CUL-2 in vivo and binds to the complex adaptor protein Elongin C using a nematode variant of the VHL-box motif. We show that the ZYG11 gene family encompasses two main branches in metazoa, and provide evidence that members of the extended ZYG11 family in nematodes and humans are conserved components of CUL2-based ubiquitin-ligases.     

Cullin 4A-mediated proteolysis of DDB2 protein at DNA damage sites regulates in vivo lesion recognition by XPC

Xeroderma pigmentosum (XP) complementation group E gene product, damaged DNA-binding protein 2 (DDB2), is a subunit of the DDB heterodimeric protein complex with high specificity for binding to a variety of DNA helix-distorting lesions. DDB is believed to play a role in the initial step of damage recognition in mammalian nucleotide excision repair (NER) of ultraviolet light (UV)-induced photolesions. It has been shown that DDB2 is rapidly degraded after cellular UV irradiation. However, the relevance of DDB2 degradation to its functionality in NER is still unknown. Here, we have provided evidence that Cullin 4A (CUL-4A), a key component of CUL-4A-based ubiquitin ligase, mediates DDB2 degradation at the damage sites and regulates the recruitment of XPC and the repair of cyclobutane pyrimidine dimers. We have shown that CUL-4A can be identified in a UV-responsive protein complex containing both DDB subunits. CUL-4A was visualized in localized UV-irradiated sites together with DDB2 and XPC. Degradation of DDB2 could be blocked by silencing CUL-4A using small interference RNA or by treating cells with proteasome inhibitor MG132. This blockage resulted in prolonged retention of DDB2 at the subnuclear DNA damage foci within micropore irradiated cells. Knock down of CUL-4A also decreased recruitment of the damage recognition factor, XPC, to the damaged foci and concomitantly reduced the removal of cyclobutane pyrimidine dimers from the entire genome. These results suggest that CUL-4A mediates the proteolytic degradation of DDB2 and that this degradation event, initiated at the lesion sites, regulates damage recognition by XPC during the early steps of NER.