SCF和APC/C的结构及功能

细胞周期蛋白依赖性蛋白激酶(cyclin dependent kinases,CDKs)是细胞周期进行的推动力,泛素-蛋白酶体途径(ubiquitin-proteasome pathway,UPP)通过对细胞周期蛋白(cyclin)和CDK抑制物(CDK inhibitors,CKIs)的蛋白质水解作用来实现对CDKs活性的调控。SCF(Skp1-Cul1-F-box protein)和APC/C(anaphase-promoting complex/cyclosome)这两个泛素连接酶复合物参与了很多细胞周期调节因子的泛素化作用。它们参与的蛋白质降解系统的功能失调可能导致细胞增殖紊乱、基因组不稳定和肿瘤的发生。现对这两个泛素连接酶复合物的结构以及它们在细胞周期调控和肿瘤发生机制中的作用进行综述。     

Inhibition of Polo kinase by BI2536 affects centriole separation during Drosophila male meiosis

Pharmacological inhibition of Drosophila Polo kinase with BI2536 has allowed us to re-examine the requirements for Polo during Drosophila male gametogenesis. BI2536-treated spermatocytes persisted in a pro-metaphase state without dividing and had condensed chromosomes that did not separate. Centrosomes failed to recruit γ-tubulin and centrosomin (Cnn) and were not associated with microtubule arrays that were abnormal and did not form proper bipolar spindles. Centrioles, which usually separate during the anaphase of the first meiosis, remained held together in a V-shaped configuration suggesting that Polo kinase regulates the proteolysis that breaks centriole linkage to ensure their disengagement. Despite these defects spermatid differentiation proceeds, leading to axoneme formation.     

Impressionist portraits of mitotic exit: APC/C,K11-linked ubiquitin chains and Cezanne

The Anaphase-Promoting Complex/Cyclosome (APC/C) is an E3 ubiquitin ligase and a key regulator of cell cycle progression. By triggering the degradation of mitotic cyclins, APC/C controls cell cycle-dependent oscillations in cyclin-dependent kinase (CDK) activity. Thus, the dynamic activities of both APC/C and CDK sit at the core of the cell cycle oscillator. The APC/C controls a large number of substrates and is regulated through multiple mechanisms, including cofactor-dependent activation. These cofactors, Cdc20 and Cdh1, recognize substrates, while the specific E2 enzymes UBE2C/UbcH10 and UBE2S cooperate with APC/C to build K11-linked ubiquitin chains on substrates to target them for proteasomal degradation. However, whether deubiquitinating enzymes (DUBs) can antagonize APC/C substrate ubiquitination during mitosis has remained largely unknown. We recently demonstrated that Cezanne/OTUD7B is a cell cycle-regulated DUB that opposes the ubiquitination of APC/C substrates. Cezanne binds APC/C substrates, reverses their ubiquitination and protects them from degradation. Accordingly, Cezanne depletion accelerates APC/C substrate degradation, leading to errors in mitotic progression and formation of micronuclei. Moreover, Cezanne is significantly amplified and overexpressed in breast cancers. This suggests a potential role for APC/C antagonism in the pathogenesis of disease. APC/C contributes to chromosome segregation fidelity in mitosis raising the possibility that copy-number and expression changes in Cezanne observed in cancer contribute to the etiology of disease. Collectively, these observations identify a new player in cell cycle progression, define mechanisms of tempered APC/C substrate destruction and highlight the importance of this regulation in maintaining chromosome stability.     

Dietary restriction enhances germline stem cell maintenance

Dietary restriction (DR) increases lifespan in species ranging from yeast to primates, maintaining tissues in a youthful state and delaying reproductive senescence. However, little is known about the mechanisms by which this occurs. Here we demonstrate that, concurrent with extending lifespan, DR attenuates the age‐related decline in male germline stem cell (GSC) number in Drosophila. These data support a model whereby DR enhances maintenance of GSCs to extend the reproductive period of animals subjected to adverse nutritional conditions. This represents the first example of DR maintaining an adult stem cell pool and suggests a potential mechanism by which DR might delay aging in the tissues of higher organisms.     

Diazepam induces abnormal mitosis in the early Drosophila embryo

Drosophila embryos, because of their high proportion of dividing nuclei, offer many advantages for the study of the mitotic cycle. In the present study we combined immunofluorescence with interference contrast techniques to follow centrosome and spindle behavior in embryos exposed to diazepam during the first stages of development. Exposure to 100 micrograms/ml of diazepam produced polyploid and aneuploid figures resulting from the unusual fusion of one or more adjacent spindles. Diazepam also causes the inhibition of centrosome shifting and induces the formation of monopolar spindles during the metaphase-anaphase transition.     

Functional Heterogeneity of Alternatively Spliced Isoforms of Drosophila Ca2+/Calmodulin-Dependent Protein Kinase II

Abstract: The gene for Drosophila calcium/calmodulin-dependent protein kinase II is alternatively spliced to generate up to 18 different proteins that vary only in a region analogous to the point where mammalian α, β, γ, and δ isozymes show the greatest divergence from each other. To investigate the function of this variable region, we have characterized the catalytic and structural properties of six of the Drosophila isoforms. By several criteria (domain organization, low affinity for calmodulin, holoenzyme structure, and ability to autophosphorylate and become independent of calcium), these proteins are functional homologues of the mammalian calcium/calmodulin-dependent protein kinase II. Two major isoform-specific catalytic differences were observed. First, the R3A isoform was found to have a significantly higher K _act for calmodulin than the other isoforms. This indicates that the variable region, which is located distal to the calmodulin-binding domain, may play a role in activation of the enzyme by calmodulin. Decreased sensitivity to calmodulin may be biologically important if free calmodulin is limiting within the neuron. The second catalytic difference noted was that the R6 isoform had a significantly lower K _m for the peptide substrate used in this study. Although the variable region is not in the catalytic part of the enzyme, it may have an indirect function in substrate selectivity.     

CDC20在细胞分裂周期中的作用

CDC20是细胞周期相关蛋白之一。在细胞分裂周期中,CDC20是纺锤体组装检查点的靶向物和有丝分裂后期促进复合体的正调控因子,在引导细胞周期中某些蛋白质的泛素化降解和确保染色体正常分离的过程中起着重要的作用。     

果蝇生殖腺干细胞和它们的微环境

干细胞微环境是由容纳一个或多个干细胞,并控制干细胞自我更新和子代细胞产生的组织细胞以及细胞外基质组成。干细胞必须在微环境内才能增殖,才能保持自我更新的特性。通过对果蝇生殖腺干细胞微环境的结构及其产生的信号路径（该路径可以调节干细胞自我更新）的研究,发现微环境中支持细胞和它们发出的信号路径在调节干细胞的增殖和分化中起重要的作用。     

The end of a monolith: Deconstructing the Cnn-Polo interaction

In Drosophila melanogaster a functional pericentriolar matrix (PCM) at mitotic centrosomes requires Centrosomin-Long Form (Cnn-LF) proteins. Moreover, tissue culture cells have shown that the centrosomal localization of both Cnn-LF and Polo kinase are co-dependent, suggesting a direct interaction. Our recent study found Cnn potentially binds to and is phosphorylated by Polo kinase at 2 residues encoded by Exon1A, the initiating exon of a subset of Cnn isoforms. These interactions are required for the centrosomal localization of Cnn-LF in syncytial embryos and a mutation of either phosphorylation site is sufficient to block localization of both mutant and wild-type Cnn when they are co-expressed. Immunoprecipitation experiments show that Cnn-LF interacts directly with mitotically activated Polo kinase and requires the 2 phosphorylation sites in Exon1A. These IP experiments also show that Cnn-LF proteins form multimers. Depending on the stoichiometry between functional and mutant peptides, heteromultimers exhibit dominant negative or positive trans-complementation (rescue) effects on mitosis. Additionally, following the completion of meiosis, Cnn-Short Form (Cnn-SF) proteins are required for polar body formation in embryos, a process previously shown to require Polo kinase. These findings, when combined with previous work, clearly demonstrate the complexity of cnn and show that a view of cnn as encoding a single peptide is too simplistic.     

Stable MCC binding to the APC/C is required for a functional spindle assembly checkpoint

The spindle assembly checkpoint (SAC) delays progression into anaphase until all chromosomes have aligned on the metaphase plate by inhibiting Cdc20, the mitotic co‐activator of the APC/C. Mad2 and BubR1 bind and inhibit Cdc20, thereby forming the mitotic checkpoint complex (MCC), which can bind stably to the APC/C. Whether MCC formation per se is sufficient for a functional SAC or MCC association with the APC/C is required remains unclear. Here, we analyze the role of two conserved motifs in Cdc20, IR and C‐Box, in binding of the MCC to the APC/C. Mutants in both motifs assemble the MCC normally, but IR motif integrity is particularly important for stable binding to the APC/C. Cells expressing Cdc20 with a mutated IR motif have a compromised SAC, as uninhibited Cdc20 can compete with the MCC for APC/C binding and activate it. We thus show that stable MCC association with the APC/C is critical for a functional SAC.     

Differential control of Eg5-dependent centrosome separation by Plk1 and Cdk1

Cyclin-dependent kinase 1 (Cdk1) is thought to trigger centrosome separation in late G2 phase by phosphorylating the motor protein Eg5 at Thr927. However, the precise control mechanism of centrosome separation remains to be understood. Here, we report that in G2 phase polo-like kinase 1 (Plk1) can trigger centrosome separation independently of Cdk1. We find that Plk1 is required for both C-Nap1 displacement and for Eg5 localization on the centrosome. Moreover, Cdk2 compensates for Cdk1, and phosphorylates Eg5 at Thr927. Nevertheless, Plk1-driven centrosome separation is slow and staggering, while Cdk1 triggers fast movement of the centrosomes. We find that actin-dependent Eg5-opposing forces slow down separation in G2 phase. Strikingly, actin depolymerization, as well as destabilization of interphase microtubules (MTs), is sufficient to remove this obstruction and to speed up Plk1-dependent separation. Conversely, MT stabilization in mitosis slows down Cdk1-dependent centrosome movement. Our findings implicate the modulation of MT stability in G2 and M phase as a regulatory element in the control of centrosome separation.