retina aberrant in pattern/fizzy-related


Activation of yeast Fizzy-related homologs

Proteolysis of mitotic cyclins depends on a multisubunit ubiquitin-protein ligase, the anaphase promoting complex (APC). Proteolysis commences during anaphase, persisting throughout G1 until it is terminated by cyclin-dependent kinases (CDKs) as cells enter S phase. Proteolysis of mitotic cyclins in yeast was shown to require association of the APC with the substrate-specific activator Hct1 (also called Cdh1). Phosphorylation of Hct1 by CDKs blocked the Hct1-APC interaction. The mutual inhibition between APC and CDKs explains how cells suppress mitotic CDK activity during G1 and then establish a period with elevated kinase activity from S phase until anaphase (Zachariae, 1998).

Ubiquitin-mediated proteolysis due to the anaphase-promoting complex/cyclosome is essential for separation of sister chromatids, requiring degradation of the anaphase inhibitor Pds1, and for exit from mitosis, requiring inactivation of cyclin B Cdk1 kinases. Exit from mitosis in yeast involves accumulation of the cyclin kinase inhibitor Sic1 as well as cyclin proteolysis mediated by APC/C bound by the activating subunit Cdh1/Hct1 [APC(Cdh1)]. Both processes require the Cdc14 phosphatase, whose release from the nucleolus during anaphase causes dephosphorylation and thereby activation of Cdh1 and accumulation of another protein, Sic1. It is not known what determines the release of Cdc14 and enables it to promote Cdk1 inactivation, but it is known to be dependent on APC/C bound by Cdc20 [APC(Cdc20)]. APC(Cdc20) allows activation of Cdc14 and promotes exit from mitosis by mediating proteolysis of Pds1 and the S phase cyclin Clb5 in the yeast Saccharomyces cerevisiae. Degradation of Pds1 is necessary for release of Cdc14 from the nucleolus, whereas degradation of Clb5 is crucial if Cdc14 is to overwhelm Cdk1 and activate its foes (Cdh1 and Sic1). Remarkably, cells lacking both Pds1 and Clb5 can proliferate in the complete absence of Cdc20 (Shirayama, 1999).

Exit from mitosis requires inactivation of mitotic cyclin-dependent kinases (CDKs). A key mechanism of CDK inactivation is ubiquitin-mediated cyclin proteolysis, which is triggered by the late mitotic activation of a ubiquitin ligase known as the anaphase-promoting complex (APC). Activation of the APC requires its association with substoichiometric activating subunits termed Cdc20 and Hct1 (also known as Cdh1). This study explores the molecular function and regulation of the APC regulatory subunit Hct1 in Saccharomyces cerevisiae. Recombinant Hct1 activates the cyclin-ubiquitin ligase activity of APC isolated from multiple cell cycle stages. APC isolated from cells arrested in G1, or in late mitosis due to the cdc14-1 mutation, is more responsive to Hct1 than APC isolated from other stages. Hct1 is phosphorylated in vivo at multiple CDK consensus sites during cell cycle stages when activity of the cyclin-dependent kinase Cdc28 is high and APC activity is low. Purified Hct1 is phosphorylated in vitro at these sites by purified Cdc28-cyclin complexes, and phosphorylation abolishes the ability of Hct1 to activate the APC in vitro. The phosphatase Cdc14, which is known to be required for APC activation in vivo, is able to reverse the effects of Cdc28 by catalyzing Hct1 dephosphorylation and activation. It is concluded that Hct1 phosphorylation is a key regulatory mechanism in the control of cyclin destruction. Phosphorylation of Hct1 provides a mechanism by which Cdc28 blocks its own inactivation during S phase and early mitosis. Following anaphase, dephosphorylation of Hct1 by Cdc14 may help initiate cyclin destruction (Jaspersen, 1999).

Progression through mitosis is controlled by protein degradation that is mediated by the anaphase-promoting complex/cyclosome and its associated specificity factors. In budding yeast, APC/C(Cdc20) promotes the degradation of the Pds1p anaphase inhibitor at the metaphase-to-anaphase transition, whereas APC/C(Cdh1) promotes the degradation of the mitotic cyclins at the exit from mitosis. Pds1p has a novel activity as an inhibitor of mitotic cyclin destruction, apparently by preventing the activation of APC/C(Cdh1). This activity of Pds1p is independent of its activity as an anaphase inhibitor. It is proposed that the dual role of Pds1p as an inhibitor of anaphase and of cyclin degradation allows the cell to couple the exit from mitosis to the prior completion of anaphase. Finally, these observations provide a novel regulatory paradigm in which the sequential degradation of two substrates is determined by the substrates themselves, such that an early substrate inhibits the degradation of a later one (Cohen-Fix, 1999).

Yeast Fizzy-related homologs, spindle assembly and mitotic checkpoints

Saccharomyces cerevisiae Cin8p belongs to the BimC family of kinesin-related motor proteins that are essential for spindle assembly. Cin8p levels were found to oscillate in the cell cycle due in part to a high rate of degradation imposed from the end of mitosis through the G1 phase. Cin8p degradation requires the anaphase-promoting complex ubiquitin ligase and its late mitosis regulator Cdh1p but not the early mitosis regulator Cdc20p. Cin8p lacks a functional destruction box sequence that is found in the majority of anaphase-promoting complex substrates. An extensive mutagenesis study was carried out to define the cis-acting sequence required for Cin8p degradation in vivo. The C terminus of Cin8p contains two elements required for its degradation: (1) a bipartite destruction sequence composed of a KEN-box plus essential residues within the downstream 22 amino acids and (2) a nuclear localization signal. The bipartite destruction sequence appears in other BimC kinesins as well. Expression of nondegradable Cin8p shows very mild phenotypic effects, with an increase in the fraction of mitotic cells with broken spindles (Hildebrandt, 2001).

Yeast Fizzy-related homologs: targets at mitotic exit and in G1

When proliferating fission yeast cells are exposed to nitrogen starvation, they initiate conjugation and differentiate into ascospores. Cell cycle arrest in the G1-phase is one of the prerequisites for cell differentiation, because conjugation occurs only in the pre-Start G1-phase. The role of ste9(+) in the cell cycle progression was investigated. Ste9 is a WD-repeat protein that is highly homologous to Hct1/Cdh1 and Fizzy-related. ste9 mutants are sterile because they are defective in cell cycle arrest in the G1-phase upon starvation. Sterility is partially suppressed by the mutation in cig2 that encodes the major G1/S cyclin. Although cells lacking Ste9 function grow normally, the ste9 mutation is synthetically lethal with the wee1 mutation. In the double mutants of ste9 cdc10(ts), cells arrest in G1-phase at the restrictive temperature, but the level of mitotic cyclin (Cdc13) does not decrease. In these cells, abortive mitosis occurs from the pre-Start G1-phase. Overexpression of Ste9 decreases the Cdc13 protein level and the H1-histone kinase activity. In these cells, mitosis is inhibited and an extra round of DNA replication occurs. Ste9 regulates G1 progression, possibly by controlling the amount of the mitotic cyclin in the G1-phase (Kitamura, 1999).

Fission yeast ste9/srw1 is a WD-repeat protein highly homologous to budding yeast Hct1/Cdh1 and Drosophila Fizzy-related, which are both involved in activating APC/C. APC(ste9/srw1) specifically promotes the degradation of mitotic cyclins cdc13 and cig1 but not the S-phase cyclin cig2. APC(ste9/srw1) is not necessary for the proteolysis of cdc13 and cig1 that occurs at the metaphase-anaphase transition but it is absolutely required for their degradation in G1. Therefore, it is proposed that the main role of APC(ste9/srw1) is to promote degradation of mitotic cyclins when cells need to delay or arrest the cell cycle in G1. ste9/srw1 is negatively regulated by cdc2-dependent protein phosphorylation. In G1, when cdc2-cyclin kinase activity is low, unphosphorylated ste9/srw1 interacts with APC/C. In the rest of the cell cycle, phosphorylation of ste9/srw1 by cdc2-cyclin complexes both triggers proteolysis of ste9/srw1 and causes its dissociation from the APC/C. This mechanism provides a molecular switch to prevent inactivation of cdc2 in G2 and early mitosis and to allow its inactivation in G1 (Blanco, 2000).

Cdc20, an activator of the anaphase-promoting complex (APC), is also required for the exit from mitosis in Saccharomyces cerevisiae. During mitosis, both the inactivation of Cdc28-Clb2 kinase and the degradation of mitotic cyclin Clb2 occur in two steps. The first phase of Clb2 proteolysis, which commences at the metaphase-to-anaphase transition when Clb2 abundance is high, is dependent on Cdc20. The second wave of Clb2 destruction in telophase requires activation of the Cdc20 homolog, Hct1/Cdh1. The first phase of Clb2 destruction, which lowers the Cdc28-Clb2 kinase activity, is a prerequisite for the second. Thus, Clb2 proteolysis is not solely mediated by Hct1 as generally believed; instead, it requires a sequential action of both Cdc20 and Hct1 (Yeong, 2000).

An architectural map of the anaphase-promoting complex

The anaphase-promoting complex or cyclosome (APC) is an unusually complicated ubiquitin ligase, composed of 13 core subunits and either of two loosely associated regulatory subunits, Cdc20 and Cdh1. The architecture of the APC was analyzed using a recently constructed budding yeast strain that is viable in the absence of normally essential APC subunits. The largest subunit, Apc1, serves as a scaffold that associates independently with two separable subcomplexes, one that contains Apc2 (Cullin), Apc11 (RING), and Doc1/Apc10, and another that contains the three TPR subunits (Cdc27, Cdc16, and Cdc23). The three TPR subunits display a sequential binding dependency, with Cdc27 the most peripheral, Cdc23 the most internal, and Cdc16 between. Apc4, Apc5, Cdc23, and Apc1 associate interdependently, such that loss of any one subunit greatly reduces binding between the remaining three. Intriguingly, the cullin and TPR subunits both contribute to the binding of Cdh1 to the APC. Enzymatic assays performed with APC purified from strains lacking each of the essential subunits revealed that only cdc27Δ complexes retain detectable activity in the presence of Cdh1. This residual activity depends on the C-box domain of Cdh1, but not on the C-terminal IR domain, suggesting that the C-box mediates a productive interaction with an APC subunit other than Cdc27. The IR domain of Cdc20 is dispensable for viability, suggesting that Cdc20 can activate the APC through another domain. This study has provided an updated model for the subunit architecture of the APC (Thornton, 2006).

A C. elegans Fizzy-related homolog

A synthetic-lethal screen in Caenorhabditis elegans is describes that overcomes a number of obstacles associated with the analysis of functionally redundant genes. Using this approach, mutations that synthetically interact with lin-35/Rb, a SynMuv gene and the sole member of the Rb/pocket protein family in C. elegans have been identified. Unlike the original SynMuv screens, this approach is completely nonbiased and can theoretically be applied to any situation in which a mutation fails to produce a detectable phenotype. This screen has identiifed fzr-1, a gene that synthetically interacts with lin-35 to produce global defects in cell proliferation control. fzr-1 encodes the C. elegans homolog of Cdh1/Hct1/FZR, a gene product shown in other systems to regulate the APC cyclosome. Genetic interactions between fzr-1 and a subset of class B SynMuv genes, and between lin-35 and the putative SCF regulator lin-23, have been uncovered. It is proposed that lin-35, fzr-1, and lin-23 function redundantly to control cell cycle progression through the regulation of cyclin levels (Fay, 2002).

fzr-1 cooperates with lin-35 to control cell proliferation. A relatively small number of genes have been described that cause widespread hyperproliferation in C. elegans. These include the putative SCF components cul-1 and lin-23 , the CIP/KIP family member cki-1, and the CBP/p300 homolog cbp-1 . In the cases examined thus far, a hyperproliferation phenotype is observed following inactivation of a single gene product. Loss of cell proliferation control can also result from a synthetic genetic interaction. Although single mutants of lin-35 and fzr-1 show only subtle or low-penetrance phenotypes, lin-35; fzr-1 double mutants showed extensive tissue hyperproliferation affecting a wide range of cell types. Thus, uncontrolled proliferation in C. elegans can in essence follow the same genetic pattern as multistep carcinogenesis in mammals. Namely, proliferation control is abolished through the sequential loss of genes that function to restrain cell cycle progression (Fay, 2002).

Given these findings, and the large body of evidence implicating Rb in human cancers, it seems reasonable to suggest that this technical approach may facilitate the study of multistep carcinogenesis using C. elegans. Along these lines, it will be interesting to determine whether the human homolog of fzr-1, hCDH1, can function as a tumor-suppressor gene, and if so, whether it does so in cooperation with Rb (Fay, 2002).

Inactivation of fzr-1 function using RNAi injection leads to sterility and aberrancies in germ cell proliferation. Although the specific cause of this phenotype has not been determined, previous studies would implicate defects in either the execution of G1 arrest or in late-stage mitotic events such as cytokinesis. Embryonic lethality is also observed when fzr-1 is inactivated using RNAi injection in a lin-35 mutant background. The cause for this lethality is presently unknown. These embryos do not show obvious hallmarks associated with either excess cellular proliferation or grossly elevated levels of apoptosis. Although additional work will be necessary to determine the nature of this embryonic requirement, a role during embryonic development is consistent with the expression patterns observed for both fzr-1 and lin-35. The lack of an apparent hyperproliferation phenotype in embryos likely reflects significant differences in the means by which embryonic and postembryonic cell cycles are regulated. For example, cyclin D, an upstream regulator of Rb, has been shown to be required exclusively for the execution of postembryonic division cycles in C. elegans (Fay, 2002).

Work carried out over the past several years has produced an explosion in the number of identified SynMuv genes. Although certain functional classifications, such as transcriptional repressors, may accurately describe some of the SynMuv genes, others clearly defy straightforward categorization. This fact alone suggests that SynMuv genes most likely do not all act through the same mechanisms or pathways (Fay, 2002).

Both lin-36 and efl-1(RNAi) can phenocopy the effect of lin-35 LOF in an fzr-1 mutant background. However other class B genes, including lin-53, hda-1, and chd-4, did not show genetic interactions with fzr-1, nor did the class A gene lin-15a. These experiments are complicated by the fact that lin-53, hda-1, and chd-4 encode for essential genes, and RNAi leads to a highly penetrant sterile or lethal phenotype within several generations. Nevertheless, no evidence was seen for hyperproliferation in either the affected or unaffected classes of RNAi-treated animals. This suggests that neither a weak nor a severe reduction in the function of these genes is capable of producing a synthetic hyperproliferation phenotype with fzr-1. In addition, no evidence was found for an interaction in lin-53(n833); fzr-1 double-mutant animals. Although n833 results in only a partial loss of LIN-53, this allele does lead to a highly penetrant Muv phenotype in conjunction with class A SynMuv mutations (Fay, 2002).

Rb and its family members p107 and p130 have been shown in multiple systems to modulate transcription through direct interactions with a variety of transcriptional regulators. The majority of work indicates that Rb serves primarily as a transcriptional repressor, acting through a number of mechanisms including the recruitment of chromatin-modifying enzymes and the steric interference of transactivation domains. Acting as transcriptional corepressors with E2F, Rb and its family members regulate the expression of many key genes required for entry and progression through S-phase, including cyclin E and cyclin A. Consistent with these reports, a significant increase in the levels of ribonucleotide reductase mRNA, an E2F-regulated gene, is seen in lin-35 mutant animals (Fay, 2002).

In Drosophila, loss of fzr function leads to reentry into the cell cycle following embryonic cycle 16, thereby bypassing the normal G1 arrest. This ectopic division cycle is correlated with excess levels of cyclin A, which when overexpressed during G1 can lead to ectopic entry into S-phase. Interestingly, mutations in the Drosophila Rb homolog rbf , as well as in the CDK inhibitor decapo, show cell cycle defects similar to those of fzr mutants, suggesting complementary roles in G1/S-phase regulation. However, it is noted that conclusions regarding fzr functions were inferred from the analysis of a large deletion that removed several genes in addition to fzr. Therefore, fzr-1(ku298) is the first reported mutation in metazoans that specifically reduces CDH1/HCT1/fzr activity (Fay, 2002).

An analysis of distal-tip cell (DTC) hyperproduction in strains that overexpress either cyclin A or cyclin E mRNA supports the model that lin-35 and fzr-1 are likely to coregulate cyclin levels during G1. In addition, the E2F homolog efl-1 synergizes with fzr-1, adding further credence to this model. The ability of both cyclin A and cyclin E to induce extra DTCs in fzr-1 mutants could indicate that these cyclins may be functionally interchangeable and that sufficient levels of either cyclin A or cyclin E, or possibly both in combination, can work to override G1 arrest (Fay, 2002).

By screening ~3500 haploid genomes, seven synthetic with lin-35/Rb (Slr) mutations were uncovered that show synthetic lethality or inviability with mutations in lin-35. Other than fzr-1 and lin-23, no Slr mutations have been identified that produce an obvious synthetic hyperproliferation phenotype with lin-35. The means used to uncover the genetic interaction between lin-35 and fzr-1 will be of general use for those wishing to assign functions to genes lacking known biological roles or to identify novel functions for genes with previously characterized activities. In addition, this genetic approach serves to identify functional copartners through the isolation and cloning of the affected second-site mutations. Importantly, this method in no way depends on prior knowledge of the synthetic double-mutant phenotype, thereby permitting a nonbiased search for genetic modifiers of any gene of interest (Fay, 2002).

Given the inevitable saturation of the genome for mutations that cause easily detectable phenotypes, the ability to identify synthetic mutations will become increasingly important. Large-scale analyses carried out in yeast and C. elegans suggest that a large percentage of genes in higher organisms may fail to show easily discernable phenotypes when mutated. It is likely that the vast majority of these no-phenotype genes may nevertheless confer a weak selective advantage to the organism, thus accounting for their presence in the genome. At the same time, it can be argued that many of these genes fail to show mutational effects owing to genetic redundancy. Importantly, these two explanations are in no way mutually exclusive. By devising methods to experimentally address this latter issue, biological roles may be assignable to many genes that would normally not be amenable to straightforward functional analyses (Fay, 2002).

Identification and expression of Fizzy-related homologs in vertebrates

The anaphase-promoting complex/cyclosome is a tightly cell cycle-regulated ubiquitin-protein ligase that targets cyclin B and other destruction box-containing proteins for proteolysis at the end of mitosis and in G1. Activation of the APC in mitosis depends on CDC20, whereas APC is maintained active in G1 via association with the CDC20-related protein CDH1. The mitotic activator CDC20 is the only component of the APC ubiquitination pathway whose expression is restricted to proliferating cells, whereas the APC and CDH1 are also expressed in several mammalian tissues that predominantly contain differentiated cells, such as adult brain. Immunocytochemical analyses of cultured rat hippocampal neurons and of mouse and human brain sections indicate that the APC and CDH1 are ubiquitously expressed in the nuclei of postmitotic terminally differentiated neurons. The APC purified from brain contains all core subunits known from proliferating cells and is tightly associated with CDH1. Purified brain APC(CDH1) has a high cyclin B ubiquitination activity that depends less on the destruction box than on the activity of mitotic APC(CDC20). On the basis of these results, it is proposed that the functions of APC(CDH1) are not restricted to controlling cell-cycle progression but may include the ubiquitination of yet unidentified substrates in differentiated cells (Gieffers, 1999).

CDC20/CDH1 activates the anaphase-promoting complex and targets various substrates for degradation, thereby allowing the ordered progression through mitosis and G1. Multiple functional CDH1 homologs have been found in the chick. The transcripts of these novel genes are differentially localized to proliferating, differentiated, and postmitotic tissues. All four proteins bind and form a complex with APC in vitro and in cultured cells and have quantitatively different activities in mediating ubiquitination of various APC substrates. These results suggest that multiple CDH1s may temporally and spatially regulate APC activity both within and outside of the cell cycle (Wan, 2001a).

Regulation of APC by Fizzy-related homologs

The cyclosome/APC (anaphase-promoting complex), the major component of cell-cycle-specific ubiquitin-mediated proteolysis of mitotic cyclins and of other cell cycle proteins, is essential for sister chromatid separation and for exit from mitosis. Cyclosome activity and substrate specificity are modulated by phosphorylation and by transient interactions with Fizzy/cdc20 (Fzy) and Fizzy-related/Hct1/Cdh1 (Fzr). This regulation has been studied so far in Drosophila embryos, in yeast, and in cell-free extracts in vitro. Studies of cyclosome regulation in mammalian cells in vivo have shown that both Fzr overexpression and Cdk1 inhibition can override the prometaphase checkpoint. Fzr activation of the cyclosome is negatively regulated by Cdk1. The mammalian cdc14 phosphatase, like its budding yeast homolog, plays a role in cyclosome pathway regulation. These results suggest that Cdk1 is essential for coupling various activities of the cyclosome and in particular for preventing Fzr from short-circuiting the spindle pole checkpoint. Cdk1-cyclin B is thus an inhibitor, activator, and substrate of the cyclosome (Listovsky, 2000).

The ordered activation of the ubiquitin protein ligase anaphase-promoting complex or cyclosome by CDC20 in metaphase and by CDH1 in telophase is essential for anaphase and for exit from mitosis, respectively. CDC20 can only bind to and activate the mitotically phosphorylated form of the Xenopus and the human APC in vitro. In contrast, the analysis of phosphorylated and nonphosphorylated forms of CDC20 suggests that CDC20 phosphorylation is neither sufficient nor required for APC activation. On the basis of these results and the observation that APC phosphorylation correlates with APC activation in vivo, it was proposed that mitotic APC phosphorylation is an important mechanism that controls the proper timing of APC(CDC20) activation. CDH1 is phosphorylated in vivo during S, G2, and M phase, and CDH1 levels fluctuate during the cell cycle. In vitro, phosphorylated CDH1 neither binds to nor activates the APC as efficiently as does nonphosphorylated CDH1. Nonphosphorylatable CDH1 mutants constitutively activate APC in vitro and in vivo, whereas mutants mimicking the phosphorylated form of CDH1 are constitutively inactive. These results suggest that mitotic kinases have antagonistic roles in regulating APC(CDC20) and APC(CDH1); the phosphorylation of APC subunits is required to allow APC activation by CDC20, whereas the phosphorylation of CDH1 prevents activation of the APC by CDH1. These mechanisms can explain the temporal order of APC activation by CDC20 and CDH1 and may help to ensure that exit from mitosis is not initiated before anaphase has occurred (Kramer, 2000).

Cell cycle progression is driven by waves of cyclin expression coupled with regulated protein degradation. An essential step for initiating mitosis is the inactivation of proteolysis mediated by the anaphase-promoting complex/cyclosome bound to its regulator Cdh1p/Hct1p. Yeast APC(Cdh1) has been proposed to be inactivated at Start by G1 cyclin/cyclin-dependent kinase (CDK). In a normal cell cycle APC(Cdh1) is inactivated in a graded manner and is not extinguished until S phase. Complete inactivation of APC(Cdh1) requires S phase cyclins. Further, persistent APC(Cdh1) activity throughout G1 helps to ensure the proper timing of Cdc20p expression. This suggests that S phase cyclins have an important role in allowing the accumulation of mitotic cyclins and further suggests a regulatory loop among S phase cyclins, APC(Cdh1), and APC(Cdc20) (Huang, 2001).

Chromosome segregation and mitotic exit depend on activation of the anaphase-promoting complex (APC) by the substrate adaptor proteins CDC20 and CDH1. The APC is a ubiquitin ligase composed of at least 11 subunits. The interaction of APC2 and APC11 with E2 enzymes is sufficient for ubiquitination reactions, but the functions of most other subunits are unknown. Subcomplexes of the human APC have been biochemically characterized. One subcomplex, containing APC2/11, APC1, APC4, and APC5, can assemble multiubiquitin chains but is unable to bind CDH1 and to ubiquitinate substrates. The other subcomplex contains all known APC subunits except APC2/11. This subcomplex can recruit CDH1 but fails to support any ubiquitination reaction. In vitro, the C termini of CDC20 and CDH1 bind to the closely related TPR subunits APC3 and APC7. Homology modeling predicts that these proteins are similar in structure to the peroxisomal import receptor PEX5, which binds cargo proteins via their C termini. APC activation by CDH1 depends on a conserved C-terminal motif that is also found in CDC20 and APC10. It is concluded that APC1, APC4, and APC5 may connect APC2/11 with TPR subunits. TPR domains in APC3 and APC7 recruit CDH1 to the APC and may thereby bring substrates into close proximity of APC2/11 and E2 enzymes. By analogy to PEX5, the different TPR subunits of the APC might function as receptors that interact with the C termini of regulatory proteins such as CDH1, CDC20, and APC10 (Vodermaier, 2003).

The isolation and characterization of two subcomplexes of the human APC have provided first insight into the molecular interactions between APC's many subunits. The cullin subunit APC2 and its binding partner, the RING finger protein APC11, are found in a subcomplex with APC1, APC4, and APC5 and are essential for the assembly of multiubiquitin chains from ubiquitin residues donated by E2 enzymes. Substrate ubiquitination requires the activator proteins CDH1 and CDC20, which interact via their C termini with the TPR subunits APC3 and APC7. APC's TPR subunits are predicted to form structures that are similar to the one of the peroxisomal import receptor PEX5, which binds cargo proteins via their C termini. The APC may therefore contain multiple TPR subunits to allow modular interactions with different regulatory proteins. These results reveal a function for the TPR subunits of the APC, and they provide insight into how substrates are recruited to the ubiquitin ligase (Vodermaier, 2003).

Protein interactions of Fizzy-related homologs

Exit from mitosis requires the degradation of regulatory proteins including the mitotic cyclins and securin through ubiquitination by the anaphase promoting complex bound to Cdc20 or Cdh1. Cdc20-APC is regulated through inhibition by the spindle assembly checkpoint protein MAD2. Knowledge of Cdh1-APC regulation is limited to the phosphorylation-dependent dissociation of Cdh1 from APC. A novel means of regulating Cdh1 by the MAD2-related gene, MAD2L2, is reported. MAD2L2 specifically binds and inhibits Cdh1-APC, paralleling the effect of MAD2 on Cdc20. It is suggested that MAD2L2 and MAD2 inhibit the release of substrates from APC and a mechanism of inhibition is proposed (Pfleger, 2001a).

The specificity of ubiquitin-mediated protein degradation with regard to the selection of substrates to be polyubiquitinated has only been determined rather recently. Substrate targeting by the N-end rule and HECT (homology to E6AP carboxyl terminus) domain ubiquitin ligases occurs through substrate-specific binding domains. In contrast, the SCF complex recruits substrates through a substrate adaptor protein, the F-box subunit. Despite evidence showing that Cdc20 and Cdh1 bind and activate the anaphase-promoting complex in a substrate-specific manner, there is no evidence that the activating protein and substrate interact directly; hence, no clear model exists for the mechanism of APC activation or recruitment of substrates. The activators Cdc20 and Cdh1 can associate with substrates via their N termini. In the absence of APC, Cdc20 and Cdh1 bind substrates reflecting Cdc20-APC and Cdh1-APC specificity. The N termini of Cdc20 and Cdh1 provide specificity functionally, as demonstrated by the generation of active chimeras that display the specificity corresponding to their N termini. Thus, Cdc20 and Cdh1 act as both substrate recognition and activating modules for APC (Pfleger, 2001b).

Two forms of the anaphase-promoting complex mediate the degradation of critical cell cycle regulators. APC(Cdc20) promotes sister-chromatid separation by ubiquitinating securin, whereas APC(Cdh1) ubiquitinates mitotic cyclins, allowing the exit from mitosis. Phosphorylation of human Cdh1 (hCdh1) by cyclin B-Cdc2 alters the conformation of hCdh1 and prevents it from activating APC. A human homolog of yeast Cdc14, human Cdc14a (hCdc14a), dephosphorylates hCdh1 and activates APC(Cdh1). In contrast, hCdc14a does not affect the activity of APC(Cdc20). hCdc14a is a major phosphatase for hCdh1 and localizes to centrosomes in HeLa cells. Therefore, hCdc14a may promote the activation of APC(Cdh1) and exit from mitosis in mammalian cells (Bembenek, 2001).

The precise order of molecular events during cell cycle progression depends upon ubiquitin-mediated proteolysis of cell cycle regulators. Hsl1p, a protein kinase that inhibits the Swe1p protein kinase in a bud morphogenesis checkpoint, is targeted for ubiquitin-mediated turnover by the anaphase-promoting complex (APC). Regions of Hsl1p that are critical both for binding to the APC machinery and for APC-mediated degradation have been investigated. Hsl1p contains both a destruction box (D box) and a KEN box motif that are necessary for Hsl1p turnover with either APC(Cdc20) or APC(Cdh1). In coimmunoprecipitation studies, the D box of full-length Hsl1p is critical for association with Cdc20p, whereas the KEN box is important for association with Cdh1p. Fusion of a 206-amino-acid fragment of Hsl1p containing these motifs to a heterologous protein results in APC-dependent degradation of the fusion protein that requires intact D box and KEN box motifs. Finally, this bacterially expressed Hsl1p fusion protein interacts with Cdc20p and Cdh1p either translated in vitro or expressed in and purified from insect cells. Binding to Cdc20p and Cdh1p is disrupted completely by a D box/KEN box double mutant. These results indicate that D box and KEN box motifs are important for direct binding to the APC machinery, leading to ubiquitination and subsequent protein degradation (Burton, 2001).

Anaphase-promoting complex (APC), a ubiquitin ligase, controls both sister chromatid separation and mitotic exit. The APC is activated in mitosis and G1 by CDC20 and CDH1, and inhibited by the checkpoint protein MAD2, a specific inhibitor of CDC20. A MAD2 homolog MAD2B also inhibits APC. In contrast to MAD2, MAD2B inhibits both CDH1-APC and CDC20-APC. This inhibition is targeted to CDH1 and CDC20, but not directly to APC. Unlike MAD2, whose interaction with MAD1 is required for mitotic checkpoint control, MAD2B does not interact with MAD1, suggesting that MAD2B may relay a different cellular signal to APC (Chen, 2001).

The anaphase promoting complex/cyclosome (APC/C), activated by fzy and fzr (fizzy and fizzy-related), degrades cell cycle proteins that carry RXXL or KEN destruction boxes (d-boxes). APC/C substrates regulate sequential events and must be degraded in the correct order during mitosis and G1. How d-boxes determine APC/Cfzy/APC/Cfzr specificity and degradation timing was studied. Cyclin B1 has an RXXL box and is degraded by both APC/Cfzy and APC/Cfzr; fzy has a KEN box and is degraded by APC/Cfzr only. The degradation of substrates with swapped d-boxes was characterized. Cyclin B1 with KEN is degraded by APC/Cfzr only. Fzy with RXXL can be degraded by APC/Cfzy and APC/Cfzr. Interestingly, APC/Cfzy-but not APC/Cfzr-specific degradation is highly dependent on the location of RXXL. Degradation of tagged substrates was studied in real time and it was observed that APC/Cfzr is activated in early G1. These observations demonstrate how d-box specificities of APC/Cfzy and APC/Cfzr, and the successive activation of APC/Cby fzy and fzr, establish the temporal degradation pattern. These observations can explain further why some endogenous RXXL substrates are degraded by APC/Cfzy, while others are restricted to APC/Cfzr (Zur, 2002).

More than a dozen different groups of proteins are degraded by the APC/C pathway, including mitotic A and B type cyclins, fzy, securin, E2-C, polo kinase, nek2A, hsl1, cdc6 and geminin. While all these proteins are degraded by the APC/C, they start to be degraded at different time points, such as prometaphase for cyclin A and nek2A, metaphase for cyclin B1, securin and xkid, and G1 for cdc6. APC/C substrates carry conserved motifs, so-called destruction boxes (d-boxes), which are required for their degradation. The cyclin B1 d-box (RTALGDIGN) is crudely shared by many of the other APC/C substrates. The arginine (R) and the leucine (L) are conserved in almost all substrates except in pim1, where arginine is replaced by lysine, and in cyclin B3, where leucine is replaced by phenylalanine. The asparagine (N) at position 9 is conserved in a subset of substrates and is required for the degradation of cyclin B1 in Xenopus extracts. Other residues of this RXXL box are much less conserved and it is virtually impossible to identify such a box merely by its sequence. However, the APC/C is evidently able to identify real RXXL boxes because not every protein that carries an RXXL is degraded. Moreover, fine differences in this box can contribute to changes in degradation, as is the case for cyclins A and B. An important recent advance is the identification of the KEN box as a targeting signal of some APC/C substrates. The discovery of this motif explained how vertebrate fzy, which lacks an RXXL box, is targeted for degradation by the APC/C. This box also plays a role in the degradation of substrates that do have an RXXL box, such as securin, clb2, hsl1 and nek2A. However, the KEN motif is also abundant in many proteins that are not APC/C substrates (Zur, 2002 and references therein).

The APC/C is activated by two WD repeat proteins: fzy/cdc20 and fzr/cdh1/hct1. In yeast, these two proteins confer some substrate specificity on the APC/C: pds1 is ubiquitylated by APC/Ccdc20, and clb2 by APC/Ccdh1. A similar specificity has been suggested in mammalian cells, and it was shown that fzy is ubiquitylated by APC/Cfzr only. Fzy and fzr directly bind different APC/C substrates in vitro. Fzy is restricted to substrates that have an RXXL box, and fzr binds both RXXL and KEN box substrates (Zur, 2002 and references therein).

In order to study the signal specificity of the RXXL and KEN boxes, artificial motifs were inserted into known substrates and their degradation was studied in vivo. The degradation of cyclin B1 with a mutated RXXL box can be restored by the insertion of an artificial KEN box close to the N-terminus of the mutated RXXL box. The location of this KEN is critical for its capacity to support degradation. Strikingly, cyclin B1 with a KEN box and a mutated RXXL box is ubiquitylated in vitro and degraded in vivo by APC/Cfzr only. This is in contrast to cyclin B1 with a wild-type RXXL box, which is degraded by both APC/Cfzy and APC/Cfzr (Zur, 2002).

The degradation of fzy, which is targeted for degradation by a KEN box and is an APC/Cfzr-specific substrate, was studied. Mutation of the KEN box stabilizes fzy, and its degradation can be restored by the insertion of an RXXL box. Following the replacement of KEN with RXXL, fzy is targeted by APC/Cfzy, as well as by APC/Cfzr. RXXL inserted anywhere into the N-terminus of fzy can support APC/Cfzr-specific degradation. Degradation by APC/Cfzy is, however, highly dependent on the location of the RXXL, suggesting that flanking sequences or conformation influence the APC/Cfzr/APC/Cfzy specificity of the RXXL box. This could explain why certain RXXL substrates are degraded by APC/Cfzr only while others are degraded by both APC/Cfzr and APC/Cfzy (Zur, 2002)

The degradation of green fluorescent protein (GFP)-tagged versions of APC/Cfzy and APC/Cfzr-specific substrates was studied in real time. APC/Cfzy-specific degradation starts upon sister chromatid separation, and APC/Cfzr-specific degradation starts in early G1 (Zur, 2002).

These results show that d-box type and location determine APC/Cfzy and APC/Cfzr specificity, and that fzy and fzr sequentially activate the APC/C. This specificity could thus form the basis of the ordered degradation of APC/C substrates during the different stages of mitosis and G1 (Zur, 2002).

Fizzy-related homologs and the degradation of cyclins

Cyclin A is a stable protein in S and G2 phases, but is destabilized when cells enter mitosis and it is almost completely degraded before the metaphase to anaphase transition. Microinjection of antibodies against subunits of the anaphase-promoting complex/cyclosome or against human Cdc20 (fizzy) arrests cells at metaphase and stabilizes both cyclins A and B1. Cyclin A is efficiently polyubiquitylated by Cdc20 or Cdh1-activated APC/C in vitro, but in contrast to cyclin B1, the proteolysis of cyclin A is not delayed by the spindle assembly checkpoint. The degradation of cyclin B1 is accelerated by inhibition of the spindle assembly checkpoint. These data suggest that the APC/C is activated as cells enter mitosis and immediately targets cyclin A for degradation, whereas the spindle assembly checkpoint delays the degradation of cyclin B1 until the metaphase to anaphase transition. The 'destruction box' (D-box) of cyclin A is 10-20 residues longer than that of cyclin B. Overexpression of wild-type cyclin A delays the metaphase to anaphase transition, whereas expression of cyclin A mutants lacking a D-box arrest cells in anaphase (Geley, 2001).

Within the mammalian ovary, oocytes remain arrested at G2 for several years. Then a peri-ovulatory hormonal cue triggers meiotic resumption by releasing an inhibitory phosphorylation on the kinase Cdk1. G2 arrest, however, also requires control in the concentrations of the Cdk1-binding partner cyclin B1, a process achieved by anaphase-promoting complex (APCCdh1) activity, which ubiquitylates and so targets cyclin B1 for degradation. Thus, APCCdh1 activity prevents precocious meiotic entry by promoting cyclin B1 degradation. However, it remains unresolved how cyclin B1 levels are suppressed sufficiently to maintain arrest but not so low that they make oocytes hormonally insensitive. This study examined spatial control of this process by determining the intracellular location of the proteins involved and using nuclear-targeted cyclin B1. It was found that raising nuclear cyclin B1 concentrations, an event normally observed in the minutes before nuclear envelope breakdown, was a very effective method of inducing the G2/M transition. Oocytes expressed only the alpha-isoform of Cdh1, which was predominantly nuclear, as were Cdc27 and Psmd11, core components of the APC and the 26S proteasome, respectively. Furthermore, APCCdh1 activity appeared higher in the nucleus, as nuclear-targeted cyclin B1 was degraded at twice the rate of wild-type cyclin B1. A simple spatial model of G2 arrest is proposed in which nuclear APCCdh1-proteasomal activity guards against any cyclin B1 accumulation mediated by nuclear import (Holt, 2010).

The APC/C activator FZR1 is essential for meiotic prophase I in mice

Fizzy-related 1 (FZR1) is an activator of the Anaphase promoting complex/cyclosome (APC/C) and an important regulator of the mitotic cell division cycle. Using a germ-cell-specific conditional knockout model this study examined its role in entry into meiosis and early meiotic events in both sexes. Loss of APC/C(FZR1) activity in the male germline led to both a mitotic and a meiotic testicular defect resulting in infertility due to the absence of mature spermatozoa. Spermatogonia in the prepubertal testes of such mice had abnormal proliferation and delayed entry into meiosis. Although early recombination events were initiated, male germ cells failed to progress beyond zygotene and underwent apoptosis. Loss of APC/C(FZR1) activity was associated with raised cyclin B1 levels, suggesting that CDK1 may trigger apoptosis. By contrast, female FZR1Delta mice were subfertile, with premature onset of ovarian failure by 5 months of age. Germ cell loss occurred embryonically in the ovary, around the time of the zygotene-pachytene transition, similar to that observed in males. In addition, the transition of primordial follicles into the growing follicle pool in the neonatal ovary was abnormal, such that the primordial follicles were prematurely depleted. It is concluded that APC/C(FZR1) is an essential regulator of spermatogonial proliferation and early meiotic prophase I in both male and female germ cells and is therefore important in establishing the reproductive health of adult male and female mammals (Holt, 2014).

Vertebrate Fizzy-related homologs and mitotic checkpoints

The ubiquitination and degradation patterns of the human securin/PTTG protein have been studied. In contrast to budding yeast pds1, securin degradation is catalyzed by both fzy (fizzy/cdc20) and fzr (fizzy-related/cdh1/hct1). Both fzy and fzr also induce the APC/C to ubiquitinate securin in vitro. Securin degradation is mediated by an RXXL destruction box and a KEN box, and is inhibited only when both sequences are mutated. Interestingly, the non-degradable securin mutant is also partially ubiquitinated by fzy and fzr in vitro. Expressing the non-degradable securin mutant in cells frequently results in incomplete chromatid separation and gives rise to daughter cells connected by a thin chromatin fiber, presumably of chromosomes that failed to split completely. Strikingly, the mutant securin does not prevent the majority of sister chromatids from separating completely, nor does it prevent mitotic cyclin degradation and cytokinesis. This phenotype, reminiscent of the fission yeast cut (cells untimely torn) phenotype, is reported here for the first time in mammals (Zur, 2001).

The spindle checkpoint is a cell cycle surveillance mechanism that ensures the fidelity of chromosome segregation during mitosis and meiosis. Bub1 is a protein serine-threonine kinase that plays multiple roles in chromosome segregation and the spindle checkpoint. In response to misaligned chromosomes, Bub1 directly inhibits the ubiquitin ligase activity of the anaphase-promoting complex or cyclosome (APC/C) by phosphorylating its activator Cdc20. The protein level and the kinase activity of Bub1 are regulated during the cell cycle; they peak in mitosis and are low in G1/S phase. Bub1 is degraded during mitotic exit and degradation of Bub1 is mediated by APC/C in complex with its activator Cdh1 [APC/C(Cdh1)]. Overexpression of Cdh1 reduces the protein levels of ectopically expressed Bub1, whereas depletion of Cdh1 by RNA interference increases the level of the endogenous Bub1 protein. Bub1 is ubiquitinated by immunopurified APC/C(Cdh1) in vitro. Two KEN-box motifs on Bub1 were identified that are required for its degradation in vivo and ubiquitination in vitro. A Bub1 mutant protein with both KEN-boxes mutated is stable in cells but fails to elicit a cell cycle phenotype, indicating that degradation of Bub1 by APC/C(Cdh1) is not required for mitotic exit. Nevertheless, this study clearly demonstrates that Bub1, an APC/C inhibitor, is also an APC/C substrate. The antagonistic relationship between Bub1 and APC/C may help to prevent the premature accumulation of Bub1 during G1 (Qi, 2007).

DNA damage checkpoint prevents segregation of damaged chromosomes by imposing cell-cycle arrest. In budding yeast, Mec1, Chk1, and Rad53 (homologous to human ATM/ATR, Chk1, and Chk2 kinases, respectively) are among the main effectors of this pathway. The DNA damage checkpoint is thought to inhibit chromosome segregation by preventing separase-mediated cleavage of cohesins. This study describes a regulatory network that prevents segregation of damaged chromosomes by restricting spindle elongation and acts in parallel with inhibition of cohesin cleavage. This control circuit involves Rad53, polo kinase, the anaphase-promoting complex activator Cdh1, and the bimC kinesin family proteins Cin8 and Kip1. The inhibition of polo kinase by Rad53-dependent phosphorylation prevents it from inactivating Cdh1. As a result, Cdh1 remains in a partially active state and limits Cin8 and Kip1 accumulation, thereby restraining spindle elongation. Hence, the DNA damage checkpoint suppresses both cohesin cleavage and spindle elongation to preserve chromosome stability (Zhang, 2009).

Fizzy-related homologs function in G1: Targets of the proteosome

In mammalian somatic-cell cycles, progression through the G1-phase restriction point and initiation of DNA replication are controlled by the ability of the retinoblastoma tumor-suppressor protein (pRb) family to regulate the E2F/DP transcription factors. Continuing transcription of E2F target genes beyond the G1/S transition is required for coordinating S-phase progression with cell division, a process driven by cyclin-B-dependent kinase and anaphase-promoting complex (APC)-mediated proteolysis. How E2F-dependent events at G1/S transition are orchestrated with cyclin B and APC activity remains unknown. Using an in vivo assay to measure protein stability in real time during the cell cycle, it has been shown that repression of E2F activity or inhibition of cyclin-A-dependent kinase in S phase triggers the destruction of cyclin B1 through the re-assembly of APC, the ubiquitin ligase that is essential for mitotic cyclin proteolysis, with its activatory subunit Cdh1. Phosphorylation-deficient mutant Cdh1 or immunodepletion of cyclin A results in assembly of active Cdh1-APC even in S-phase cells. These results implicate an E2F-dependent, cyclin A/Cdk2-mediated phosphorylation of Cdh1 in the timely accumulation of cyclin B1 and the coordination of cell-cycle progression during the post-restriction point period (Lukas, 1999).

The ordered progression through the cell cycle depends on regulating the abundance of several proteins through ubiquitin-mediated proteolysis. Degradation is precisely timed and specific. One key component of the degradation system, the anaphase promoting complex (APC), is a ubiquitin protein ligase. It is activated both during mitosis and late in mitosis/G1, by the WD repeat proteins Cdc20 and Cdh1, respectively. These activators target distinct sets of substrates. Cdc20-APC requires a well-defined destruction box (D box), whereas Cdh1-APC confers a different and as yet unidentified specificity. The sequence specificity for Cdh1-APC has been determined using two assays, ubiquitination in a completely defined and purified system and degradation promoted by Cdh1-APC in Xenopus extracts. Cdc20 is itself a Cdh1-APC substrate. Vertebrate Cdc20 lacks a D box and therefore is recognized by Cdh1-APC through a different sequence. By analysis of Cdc20 as a substrate, a new recognition signal has been identified. This signal, composed of K-E-N, serves as a general targeting signal for Cdh1-APC. Like the D box, it is transposable to other proteins. Using the KEN box as a template, cell cycle genes Nek2 and B99 have been identified as additional Cdh1-APC substrates. Mutation in the KEN box stabilizes all three proteins against ubiquitination and degradation (Pfleger, 2000).

Ubiquitin-proteasome-mediated destruction of rate-limiting proteins is required for timely progression through the main cell cycle transitions. The anaphase-promoting complex (APC), periodically activated by the Cdh1 subunit, represents one of the major cellular ubiquitin ligases which, in Saccharomyces cerevisiae and Drosophila, triggers exit from mitosis and during G1 prevents unscheduled DNA replication. In this study the importance of periodic oscillation of the APC-Cdh1 activity for the cell cycle progression in human cells was investigated. Conditional interference with the APC-Cdh1 dissociation at the G1/S transition results in an inability to accumulate a surprisingly broad range of critical mitotic regulators including cyclin B1, cyclin A, Plk1, Pds1, mitosin (CENP-F), Aim1, and Cdc20. Unexpectedly, although constitutively assembled APC-Cdh1 also delays G1/S transition and lowers the rate of DNA synthesis during S phase, some of the activities essential for DNA replication became markedly amplified, mainly due to a progressive increase of E2F-dependent cyclin E transcription and a rapid turnover of the p27(Kip1) cyclin-dependent kinase inhibitor. Consequently, failure to inactivate APC-Cdh1 beyond the G1/S transition not only inhibits productive cell division but also supports slow but uninterrupted DNA replication, precluding S-phase exit and causing massive overreplication of the genome. These data suggest that timely oscillation of the APC-Cdh1 ubiquitin ligase activity represents an essential step in coordinating DNA replication with cell division and that failure of mechanisms regulating association of APC with the Cdh1 activating subunit can undermine genomic stability in mammalian cells (Sorensen, 2000).

CDC6 is conserved during evolution and is essential and limiting for the initiation of eukaryotic DNA replication. Human CDC6 activity is regulated by periodic transcription and CDK-regulated subcellular localization. In addition to being absent from nonproliferating cells, CDC6 is targeted for ubiquitin-mediated proteolysis by the anaphase promoting complex (APC)/cyclosome in G1. A combination of point mutations in the destruction box and KEN-box motifs in CDC6 stabilizes the protein in G1 and in quiescent cells. Furthermore, APC, in association with CDH1, ubiquitinates CDC6 in vitro, and both APC and CDH1 are required and limiting for CDC6 proteolysis in vivo. Although a stable mutant of CDC6 is biologically active, overexpression of this mutant or wild-type CDC6 is not sufficient to induce multiple rounds of DNA replication in the same cell cycle. The APC-CDH1-dependent proteolysis of CDC6 in early G1 and in quiescent cells suggests that this process is part of a mechanism that ensures the timely licensing of replication origins during G1 (Petersen, 2000).

Progress through mitosis is controlled by the sequential destruction of key regulators including the mitotic cyclins and securin, an inhibitor of anaphase whose destruction is required for sister chromatid separation. Live cell imaging was used to determine the exact time when human securin is degraded in mitosis. The timing of securin destruction is set by the spindle checkpoint; securin destruction begins at metaphase once the checkpoint is satisfied. Furthermore, reimposing the checkpoint rapidly inactivates securin destruction. Thus, securin and cyclin B1 destruction have very similar properties. Moreover, both cyclin B1 and securin have to be degraded before sister chromatids can separate. A mutant form of securin that lacks its destruction box (D-box) is still degraded in mitosis, but now this is in anaphase. This destruction requires a KEN box in the NH2 terminus of securin and may indicate the time in mitosis when ubiquitination switches from APCCdc20 to APCCdh1. Lastly, a D-box mutant of securin that cannot be degraded in metaphase inhibits sister chromatid separation, generating a cut phenotype where one cell can inherit both copies of the genome. Thus, defects in securin destruction alter chromosome segregation and may be relevant to the development of aneuploidy in cancer (Hagting, 2002).

Interaction of Cdh1 with cyclin A-Cdk2

Periodic activity of the anaphase-promoting complex (APC) ubiquitin ligase determines progression through multiple cell cycle transitions by targeting cell cycle regulators for destruction. At the G(1)/S transition, phosphorylation-dependent dissociation of the Cdh1-activating subunit inhibits the APC, allowing stabilization of proteins required for subsequent cell cycle progression. Cyclin-dependent kinases (CDKs) that initiate and maintain Cdh1 phosphorylation have been identified. However, the issue of which cyclin-CDK complexes are involved has been a matter of debate, and the mechanism of how cyclin-CDKs interact with APC subunits remains unresolved. This study substantiates the evidence that mammalian cyclin A-Cdk2 prevents unscheduled APC reactivation during S phase by demonstrating the Cdk2 periodic interaction with Cdh1 at the level of endogenous proteins. Moreover, a conserved cyclin-binding motif has been identified within the Cdh1 WD-40 domain; its disruption abolishes the Cdh1-cyclin A-Cdk2 interaction, eliminates Cdh1-associated histone H1 kinase activity, and impairs Cdh1 phosphorylation by cyclin A-Cdk2 in vitro and in vivo. Overexpression of cyclin binding-deficient Cdh1 stabilizes the APC-Cdh1 interaction and induces prolonged cell cycle arrest at the G(1)/S transition. Conversely, cyclin binding-deficient Cdh1 loses its capability to support APC-dependent proteolysis of cyclin A but not that of other APC substrates such as cyclin B and securin Pds1. Collectively, these data provide a mechanistic explanation for the mutual functional interplay between cyclin A-Cdk2 and APC-Cdh1 and the first evidence that Cdh1 may activate the APC by binding specific substrates (Sorensen, 2001).

The anaphase-promoting complex (APC) coordinates mitosis and G1 by sequentially promoting the degradation of key cell-cycle regulators. Following the degradation of its substrates in G1, the APC catalyzes the autoubiquitination of its E2 UbcH10. This stabilizes cyclin A and allows it to inactivate APC(Cdh1). How the APC establishes this complex temporal sequence of ubiquitinations, referred to as substrate ordering, is not understood. This study shows that substrate ordering depends on the relative processivity of substrate multiubiquitination by the APC. Processive substrates obtain ubiquitin chains in a single APC binding event. The multiubiquitination of distributive substrates requires multiple rounds of APC binding, which render it sensitive to lower APC concentrations, competition by processive substrates, and deubiquitination. Consequently, more processive substrates are preferentially multiubiquitinated in vitro and degraded earlier in vivo. The processivity of multiubiquitination is strongly influenced by the D box within the substrate, suggesting that substrate ordering is established by a mechanism intrinsic to APC and its substrates and similar to kinetic proofreading (Rape, 2006).

APC/C Cdh1 targets aurora kinase to control reorganization of the mitotic spindle at anaphase

Control of mitotic cell cycles by the anaphase-promoting complex or cyclosome (APC/C) ubiquitin ligase depends on its coactivators Cdc20 and Cdh1. APC/C(Cdc20) is active during mitosis and promotes anaphase onset by targeting mitotic cyclins and securin. APC/C(Cdh1) becomes active during mitotic exit and has essential targets in G1 phase. It is not known whether targeting of substrates by APC/C(Cdh1) plays any role in the final stages of mitosis. This study has investigated the role of APC/C(Cdh1) at this time in the cell cycle by using siRNA-mediated depletion of Cdh1 in human cells. In contrast to the current view that Cdh1 takes over from Cdc20 at anaphase, it was shown that reduced Cdh1 levels have no effect on destruction of many APC/C substrates during mitotic exit but strongly and specifically stabilize Aurora kinases. APC/C(Cdh1) is required for assembly of a robust spindle midzone at anaphase and for normal timings of spindle elongation and cytokinesis. The effect of Cdh1 siRNA on anaphase spindle dynamics requires Aurora A, and its effect can be mimicked by nondegradable Aurora kinase. It is concluded that targeting of Aurora kinases at anaphase by APC/C(Cdh1) participates in the control of mitotic exit and cytokinesis (Floyd, 2008).

Visualizing developmentally programmed endoreplication in mammals using ubiquitin oscillators

The majority of mammalian somatic cells maintain a diploid genome. However, some mammalian cell types undergo multiple rounds of genome replication (endoreplication) as part of normal development and differentiation. For example, trophoblast giant cells (TGCs) in the placenta become polyploid through endoreduplication (bypassed mitosis), and megakaryocytes (MKCs) in the bone marrow become polyploid through endomitosis (abortive mitosis). During the normal mitotic cell cycle, geminin and Cdt1 are involved in 'licensing' of replication origins, which ensures that replication occurs only once in a cell cycle. Their protein accumulation is directly regulated by two E3 ubiquitin ligase activities, APCCdh1 and SCFSkp2, which oscillate reciprocally during the cell cycle. Although proteolysis-mediated, oscillatory accumulation of proteins has been documented in endoreplicating Drosophila cells, it is not known whether the ubiquitin oscillators that control normal cell cycle transitions also function during mammalian endoreplication. In this study, transgenic mice were used expressing Fucci fluorescent cell-cycle probes that report the activity of APCCdh1 and SCFSkp2. By performing long-term, high temporal-resolution Fucci imaging, it was possible to visualize reciprocal activation of APCCdh1 and SCFSkp2 in differentiating TGCs and MKCs grown in custom-designed culture wells. TGCs and MKCs were found to both skip cytokinesis, but in different ways, and that the reciprocal activation of the ubiquitin oscillators in MKCs varies with the polyploidy level. Three-dimensional reconstructions were obtained of highly polyploid TGCs in whole, fixed mouse placentas. Thus, the Fucci technique is able to reveal the spatiotemporal regulation of the endoreplicative cell cycle during differentiation (Sakaue-Sawano, 2013).

Mutation of Fizzy-related homologs

Anaphase-promoting complex is activated by two regulatory proteins, Cdc20 and Cdh1. In yeast and Drosophila, Cdh1-dependent APC (Cdh1-APC) activity targets mitotic cyclins from the end of mitosis to the G1 phase. To investigate the function of Cdh1 in vertebrate cells, clones of chicken DT40 cells disrupted in their Cdh1 loci were generated. Cdh1 is dispensable for viability and cell cycle progression. However, similarly to yeast and Drosophila, loss of Cdh1 induced unscheduled accumulation of mitotic cyclins in G1, resulting in abrogation of G1 arrest caused by treatment with rapamycin, an inducer of p27(Kip1). Cdh1(-/-) cells fail to maintain DNA damage-induced G2 arrest and Cdh1-APC is activated by X-irradiation-induced DNA damage. Thus, activation of Cdh1-APC plays a crucial role in both cdk inhibitor-dependent G1 arrest and DNA damage-induced G2 arrest (Sudo, 2001).

Fizzy-related homologs and TGF-beta signaling

Degradation of SnoN is thought to play an important role in the transactivation of TGF-beta responsive genes. The anaphase-promoting complex (APC) is a ubiquitin ligase required for the destruction of SnoN and the APC pathway is regulated by TGF-beta. The destruction box of SnoN is required for its degradation in response to TGF-beta signaling. Furthermore, the APC activator CDH1 and Smad3 synergistically regulate SnoN degradation. Under these circumstances, CDH1 forms a quaternary complex with SnoN, Smad3, and APC. These results suggest that APC(CDH1) and SnoN play central roles in regulating growth through the TGF-beta signaling system (Wan, 2001b).

AML1/RUNX1 phosphorylation by cyclin-dependent kinases regulates the degradation of AML1/RUNX1 by the anaphase-promoting complex

AML1 (RUNX1) regulates hematopoiesis, angiogenesis, muscle function, and neurogenesis. Previous studies have shown that phosphorylation of AML1, particularly at serines 276 and 303, affects its transcriptional activation. Phosphorylation of AML1 serines 276 and 303 can be blocked in vivo by inhibitors of the cyclin-dependent kinases (CDKs) Cdk1 and Cdk2. Furthermore, these residues can be phosphorylated in vitro by purified Cdk1/cyclin B and Cdk2/cyclin A. Mutant AML1 protein that cannot be phosphorylated at these sites (AML1-4A) is more stable than wild-type AML1. AML-4A is resistant to degradation mediated by Cdc20, one of the substrate-targeting subunits of the anaphase-promoting complex (APC). However, Cdh1, another targeting subunit used by the APC, can mediate the degradation of AML1-4A. A phospho-mimic protein, AML1-4D, can be targeted by Cdc20 or Cdh1. These observations suggest that both Cdc20 and Cdh1 can target AML1 for degradation by the APC but that AML1 phosphorylation may affect degradation mediated by Cdc20-APC to a greater degree (Biggs, 2006).

The Cdc14B-Cdh1-Plk1 axis controls the G2 DNA-damage-response checkpoint

In response to DNA damage in G2, mammalian cells must avoid entry into mitosis and instead initiate DNA repair. This study shows that in response to genotoxic stress in G2, the phosphatase Cdc14B translocates from the nucleolus to the nucleoplasm and induces the activation of the ubiquitin ligase APC/CCdh1, with the consequent degradation of Plk1, a prominent mitotic kinase. This process induces the stabilization of Claspin, an activator of the DNA-damage checkpoint, and Wee1, an inhibitor of cell-cycle progression, and allows an efficient G2 checkpoint. As a by-product of APC/CCdh1 reactivation in DNA-damaged G2 cells, Claspin, which is shown in this study to be an APC/CCdh1 substrate in G1, is targeted for degradation. However, this process is counteracted by the deubiquitylating enzyme Usp28 to permit Claspin-mediated activation of Chk1 in response to DNA damage. These findings define a novel pathway that is crucial for the G2 DNA-damage-response checkpoint (Bassermann, 2008).

Irreversible APC(Cdh1) inactivation underlies the point of no return for cell-cycle entry

Proliferating cells must cross a point of no return before they replicate their DNA and divide. This commitment decision plays a fundamental role in cancer and degenerative diseases and has been proposed to be mediated by phosphorylation of retinoblastoma (Rb; see Drosophila Rb) protein. This study shows that inactivation of the anaphase-promoting complex/cyclosome (APC(Cdh1)) in a cultured mammalian epithelial cell line has the necessary characteristics to be the point of no return for cell-cycle entry. APC(Cdh1) inactivation is shown to be a rapid, bistable switch initiated shortly before the start of DNA replication by cyclin E/Cdk2 (see Drosophila Cyclin E) and made irreversible by Emi1 (see Drosophila Rca1). Exposure to stress between Rb phosphorylation and APC(Cdh1) inactivation, but not after APC(Cdh1) inactivation, reverted cells to a mitogen-sensitive quiescent state, from which they can later re-enter the cell cycle. Thus, APC(Cdh1) inactivation is the commitment point when cells lose the ability to return to quiescence and decide to progress through the cell cycle (Cappell, 2016).

APC/CCdh1 enables removal of Shugoshin-2 from the arms of bivalent chromosomes by moderating Cyclin-dependent kinase activity

In mammalian females, germ cells remain arrested as primordial follicles. Resumption of meiosis is heralded by germinal vesicle breakdown, condensation of chromosomes, and their eventual alignment on metaphase plates. At the first meiotic division, anaphase-promoting complex/cyclosome associated with Cdc20 (APC/CCdc20; see Drosophila Cdc20) activates separase (see Drosophila Separase) and thereby destroys cohesion along chromosome arms. Because cohesion around centromeres is protected by shugoshin-2 (see Drosophila mei-S332), sister chromatids remain attached through centromeric/pericentromeric cohesin. This study shows that, by promoting proteolysis of cyclins and Cdc25B (see Drosophila String) at the germinal vesicle (GV) stage, APC/C associated with the Cdh1 protein (APC/CCdh1; see Drosophila Fizzy-related) delays the increase in Cdk1 (see Drosophila Cdk2) activity, leading to germinal vesicle breakdown (GVBD). More surprisingly, by moderating the rate at which Cdk1 is activated following GVBD, APC/CCdh1 creates conditions necessary for the removal of shugoshin-2 from chromosome arms by the Aurora B/C kinase (see Drosophila Aurora B), an event crucial for the efficient resolution of chiasmata (Rattani, 2017).

EMI1 switches from being a substrate to an inhibitor of APC/C(CDH1) to start the cell cycle

Mammalian cells integrate mitogen and stress signalling before the end of G1 phase to determine whether or not they enter the cell cycle. Before cells can replicate their DNA in S phase, they have to activate cyclin-dependent kinases (CDKs), induce an E2F transcription program and inactivate the anaphase-promoting complex (APC/C(CDH1), also known as the cyclosome), which is an E3 ubiquitin ligase that contains the co-activator CDH1 (also known as FZR, encoded by FZR1; see Drosophila Fizzy related). It was recently shown that stress can return cells to quiescence after CDK2 activation and E2F induction but not after inactivation of APC/C(CDH1), which suggests that APC/C(CDH1) inactivation is the point of no return for cell-cycle entry. Rapid inactivation of APC/C(CDH1) requires early mitotic inhibitor 1 (EMI1; see Drosophila Regulator of cyclin A1), but the molecular mechanism that controls this cell-cycle commitment step is unknown. This study shows using human cell models that cell-cycle commitment is mediated by an EMI1-APC/C(CDH1) dual-negative feedback switch, in which EMI1 is both a substrate and an inhibitor of APC/C(CDH1). The inactivation switch triggers a transition between a state with low EMI1 levels and high APC/C(CDH1) activity during G1 and a state with high EMI1 levels and low APC/C(CDH1) activity during S and G2. Cell-based analysis, in vitro reconstitution and modelling data show that the underlying dual-negative feedback is bistable and represents a robust irreversible switch. This study suggests that mammalian cells commit to the cell cycle by increasing CDK2 activity and EMI1 mRNA expression to trigger a one-way APC/C(CDH1) inactivation switch that is mediated by EMI1 transitioning from acting as a substrate of APC/C(CDH1) to being an inhibitor of APC/C(CDH1) (Cappell, 2018).

De novo FZR1 loss-of-function variants cause developmental and epileptic encephalopathies

FZR1, which encodes the Cdh1 subunit of the Anaphase Promoting Complex, plays an important role in neurodevelopment by regulating the cell cycle and by its multiple post-mitotic functions in neurons. In this study, evaluation of 250 unrelated patients with developmental and epileptic encephalopathies and a connection on GeneMatcher led to the identification of three de novo missense variants in FZR1. Functional studies in Drosophila were performed using three different mutant alleles of the Drosophila homolog of FZR1 fzr. All three individuals carrying de novo variants in FZR1 had childhood onset generalized epilepsy, intellectual disability, mild ataxia and normal head circumference. Two individuals were diagnosed with the developmental and epileptic encephalopathy subtype Myoclonic Atonic Epilepsy. Functional evidence is provided that the missense variants are loss-of-function alleles using Drosophila neurodevelopment assays. Using three fly mutant alleles of the Drosophila homolog fzr and overexpression studies, it was shown that patient variants can affect proper neurodevelopment. This study consolidates the relationship between FZR1 and developmental epileptic encephalopathy, and expands the associated phenotype. It is concluded that heterozygous loss-of-function of FZR1 leads to developmental epileptic encephalopathies associated with a spectrum of neonatal to childhood onset seizure types, developmental delay and mild ataxia. In summary, this approach of targeted sequencing using novel gene candidates and functional testing in Drosophila will help solve undiagnosed myoclonic atonic epilepsy or developmental epileptic encephalopathy cases (Manivannan, 2021).

retina aberrant in pattern/fizzy-related: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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