InteractiveFly: GeneBrief

vihar: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References


Gene name - vihar

Synonyms -

Cytological map position - 69C4

Function - enzyme

Keywords - protein degradation, mitosis, cell cycle

Symbol - vihar

FlyBase ID: FBgn0264848

Genetic map position - 3L

Classification - E2 ubiquitin-conjugating enzyme

Cellular location - cytoplasmic



NCBI links: Precomputed BLAST | Entree Gene
BIOLOGICAL OVERVIEW

Proteolytic degradation of mitotic regulatory proteins first requires these targets to be ubiquitinated. This is regulated at the level of conjugation of ubiquitin to substrates by the anaphase-promoting complex/cyclosome (APC/C) ubiquitin-protein ligase. Substrate specificity and temporal activity of the APC/C has been thought to lie primarily with its two activators, Cdc20/Fizzy and Cdh1/Fizzy-related. Reduction in the E2 ubiquitin-conjugating enzyme (UBC) of the E2-C family that is encoded by the Drosophila gene vihar (vih), by either mutation or RNAi, leads to an accumulation of cells in a metaphase-like state. Cyclin B accumulates to high levels in all mitotic vih cells, particularly at the spindle poles. Vihar E2-C is present in the cytoplasm of mitotic cells but also associates with centrosomes, and its own degradation is initiated at the metaphase-anaphase transition. Expression of destruction D box mutants of vihar in the syncytial embryo results in mitotic arrest at late anaphase. In contrast to hypomorphic mutants, Cyclin B is degraded at the spindle poles and accumulates in the equatorial region of the spindle. It is concluded that in Drosophila, the Vihar E2 UBC contributes to the spatiotemporal control of Cyclin B degradation that first occurs at the spindle poles. APC/C-mediated proteolysis of Vihar E2-C autoinactivates the APC/C at the centrosome before a second wave of proteolysis to degrade Cyclin B on the rest of the spindle and elsewhere in the cell (Mathe, 2004).

The ordered progression of cells through the division cycle is brought about by periodic series of protein modification events that involve cycles of phosphorylation that are mediated by multiple protein kinases and cycles of ubiquitination that lead to the periodic degradation of specific regulatory proteins. The ubiquitination of target proteins is achieved by three enzymes. The first ATP-dependent step is the activation of ubiquitin by the formation of a thio-ester bond between its C terminus and a cysteine residue in the activating enzyme E1 itself. In the second step, the ubiquitin is transferred as a thio-ester to a cysteine residue in a ubiquitin-conjugating enzyme (UBC) E2. In cooperation with an ubiquitin protein ligase, E3, the ubiquitin residue is then transferred to a lysine residue in the target protein. Polyubiquitinated proteins are targeted to the proteasome for their destruction. Proteolytic degradation is controlled in the cell cycle by two major classes of E3 enzyme: the Skp1 protein, Cullin, and F box (SCF) complex, which is required for the G1-S transition, and the anaphase-promoting complex/cyclosome (APC/C), which is functional during mitosis and G1. The APC/C catalyzes the ubiquitination of securin, an inhibitor of the protease that cleaves chromosome cohesion proteins. It is also responsible for degradation of the mitotic cyclins and of other regulatory molecules. The complex is comprised of 12 to 13 protein subunits and targets proteins that contain either of two types of destruction motif, the D box or the KEN box. APC/C activity is regulated in part by two related classes of WD40 repeat-containing proteins named after the Drosophila and budding yeast orthologs: Fizzy (Fzy) or Cdc20, which is required at the metaphase-anaphase transition, and Fizzy-related (Fzr) or Cdh1, which is effective toward the end of mitosisand into G1 to maintain mitotic cyclins at a low level. Activation of many APC/C functions may be delayed by the spindle assembly checkpoint that monitors microtubule attachment of and tension at kinetochores and signals Mad2 to complex with and inhibit the APC/C (Mathe, 2004 and references therein).

The mitotic cyclins are a major target of the APC/C, and in normal mitotic progression A-type cyclins are degraded ahead of the B-type cyclins. Experiments with stable forms of B-type cyclins in several organisms have shown their degradation to be required not at the time of chromatid separation but at later stages of mitosis. This is supported by real-time studies in Drosophila embryos that show stable Cyclin B1 (hereafter referred to simply as Cyclin B) functions to block spindle elongation at anaphase B, resulting in the oscillation of disjoined chromatids. Stable Cyclin B3 gave a late arrest in which anaphase and cytokinesis were completed, but chromosomes failed to decondense (Parry, 2001). Parry (2003) subsequently showed that the oscillation of disjoined chromatids resulted from the establishment of merotelic attachments of their kinetochores to both poles and that this was likely to be a consequence of the failure to release the Aurora B kinase from the kinetochore. It also had the consequence of blocking cytokinesis (Mathe, 2004 and references therein).

Early studies of Cyclin B behavior in syncytial Drosophila embryos had shown degradation to be incomplete, but these were complicated by the use of fixed preparations of embryos where the pattern of immunostaining depended upon fixation conditions. The use of GFP-tagged Cyclin B, however, has permitted time-lapse studies in both Drosophila and mammalian cells that have suggested that Cyclin B degradation begins first on the mitotic apparatus and then occurs subsequently in the cytoplasm (Huang, 1999; Clute, 1999). In Drosophila, Cyclin B is degraded on the spindle in a wave that spreads from the poles and then subsequently in the cytoplasm. Consistently, in mutant embryos derived from centrosome fall off (cfo) mothers, Cyclin B is degraded on the detached centrosomes but not on the acentrosomal spindles, as though the physical detachment presents a barrier to the wave of cyclin destruction (Wakefield, 2000). Degradation of spindle-associated Cyclin B has been attributed to APC/C associated with Fzy/Cdc20, and degradation of the cytoplasmic Cyclin B to Fzr/Cdh1, a protein that only appears to be active after cellularization (Raff, 2001). This led to the hypothesis that Fzy/Cdc20, localized on the kinetochores and centrosome prior to the metaphase-anaphase transition, might mediate the degradation of cyclin throughout the spindle once the metaphase checkpoint has been relieved at the kinetochore (Mathe, 2004 and references therein).

However, the above hypothesis does not satisfactorily account for how Fzy/Cdc20 might direct Cyclin B degradation to begin at the spindle poles rather than elsewhere on the spindle. The possibility that components of the ubiquitination pathway other than the APC/C and its associated proteins may contribute to determining the specificity of proteolytic degradation of proteins in mitosis by the APC/C was first raised by the finding of E2 enzymes that were specific for the mitotic cyclins (Aristarkhov, 1996; Yu, 1996). However, when the catalytic cysteine of the clam enzyme E2-C was changed to serine, this resulted in a dominant-negative form of the enzyme that was able to arrest mammalian cells in metaphase and inhibit destruction of both Cyclin A and Cyclin B (Townsley, 1997). Elimination of E2-C function through either mutation or RNA interference in Drosophila cells results in the accumulation of Cyclin B principally at the centrosomes and a characteristic delay of cells in a metaphase-anaphase-like state. Metazoan E2-C enzymes themselves contain putative destruction D boxes (Yamanaka, 2000). It is now shown directly that Drosophila E2-C is concentrated at the centrosome and that it is itself subject to cyclical degradation. Expression of a D box mutant form of the Vihar E2-C enzyme leads to mitotic defects in which Cyclin B is degraded at the spindle poles but not in the equatorial region of the spindle (Mathe, 2004).

This gene has been named vihar (Hungarian for storm) after the characteristic mutant phenotype in which chromosomes are scattered throughout the mitotic spindle. Similar reductions are observed in the levels of Vihar E2-C protein both in syncytial embryos derived from vih mutant mothers and S2 cells subjected to vih RNAi; both these treatments lead to comparable mitotic abnormalities. The majority of mitoses are delayed as metaphase figures in which chromosomes have undergone congression to the equator of the spindle. As seen with other mitotic mutants in Drosophila, many of these mitotic spindles lack centrosomes from one or both poles. Thus, this aspect of the phenotype cannot be attributed directly to reduction of the Vihar protein. The scattered chromosomes have the BubR1 checkpoint protein at their kinetochores, and the Aurora B passenger protein kinase appears not to have been transferred to the spindle, which does not adopted its characteristic late anaphase morphology. Such features have also been reported in cells arrested in mitosis due to the presence of nondegradable Cyclin B. Indeed, cells with reduced Vihar levels show pronounced increases in Cyclin B levels (Mathe, 2004).

The accumulation of Cyclin B at the centrosomes of vihar arrested cells is particularly striking and is an exaggeration of the association of the cdk1-Cyclin B complex with spindle poles that has been described in a wide number of organisms. The extent of degradation of Cyclin B varies during the embryonic development of Drosophila. Although the mitotic cyclins undergo extensive degradation at the metaphase-anaphase transition in cellularized Drosophila embryos and in tissues at later stages, they appear to persist throughout mitosis in the syncytial Drosophila embryo. There are successively increasing levels of Cyclin B degradation throughout the early syncytial cycles. This has been suggested to occur in restricted areas around the spindles as a result of observations of a gradient of the dephosphorylation of phospho-histone H3 along anaphase chromosomes, which is maximal near the spindle poles. Such a gradient has been interpreted as reflecting reduction of cdk1 activity near the spindle poles or centromere. Although this finding would probably now be interpreted as the activation of the protein phosphatase that opposes the B-type Aurora kinase that phosphorylates Histone H3, it nevertheless reflects a gradient of the activities of several enzymes associated with mitotic exit, which is initiated at the spindle poles. Support for this idea was provided by real-time imaging of GFP-tagged Cyclin B (Huang, 1999) that shows that its degradation begins at the spindle poles in cellularized embryos (Mathe, 2004).

Some indication of how Cyclin B degradation might be propagated along the spindle comes from studies of the localization of the Vihar E2-C protein and its own pattern of proteolysis. Immunolocalization experiments show not only that a considerable proportion of Vihar E2-C is associated with the centrosome, but also that it too undergoes degradation following the metaphase-anaphase transition. This was confirmed by the rapid destruction of accumulated Vihar E2-C protein following release of cells from a nocodazole block and also by the stabilization of the protein following treatment of embryos with a proteasome inhibitor. The Vihar centrosomal associated enzyme either dissociates or is directly degraded during anaphase, leaving a diffuse distribution of protein in the central part of the cell that is largely degraded upon mitotic exit. Thus, the spatiotemporal pattern of Vihar distribution is reminiscent of that described by Huang (1999) for Cyclin B. It suggests that degradation of the two proteins might be mediated by activated APC/C in similar subcellular compartments. However, Vihar E2-C appears to be more tightly localized to the centrosome in the syncytial embryo than Cyclin B, which has a more punctate distribution over astral microtubules and is more strongly associated with other regions of the spindle. Vihar also has a tighter distribution on the centrosome than the APC/C component Apc11 that clusters in a much larger 'cloud' of staining around the spindle poles as if associated with astral microtubules. Thus, the focus for APC/C activity may initially be the centrosome itself, where Vihar E2-C would not be rate limiting (Mathe, 2004).

Both of the Vihar D boxes appear to be required for efficient destruction of the Vihar protein, because mutations in either box alone result in defects in embryonic development, whereas expression of Vihar E2-C having mutations in both boxes gives a dominant mitotic phenotype. Previous studies with the mammalian counterpart of Vihar had shown that mutation of these sequences stabilized the protein in an in vitro assay, but it had not been possible to study effects in vivo (Yamanaka, 2000). Expression of the double D box mutant of Vihar leads to the preferential degradation of Cyclin B at the spindle poles and its continued presence in the equatorial region of the spindle, in contrast to the phenotype seen following downregulation of the enzyme when Cyclin B accumulates at the poles. How can this dominant effect be explained, and what is its relevance to wild-type Vihar function? Because the double D box mutant shows some reduction in catalytic activity (albeit measured in vitro against a nonphysiological substrate, a fragment of human Cyclin B), it is possible that the effects of downregulating Vihar function are being observed. However, it is noted that when a truly catalytically dead version of Vihar (mutated at its catalytic cysteine residue) is expressed in mitotic cells, the phenotype is similar to loss of Vihar function, metaphase arrest with accumulation of Cyclin B at the centrosomes. Nevertheless, the dominant effect of the Vihar double D box mutant is seen only at reduced concentration of the wild-type protein. In the whole organism, this is seen following reduction of the gene dosage of the wild-type allele, and in mitosis only once the wild-type protein has been partially destroyed. Thus, it may be that at this critical ratio of wild-type to D box mutant Vihar protein the dominant-negative effect comes into action to prevent the late pattern of Cyclin B degradation. However, it is felt that this explanation does not fully take into account the spatial allocation of Vihar to different subcellular compartments. There is no reason to suspect that at the onset of mitosis the ratio of wild-type to D box forms should vary whether associated with the centrosome, spindle, or cytoplasm. Thus, even though the ratio of wild-type to D box forms would first reduce at the poles, it should remain unchanged in other parts of the cell, where there should still be sufficient wild-type Vihar to give the potential for APC/C activity. Thus, the dominant-negative effect could be viewed from two perspectives, being a result either of reduced E2 activity or of continued E2 activity albeit at a reduced level, but in either event the mutant protein would persist at the spindle poles. The outcome of either of these viewpoints of the dominant effect might be similar: namely, the observed effect of preventing subsequent waves of Cyclin B proteolysis on the central part of the spindle and in the cytoplasm. Thus, the preferred hypothesis is that the continued presence of this nondegradable form of Vihar at the spindle poles could block mitotic progression, irrespective of its own level of E2 activity, by sequestering other rate-limiting components of the ubiquitination machinery. Conversely, in the wild-type situation, when the APC/C becomes competent at the metaphase-anaphase transition, the concentration of Vihar at the poles would direct ubiquitination of APC/C targets at this site, and its own polar destruction would be required to permit subsequent waves of proteolysis elsewhere in the cell (Mathe, 2004).

Perhaps the key to such a spatially regulated system lies in processes of microtubule-mediated transport. Raff (2001) has proposed that the degradation of spindle-associated Cyclin B might be mediated by APC/C associated with Fzy/Cdc20 and that Fizzy-related/Cdh1 directs more the degradation of Cyclin B in the cytoplasm. It is further suggested that once the metaphase-anaphase checkpoint is satisfied, flux of Fzy/Cdc20 from the kinetochore to the poles first mediates cyclin degradation. The finding that the Vihar E2-C is concentrated at the poles provides an explanation of why the Cyclin B degradation is initiated at the poles. The spatiotemporal pattern by which Vihar E2-C is then degraded suggests that it is ubiquitinated for degradation alongside its Cyclin B target once the APC/C has been activated in the vicinity of the spindle poles. Such autoregulatory inactivation of APC/C activity at the spindle poles might also release the APC/C or other regulatory components to mediate Cyclin B degradation within the central part of the spindle. This may be linked to a general influx of late mitotic regulators to the central spindle region. Indeed, plus end-directed motor proteins such as Pavarotti-KLP and several other regulators of cytokinesis begin to accumulate in the central spindle structure that forms at this time in preparation for cytokinesis. Once the ubiquitination process has been shut down at the poles, then similar autoinhibition could later operate upon Vihar E2-C in other regions of the cell following the subsequent wave of APC/C activity (Mathe, 2004).

Finally, it is noted that in vitro studies using Xenopus and clam extracts indicate that APC-mediated ubiquitination reactions are supported equally well by the Ubc4 and UBCx/E2-C enzymes. However, it has remained unclear whether these enzymes are redundant in vivo. A recent study from Seino (2003) in fission yeast, however, suggests that these two classes of E2 ubiquitin-conjugating enzymes are not functionally equivalent and have distinct roles in degrading the mitotic cyclin Cdc13. The phenotypes of cells deficient for Vihar E2-C enzyme indicate that it cannot be redundant with the Ubc4/5 family of E2 enzymes that remain functional in these cells. Thus, it remains of considerable interest to understand how different E2 family members cooperate in the APC/C-mediated destruction of mitotic cyclins and other mitotic regulatory proteins (Mathe, 2004).

It is concluded that the Vihar E2 UBC is concentrated at the spindle poles to facilitate the degradation of Cyclin B that first occurs at this site during the metaphase-anaphase transition. Vihar E2 UBC is also subject to ubiquitination and degradation by the APC/C. This results in the autoinactivation of APC/C-mediated ubiquitination at the spindle poles. Cyclin B degradation then takes place in the central spindle and other parts of the cell (Mathe, 2004).


REGULATION

lemmingA encodes the Apc11 subunit of the APC/C in Drosophila melanogaster that forms a ternary complex with the E2-C type ubiquitin conjugating enzyme, Vihar and Morula/Apc2

Ubiquitin-dependent protein degradation is a critical step in key cell cycle events, such as metaphase-anaphase transition and mitotic exit. The anaphase promoting complex/cyclosome (APC/C) plays a pivotal role in these transitions by recognizing and marking regulatory proteins for proteasomal degradation. Its overall structure and function has been elucidated mostly in yeasts and mammalian cell lines. The APC/C is, however, a multisubunit assembly with at least 13 subunits and their function and interaction within the complex is still relatively uncharacterized, particularly in metazoan systems. This study used lemming(lmg) mutants to study the APC/C subunit, Apc11, and its interaction partners in Drosophila. The lmg gene was initially identified through a pharate adult lethal P element insertion mutation expressing developmental abnormalities and widespread apoptosis in larval imaginal discs and pupal abdominal histoblasts. Larval neuroblasts were observed to arrest mitosis in a metaphase-like state with highly condensed, scattered chromosomes and frequent polyploidy. These neuroblasts contain high levels of both cyclin A and cyclin B. The lmg gene was cloned by virtue of the lmg03424 P element insertion which is located in the 5' untranslated region. The lemming locus is transcribed to give a 2.0 kb mRNA that contains two ORFs, lmgA and lmgB. The lmgA ORF codes for a putative protein with more than 80% sequence homology to the APC11 subunit of the human APC/C. The 85 amino acid protein also contains a RING-finger motif characteristic of known APC11 subunits. The lmgA ORF alone was sufficient to rescue the lethal and mitotic phenotypes of the lmg138 null allele and to complement the temperature sensitive lethal phenotype of the APC11-myc9 budding yeast mutant. The LmgA protein interacts with Mr/Apc2, and they together form a binding site for Vihar, the E2-C type ubiquitin conjugating enzyme. Despite being conserved among Drosophila species, the LmgB protein is not required for viability or fertility. This work provides insight into the subunit structure of the Drosophila APC/C with implications for its function. Based on the presented data, it is suggested that the Lmg/Apc11 subunit recruits the E2-C type ubiquitin conjugating enzyme, Vihar, to the APC/C together with Mr/Apc2 by forming a ternary complex (Nagy, 2012).

The APC/C belongs to the cullin-RING family of multisubunit ubiquitin ligases. Previous studies of the budding yeast and human APC/C indicated that the cullin-related Apc2 and the RING-finger-containing Apc11 subunits together form the minimal ubiquitin ligase modul. This study shows that, in Drosophila, the Apc11 subunit is encoded by the dicistronic lemming locus. The upstream ORF, lmgA, encodes a putative protein containing a RING-finger motif characteristic of known APC11 subunits and shows more than 80% sequence similarity with the APC11 subunit of the human APC/C. Since the Apc11 subunit is proposed to play a role in the catalytic center of the APC/C, mutations in lmg are expected to lead to loss of APC function, and therefore to aberrant cell cycle progression. The lmg mitotic phenotype presented in this paper is consistent with the Lmg protein being a subunit of the APC. The mitotic defects observed in lmg larval neuroblasts, including metaphase-like arrest, chromosome overcondensation and polyploidy, in addition to widespread apoptosis of mitotically-active cells, are very similar to those reported for loss of other subunits of the Drosophila APC/C. A role of LmgA in the APC/C is further supported by the elevated levels of cyclin A and B observed in lmg neuroblasts. Another line of supporting evidence comes from the synergistic genetic interaction between lmgA and mr/Apc2 and lmgA and vihar and from the physical interactions among these proteins, since it is known that, in yeasts and vertebrates, these proteins form the catalytic module of the APC/C. These data, together with its ability to complement the mutant phenotype of yeast Apc11-deficient cells support the designation of lmgA as a true Apc11 orthologue (Nagy, 2012).

The APC/C requires special E2 enzymes for activity and has been demonstrated to function with Ubc4/5 and E2-C type E2 enzymes in vitro . Whereas in yeast and human cells the E2 enzymes bind to either Apc2 or Apc11, the current data suggest that in Drosophila, both of these subunits are required for effective E2 binding. This could represent an architectural variation in the catalytic subcomplex of different APC/C ligases (Nagy, 2012).

The dicistronic nature of the lmg locus is a notable but puzzling fact. Whereas the upstream lmgA ORF encodes the Apc11 subunit of the Drosophila APC/C, the existence and function of the predicted downstream lmgB ORF product remains unknown. No sequence or functional relationship could be found between lmgA and lmgB, though such relationships are characteristic of many dicistronic genes. Genomes of other species from Drosophilidae (especially in the melanogaster group) contain both these ORFs and the intercistronic sequence in a similar arrangement. Moreover, the high evolutionary conservation of LmgA and LmgB and significant conservation of both ICS and 3'-UTR suggest functional relevance. However, it was found that the putative LmgB is dispensable for the organism and lacks known protein motifs. In addition to this, no apparent LmgB interaction partners could be found in yeast two hybrid screen and LmgB could not be efficiently translated from the dicistronic mRNA in S2 cells. lmgA contains three in-frame AUG codons in addition to its initiating AUG codon. It has been shown for two Drosophila dicistronic transcripts, of the stoned and snapin loci, that such in-frame AUG codons effectively attenuate the translation of the second ORF. However, the rationale for the dicistronic arrangement of the lmgA and lmgB cistrons and the function of the lmgB ORF remains obscure (Nagy, 2012).

The mechanism by which loss of APC/C function leads to apoptosis is unknown but it may be significant that lmg mutant cells entered apoptosis directly, and rapidly, from arrested cells, without a return to the interphase state. There is accumulating evidence that mitosis and apoptosis share components. It has been suggested that apoptosis is a default pathway and proteins such as survivin are required to counteract this pathway during mitosis. Cells treated with drugs which alter microtubule dynamics, such as paclitaxel (Taxol) also undergo mitotic arrest and enter apoptosis rapidly, and directly, from mitosis. Since these drugs are thought to trigger the spindle assembly checkpoint which in turn acts by inhibiting the APC/C , it is possible that loss of APC/C activity is responsible for triggering apoptosis. Inactivation of the APC/C by cleavage of the CDC27 component by caspases has also been shown to occur during apoptosis triggered by Fas ligand in Jurkat cells, contributing to an increase in Cdk activity. There have been several reports of increased Cdk activity during apoptosis, suggesting that these enzymes form part of the apoptotic pathway. Increased mitotic cyclin levels, and Cdk activity, may therefore play a role in apoptosis triggered by loss of APC/C function. Apoptosis, however, does not normally occur when cyclin levels are high at metaphase. This may be because of protective factors such as survivin. A loss of protective activity during anaphase may allow cells to respond to abnormally high levels of Cdk activity and undergo apoptosis. Alternatively, if the APC/C itself plays a protective role, simultaneous loss of this protection and elevated Cdk levels would result in apoptosis (Nagy, 2012).

The polyploid cells observed in larval brain squashes may be cells that have escaped apoptosis, exited mitosis without cytokinesis, and then duplicated their chromosomes before re-entering mitosis again. If so, some cells can clearly repeat the process several times, as cells were observed that were highly polyploid. Furthermore, no G2-arrested interphase larval abdominal histoblasts were observed undergoing apoptosis. This suggests that there is a phase, during mitosis, when cells are particularly sensitive to loss of lmg function and respond by undergoing apoptosis. This might be expected if loss of APC/C function is playing a relatively direct role in triggering apoptosis (Nagy, 2012).

The data presented in this paper demonstrate that the upstream member of a dicistronic gene, lmgA codes for the Apc11 subunit of the APC/C in a multicellular metazoan species, Drosophila melanogaster. Its genetic and physical interactions with Mr/Apc2 and the E2-C type ubiquitin-conjugating enzyme, Vihar, suggest that their ternary complex represents the same catalytic module of the APC/C that was identified in yeast and mammalian cells by functional means (Nagy, 2012).

lemmingA encodes the Apc11 subunit of the APC/C in Drosophila melanogaster that forms a ternary complex with the E2-C type ubiquitin conjugating enzyme, Vihar and Morula/Apc2

Ubiquitin-dependent protein degradation is a critical step in key cell cycle events, such as metaphase-anaphase transition and mitotic exit. The anaphase promoting complex/cyclosome (APC/C) plays a pivotal role in these transitions by recognizing and marking regulatory proteins for proteasomal degradation. Its overall structure and function has been elucidated mostly in yeasts and mammalian cell lines. The APC/C is, however, a multisubunit assembly with at least 13 subunits and their function and interaction within the complex is still relatively uncharacterized, particularly in metazoan systems. In this study, lemming (lmg) mutants were used to study the APC/C subunit, Apc11, and its interaction partners in Drosophila. The lmg gene was initially identified through a pharate adult lethal P element insertion mutation expressing developmental abnormalities and widespread apoptosis in larval imaginal discs and pupal abdominal histoblasts. Larval neuroblasts were observed to arrest mitosis in a metaphase-like state with highly condensed, scattered chromosomes and frequent polyploidy. These neuroblasts contain high levels of both cyclin A and cyclin B. The lmg gene was cloned by virtue of the lmg03424 P element insertion which is located in the 5' untranslated region. The lemming locus is transcribed to give a 2.0 kb mRNA that contains two ORFs, lmgA and lmgB. The lmgA ORF codes for a putative protein with more than 80% sequence homology to the APC11 subunit of the human APC/C. The 85 amino acid protein also contains a RING-finger motif characteristic of known APC11 subunits. The lmgA ORF alone is sufficient to rescue the lethal and mitotic phenotypes of the lmg138 null allele and to complement the temperature sensitive lethal phenotype of the APC11-myc9 budding yeast mutant. The LmgA protein interacts with Mr/Apc2, and they together form a binding site for Vihar, the E2-C type ubiquitin conjugating enzyme. Despite being conserved among Drosophila species, the LmgB protein is not required for viability or fertility. This work provides insight into the subunit structure of the Drosophila APC/C with implications for its function. Based on the presented data, it is suggested that the Lmg/Apc11 subunit recruits the E2-C type ubiquitin conjugating enzyme, Vihar, to the APC/C together with Mr/Apc2 by forming a ternary complex (Nagy, 2013).


DEVELOPMENTAL BIOLOGY

The E2-C family of ubiquitin-conjugating enzymes contains two destruction boxes that at least in the case of the mammalian protein are required to mediate the destruction of the protein during mitosis. It was therefore of interest to determine whether the Vihar protein of Drosophila also undergoes cyclical proteolysis. Because it is very difficult to obtain synchronized material for biochemical studies of cell cycle progression in Drosophila, this question was approached in three different ways. First immunostaining was used to localize Vihar E2-C at different mitotic stages in both cultured S2 cells and in wild-type syncytial embryos (Mathe, 2004).

In cultured S2 cells, it was found that Vihar E2-C protein accumulates in nuclei as soon as the chromosomes start to condense and on the centrosomes while the daughter centrosomes have separated and are migrating. At metaphase, it shows strong association to centrosomes. The intensity of centrosome-associated Vihar E2-C staining is distinctly lower at anaphase, but it is not until telophase/cytokinesis that its degradation appears to be maximal. The cytoplasmic levels of Vihar E2-C appear to be similar during mitosis until telophase, when Vihar E2-C, initially excluded from the telophase nuclei, appears to accumulate in the equatorial region of the cell, where the cleavage furrow is to form. By the time cytokinesis is completed, almost all the cytoplasmic Vihar E2-C has been degraded. These characteristic patterns of Vihar E2-C immunolocalization are completely abolished following vih RNAi. A similar pattern of localization is observed in syncytial embryos. Taken together, these observations indicate that Vihar E2-C appears to undergo cyclical proteolysis initiated with either release of Vihar E2-C from the centrosome or the direct degradation of the centrosome-associated protein followed by a second wave of degradation of cytoplasmic protein that is maximal later in mitosis (Mathe, 2004).

In order to rule out the possibility that the diminution of Vihar immunostaining does not simply reflect its dispersal through the cell, attempts were made to demonstrate reduction in Vihar levels by Western blotting. To this end, cells were subjected to nocodazole treatment for 24 hr, causing them to accumulate in mitosis. The drug was washed away, and cells were analyzed at 1 hr intervals for the levels of Vihar E2-C and Cyclin B as a positive control. The majority of both Vihar E2-C and Cyclin B had been degraded by 2 hr, and both were scarcely detectable at 3 hr after removal of the drug. Thus, like Cyclin B, the E2-C enzyme is subject to proteolysis once nocodazole-arrested cells are released from their metaphase-like arrest (Mathe, 2004).


EFFECTS OF MUTATION

The vihar gene was first identified through a semilethal P-lacW insertion in which about 20% of individuals die as pharate adults. The mitotic index and proportion of cells at different phases of the mitotic cycle in the brains of vih1/vih1 larvae were not significantly different from those of wild-type. However, the mutant brains displayed aberrant mitotic figures in which the chromosomes were scattered apparently randomly through the cell and in which there was overall a significant increase in polyploid mitotic figures. The vihar locus was uncovered by chromosomal deficiencies that place the gene in the 69C-D region, consistent with the insertion site of the P-lacW element. The mitotic index and proportion of mitotic defects were increased in the larval brains of hemizygous mutant larvae, indicating the hypomorphic nature of the mutation. Both homozygous and hemizygous vih1 females showed maternal effect lethality and produced syncytial embryos that failed to develop as a result of mitotic defects (Mathe, 2004).

It was possible to revert the vih1 semilethal and maternal effect lethal phenotype under dysgenic conditions to restore full viability and fertility when homozygous or when heterozygous against the original vih1 allele or an uncovering deficiency, Df(3L)iro-2. This experiment also generated one partial fully sterile revertant, vih2. The P-lacW element from the vih1 mutant was inserted 9 bp upstream of a Drosophila consensus sequence for the initiation of translation and 129 bp upstream of the start codon of the open reading frame CG10682. A germline transformant in which the CG10682 cDNA was expressed downstream of the polyubiquitin promoter fully restored the fertility of vih mothers. Thus, the vihar gene corresponds to CG10682 (Mathe, 2004).

To gain further insight into the mitotic defects, the mitotic spindles were examined of vih mutants. The central nervous systems of vih1 larvae display characteristic mitotic defects at a modest frequency consistent with those in orcein-stained squashed preparations and are mirrored by only partial reduction of the Vihar protein. Defective mitotic spindles were characterized by chromosomes being scattered throughout the length of the spindle and/or by the absence of the core centrosomal antigen CNN from one or both poles. The spindle poles that lacked centrosomes are characteristically broad, and in all such cells the chromosomes appear to be overcondensed. Embryos derived from mothers carrying either the vih1 or vih2 alleles, whether homozygous, hemizygous, or transheterozygous, show similarly aberrant mitotic spindles and have a greater reduction in levels of Vihar protein. Thus, the P insertion would appear to affect maternally driven components of the vih promoter more strongly than zygotic ones. Such embryos rarely develop beyond the third nuclear division cycle, and all the mitotic figures show these defects. The mitotic figures in embryos at these stages frequently have a polyploid complement of chromosomes (Mathe, 2004).

Since members of the E2-C family of proteins in fission yeast UbcP4/Ubc11, clam E2-C, Xenopus UBCx, mouse E2-C, and human UbcH10 participate in the degradation of mitotic cyclins (Aristarkhov, 1996; Yu, 1996; Townsley, 1997; Yamanaka, 2000; Osaka, 1997), Cyclin B levels were followed in cultured cells subjected to vih RNAi. The development of the phenotype was followed over 3 days of treatment with vih dsRNA, during which time Western blots showed substantial elimination of Vihar protein that was associated with a corresponding increase in levels of Cyclin B. In the course of such experiments, there was a 7-fold increase in the mitotic index, such that 40% of the cells arrested in mitosis with a 3-fold increase in the frequency of metaphases at the expense of a similar decrease in the proportion of cells in cytokinesis. vih dsRNA was able to phenocopy the characteristic spindle defects observed in vih mutants, and the mitotic spindle never showed prominent lengthening as normally occurs in wild-type anaphase. Immunostaining identified three categories of defective bipolar spindles. The first class features spindles with aligned metaphase chromosomes having a Cyclin B-stained centrosome at only one pole. Such figures are also present in untreated control cells. The second class shows chromosomes scattered throughout spindles that usually have just a single centrosome. Finally, spindles of the third class have chromosomes in the equatorial region but are significantly shorter (by approximately 60%) than the metaphase spindles in untreated cells. These small spindles have centrosomes at each pole. The proportions of these last two classes of defective spindle increased during the course of the RNAi experiment. In contrast to untreated cells, where Cyclin B undergoes cyclical degradation, Cyclin B seems to accumulate at the centrosomes, at the spindle, and throughout the cell following vihar RNAi. This is consistent with the increase in Cyclin B seen in many cell types after APC/C inhibition, for example, following spindle integrity checkpoint arrest (Mathe, 2004).

The scattered distribution of chromosomes in cells with reduced levels of Vihar resembles the phenotype seen following the expression of nondegradable Cyclin B in Drosophila (Parry, 2001; Sigrist, 1995; Rimmington, 1994; Echard; 2003; Parry; 2003). In such circumstances, Parry (2003) showed that chromosomes undergo an oscillating behavior due to reestablishment of microtubule connections between kinetochores and both poles with concomitant reacquisition of checkpoint proteins. The kinetochore regions of the scattered chromosomes in cells with reduced amounts of Vihar are also associated with the BubR1 checkpoint protein, suggesting that, as in the case of cells expressing nondegradable Cyclin B, they are attempting chromosome congression (Parry, 2003). Consistently, the Aurora B kinase also remains associated with the kinetochore regions, and there is no sign in these cells of any attempt to organize the central spindle (Mathe, 2004).

Since both Cyclin B and Vihar E2-C have well-defined spatiotemporal profiles of localization and destruction on the mitotic spindle, it was of interest to determine the consequences of expressing a nondegradable form of Vihar E2-C upon mitotic progression. To this end, destruction box mutant variants of Vihar E2-C were cloned downstream of the GAL4 responsive element in pUASP. Germline transformants were then established that carried transgenes with mutations in each individual D box alone (vihmD1or vihmD2) or in both D boxes (vihmD1D2). Crossing such lines with one in which the GAL4 protein is expressed from the maternal alpha-tubulin promoter activates female germline expression from the UAS-containing transgenes. Thus, the embryos derived from such mothers contain both wild-type and D box mutant Vihar E2-C protein (Mathe, 2004).

When wild-type Vihar E2-C protein was expressed from this maternal GAL4-driven system, the protein was fully able to rescue the vih1 maternal effect mutant. In contrast, neither vihmD1, vihmD2, nor vihmD1D2 is able to rescue this maternal effect mutant. This indicates that none of these constructs is fully functional. However, no significant change was seen in the embryonic phenotype above that of the background vih1 mutant. When the three constructs were expressed in a wild-type background, flies were fully fertile and displayed no maternal effect. When, however, the gene dosage of wild-type protein was reduced to 50%, it was found that the expression of vihmD1 has no effect, the expression of vihmD2 results in some mothers failing to produce viable embryos, and the expression of vihmD1D2 results in complete maternal effect lethality (Mathe, 2004).

Immunostaining experiments were carried out to determine whether expression of vihmD1D2 results in any mitotic defects and, if so, whether they are associated abnormalities in Cyclin B degradation. In wild-type embryos, Cyclin B is degraded on the mitotic spindle in an apparent wave that spreads from the poles toward the chromosomes as mitotic figures progress through the metaphase-anaphase transition. This is most clearly visualized using GFP-tagged Cyclin B in cellularized embryos but can be seen with some difficulty in immunostained fixed preparations of syncytial stage embryos. The expression of vihmD1D2 in embryos derived from hemizygous vih+/− mothers results in roughly equivalent levels of wild-type and mutant protein and leads to arrest of mitosis predominantly in a metaphase or anaphase state during the cleavage embryonic cycles in the absence of a proteasome inhibitor. It did not appear to prevent chromatid separation, because chromosomes were in either a metaphase or anaphase configuration. It was not possible to detect the His-tagged ViharmD1D2 protein at centrosomes in the arrested mitotic figures. In all cases, expression of the vihmD1D2 mutant results in a reduction of Cyclin B staining at the spindle poles and an accumulation of Cyclin B in the central part of the spindle in a manner never seen in wild-type embryos across a whole field of mitoses. Thus, stabilization of the Vihar E2-C permits Cyclin B degradation in the region of the spindle poles, but not at the spindle equator, and this prevents completion of mitosis. This contrasts to loss of Vihar function when Cyclin B accumulates at the spindle poles (Mathe, 2004).


EVOLUTIONARY HOMOLOGS

UbcP4, a yeast homolog of E2-C

A cDNA encoding a ubiquitin-conjugating enzyme designated UbcP4 in fission yeast was isolated. Disruption of its genomic gene revealed that it was essential for cell viability. In vivo depletion of the UbcP4 protein demonstrated that it was necessary for cell cycle progression at two phases, G2/M and metaphase/anaphase transitions. The G2 arrest of UbcP4-depleted cells is dependent upon chk1, which mediates checkpoint pathway. UbcP4-depleted cells arrest at metaphase, have condensed chromosomes, but are defective in separation. However, septum formation and cytokinesis are not restrained during the metaphase arrest. Overexpression of UbcP4 specifically rescues the growth defect of cut9ts cells at a restrictive temperature. cut9 encodes a component of the anaphase-promoting complex (APC) which is required for chromosome segregation at anaphase and moreover is defined as cyclin-specific ubiquitin ligase. Cdc13, a mitotic cyclin in fission yeast, accumulates in the UbcP4-depleted cells. These results strongly suggested that UbcP4 is a ubiquitin-conjugating enzyme working in conjunction with APC and mediates the ubiquitin pathway for degradation of 'sister chromatid holding protein(s)' at the onset of anaphase and possibly of mitotic cyclin at the exit of mitosis (Osaka, 1997).

UBC11 is the Saccharomyces cerevisiae gene that is most similar in sequence to E2-C, a ubiquitin carrier protein required for the destruction of mitotic cyclins and proteins that maintain sister chromatid cohesion in animal cells and in Schizosaccharomyces pombe. The UBC11 gene has been disrupted and it was found to not be essential for yeast cell viability even when combined with deletion of UBC4, a gene that has also been implicated in mitotic cyclin destruction. Ubc11p does not ubiquitinate cyclin B in clam cell-free extracts in vitro and the destruction of Clb2p is not impaired in extracts prepared from delta ubc11 or delta ubc4 delta ubc11 cells. These results suggest Ubc4p and Ubc11p together are not essential for mitotic cyclin destruction in S. cerevisiae and no evidence has been found to suggest that Ubc11p is the true functional homologue of E2-C (Townsley, 1998).

Cell cycle events are regulated by sequential activation and inactivation of Cdk kinases. Mitotic exit is accomplished by the inactivation of mitotic Cdk kinase, which is mainly achieved by degradation of cyclins. The ubiquitin-proteasome system is involved in this process, requiring APC/C (anaphase-promoting complex/cyclosome) as a ubiquitin ligase. In Xenopus and clam oocytes, the ubiquitin-conjugating enzymes that function with APC/C have been identified as two proteins, UBC4 and UBCx/E2-C. The fission yeast ubiquitin-conjugating enzyme UbcP4/Ubc11, a homologue of UBCx/E2-C, is required for mitotic transition. The other fission yeast ubiquitin-conjugating enzyme, UbcP1/Ubc4, which is homologous to UBC4, is also required for mitotic transition in the same manner as UbcP4/Ubc11. Both ubiquitin-conjugating enzymes are essential for cell division and directly required for the degradation of mitotic cyclin Cdc13. They function nonredundantly in the ubiquitination of CDC13 because a defect in ubcP1/ubc4+ cannot be suppressed by high expression of UbcP4/Ubc11 and a defect in ubcP4/ubc11+ cannot be suppressed by high expression of UbcP1/Ubc4. In vivo analysis of the ubiquitinated state of Cdc13 shows that the ubiquitin chains on Cdc13 were short in ubcP1/ubc4 mutant cells while ubiquitinated Cdc13 was totally reduced in ubcP4/ubc11 mutant cells. Taken together, these results indicate that the two ubiquitin-conjugating enzymes play distinct and essential roles in the degradation of mitotic cyclin Cdc13, with the UbcP4/Ubc11-pathway initiating ubiquitination of Cdc13 and the UbcP1/Ubc4-pathway elongating the short ubiquitin chains on Cdc13 (Seino, 2003).

Cloning and characterization of E2-C/UbcH10

Ubiquitin-dependent proteolysis of the mitotic cyclins A and B is required for the completion of mitosis and entry into the next cell cycle. This process is catalyzed by the cyclosome, an approximately 22S particle that contains a cyclin-selective ubiquitin ligase activity, E3-C, that requires a cyclin-selective ubiquitin carrier protein (UBC) E2-C. The purification and cloning of E2-C from clam oocytes is reported in this study. The deduced amino acid sequence of E2-C indicates that it is a new UBC family member. Bacterially expressed recombinant E2-C is active in in vitro cyclin ubiquitination assays, where it exhibits the same substrate specificities seen with native E2-C. These results demonstrate that E2-C is not a homolog of UBC4 or UBC9, proteins previously suggested to be involved in cyclin ubiquitination, but is a new UBC family member with unique properties (Aristarkohov, 1996).

The destruction of cyclin B is required for exit from mitosis, and is mediated by the ubiquitin pathway. A 20S complex, termed the anaphase-promoting complex (APC) or the cyclosome, has been genetically and biochemically identified as the cyclin-specific ubiquitin ligase (E3). In addition, a ubiquitin-conjugating enzyme (E2), UBC4, was shown to be involved in cyclin ubiquitination in Xenopus egg extracts. Another E2 activity, designated UBCx, can independently support cyclin ubiquitination in Xenopus. A similar activity (E2-C) has also been observed in clams. However, the molecular identity of Xenopus UBCx or clam E2-C has not been established. Xenopus UBCx has been purified and cloned. Sequence comparisons with known E2s reveal that UBCx is a novel ubiquitin-conjugating enzyme. Purified recombinant UBCx is sufficient to complement purified APC and E1 in destruction box-dependent cyclin ubiquitination. UBCx and UBC4 are active in a similar concentration range and with similar kinetics. At saturating enzyme concentrations, UBCx converts twice as much substrate into ubiquitin conjugates, but generates conjugates of lower molecular mass than UBC4. It is concluded that UBCx is a novel ubiquitin-conjugating enzyme involved in cyclin ubiquitination in Xenopus. Like UBC4, ubiquitination catalyzed by UBCx is dependent on both the destruction box and the APC, suggesting that these E2s function through a similar mechanism. However, as the patterns of conjugates generated by these E2s are distinct, these enzymes may play different roles in promoting cyclin proteolysis in mitosis (Yu, 1996).

Destruction of mitotic cyclins by ubiquitin-dependent proteolysis is required for cells to complete mitosis and enter interphase of the next cell cycle. In clam eggs, this process is catalyzed by a cyclin-selective ubiquitin carrier protein, E2-C, and the cyclosome/anaphase promoting complex (APC), a 20S particle containing cyclin-selective ubiquitin ligase activity. A human homolog of E2-C, UbcH10, shares 61% amino acid identity with clam E2-C and can substitute for clam E2-C in vitro. Dominant-negative clam E2-C and human UbcH10 proteins, created by altering the catalytic cysteine to serine, inhibit the in vitro ubiquitination and destruction of cyclin B in clam oocyte extracts. When transfected into mammalian cells, mutant UbcH10 inhibits the destruction of both cyclin A and B, arrests cells in M phase, and inhibits the onset of anaphase, presumably by blocking the ubiquitin-dependent proteolysis of proteins responsible for sister chromatid separation. Thus, E2-C/UbcH10-mediated ubiquitination is involved in both cdc2 inactivation and sister chromatid separation, processes that are normally coordinated during exit from mitosis (Townsley, 1997).

The anaphase promoting complex or cyclosome is the ubiquitin-ligase that targets destruction box-containing proteins for proteolysis during the cell cycle. Anaphase promoting complex or cyclosome and its activator (the fizzy and fizzy-related) proteins work together with ubiquitin-conjugating enzymes (UBCs) (E2s). One class of E2s (called E2-C) seems specifically involved in cyclin B1 degradation. Although it has recently been shown that mammalian E2-C is regulated at the protein level during the cell cycle, not much is known concerning the expression of these genes. Arabidopsis encodes two genes belonging to the E2-C gene family (called UBC19 and UBC20). UBC19 is able to complement fission yeast (Schizosaccharomyces pombe) UbcP4-140 mutant, indicating that the plant protein can functionally replace its yeast ortholog for protein degradation during mitosis. In situ hybridization experiments were performed to study the expression of the E2-C genes in various tissues of plants. Their transcripts were always, but not exclusively, found in tissues active for cell division. Thus, the UBC19/20 E2s may have a key function during cell cycle, but may also be involved in ubiquitylation reactions occurring during differentiation and/or in differentiated cells. Finally, a translational fusion protein between UBC19 and green fluorescent protein localizes both in the cytosol and the nucleus in stable transformed tobacco (Nicotiana tabacum cv Bright Yellow 2) cells (Criqui, 2002).

Crystal structure of E2-C/UbcH10

The destruction of the cyclin B protein is necessary for the cell to exit from mitosis. The destruction of cyclin B occurs via the ubiquitin/proteasome system and involves a specific ubiquitin-conjugating enzyme (Ubc) that donates ubiquitin to cyclin B. The crystal structure is presented of the cyclin-specific Ubc from clam, E2-C, determined at 2.0-Å resolution. The E2-C enzyme contains an N-terminal extension in addition to the Ubc core domain. The N-terminal extension is disordered, perhaps reflecting a need for flexibility as it interacts with various partners in the ubiquitination system. The overall structure of the E2-C core domain is quite similar to those in previously determined Ubc proteins. The interaction between particular pairs of E2-C proteins in the crystal has some of the hallmarks of a functional dimer, though solution studies suggest that the E2-C protein exists as a monomer. Comparison of the E2-C structure with that of the other available Ubc structures indicates conserved surface residues that may interact with common components of the ubiquitination system. Such comparison also reveals a remarkable spine of conserved hydrophobic residues in the center of the protein that may drive the protein to fold and stabilize the protein once folded. Comparison of residues conserved only among E2-C and its homologues indicates surface areas that may be involved in mitotic-specific ubiquitination (Jiang, 1999).

Cell cycle progression is controlled at several different junctures by the targeted destruction of cell cycle regulatory proteins. These carefully orchestrated events include the destruction of the securin protein to permit entry into anaphase, and the destruction of cyclin B to permit exit from mitosis. These destruction events are mediated by the ubiquitin/proteasome system. The human ubiquitin-conjugating enzyme, UbcH10, is an essential mediator of the mitotic destruction events. The 1.95-Å crystal structure of a mutant UbcH10. in which the active site cysteine has been replaced with a serine, is reported. Functional analysis indicates that the mutant is active in accepting ubiquitin, although not as efficiently as wild-type. Examination of the crystal structure reveals that the NH2-terminal extension in UbcH10 is disordered and that a conserved 3(10)-helix places a lysine residue near the active site. Analysis of relevant mutants demonstrates that for ubiquitin-adduct formation the presence or absence of the NH2-terminal extension has little effect, whereas the lysine residue near the active site has significant effect. The structure provides additional insight into UbcH10 function, including possible sites of interaction with the anaphase promoting complex/cyclosome and the disposition of a putative destruction box motif in the structure (Lin, 2002).

E2-C/UbcH10 and the degradation of Cyclin B

The ubiquitin-dependent proteolysis of mitotic cyclin B, which is catalyzed by the anaphase-promoting complex/cyclosome (APC/C) and ubiquitin-conjugating enzyme H10 (UbcH10), begins around the time of the metaphase-anaphase transition and continues through G1 phase of the next cell cycle. Cell-free systems from mammalian somatic cells collected at different cell cycle stages (G0, G1, S, G2, and M) were used to investigate the regulated degradation of four targets of the mitotic destruction machinery: cyclins A and B, geminin H (an inhibitor of S phase identified in Xenopus), and Cut2p (an inhibitor of anaphase onset identified in fission yeast). All four are degraded by G1 extracts but not by extracts of S phase cells. Maintenance of destruction during G1 requires the activity of a PP2A-like phosphatase. Destruction of each target is dependent on the presence of an N-terminal destruction box motif, is accelerated by additional wild-type UbcH10 and is blocked by dominant negative UbcH10. Destruction of each is terminated by a dominant activity that appears in nuclei near the start of S phase. Previous work indicates that the APC/C-dependent destruction of anaphase inhibitors is activated after chromosome alignment at the metaphase plate. In support of this, addition of dominant negative UbcH10 to G1 extracts is shown to block destruction of the yeast anaphase inhibitor Cut2p in vitro, and injection of dominant negative UbcH10 blocks anaphase onset in vivo. Injection of dominant negative Ubc3/Cdc34, whose role in G1-S control is well established and has been implicated in kinetochore function during mitosis in yeast, dramatically interferes with congression of chromosomes to the metaphase plate. These results demonstrate that the regulated ubiquitination and destruction of critical mitotic proteins is highly conserved from yeast to humans (Bastians, 1999).

Other targets of UbcH10

Hox proteins are transcription factors involved in controlling axial patterning, leukaemias and hereditary malformations. HOXC10 oscillates in abundance during the cell cycle, being targeted for degradation early in mitosis by the ubiquitin-dependent proteasome pathway. Among abdominal-B subfamily members, the mitotic proteolysis of HOXC10 appears unique, since the levels of the paralogous HOXD10 and the related homeoprotein HOXC13 are constant throughout the cell cycle. When two destruction box motifs (D-box) are mutated, HOXC10 is stabilized and cells accumulate in metaphase. HOXC10 appears to be a new prometaphase target of the anaphase-promoting complex (APC), since its degradation coincides with cyclin A destruction and is suppressed by expression of a dominant-negative form of UbcH10, an APC-associated ubiquitin-conjugating enzyme. Moreover, HOXC10 co-immunoprecipitates the APC subunit CDC27, and its in vitro degradation is reduced in APC-depleted extracts or by competition with the APC substrate cyclin A. These data imply that HOXC10 is a homeoprotein with the potential to influence mitotic progression, and might provide a link between developmental regulation and cell cycle control (Gabellini, 2003).

Cell cycle dependent degradation of UbcH10

Progression through mitosis requires the precisely timed ubiquitin-dependent degradation of specific substrates. E2-C is a ubiquitin-conjugating enzyme that plays a critical role with anaphase-promoting complex/cyclosome (APC/C) in progression of and exit from M phase. Mammalian E2-C is expressed in late G(2)/M phase and is degraded as cells exit from M phase. The mammalian E2-C shows an autoubiquitinating activity leading to covalent conjugation to itself with several ubiquitins. The ubiquitination of E2-C is strongly enhanced by APC/C, resulting in the formation of a polyubiquitin chain. The polyubiquitination of mammalian E2-C occurs only when cells exit from M phase. Furthermore, mammalian E2-C contains two putative destruction boxes that are believed to act as recognition motifs for APC/C. The mutation of this motif reduced the polyubiquitination of mammalian E2-C, resulting in its stabilization. These results suggest that mammalian E2-C is itself a substrate of the APC/C-dependent proteolysis machinery, and that the periodic expression of mammalian E2-C may be a novel autoregulatory system for the control of the APC/C activity and its substrate specificity (Yamanaka, 2000).

Oscillations in cyclin-dependent kinase (CDK) activity drive the somatic cell cycle. After entry into mitosis, CDKs activate the APC, which then promotes cyclin degradation and mitotic exit. The re-accumulation of cyclin A causes the inactivation of APC and entry into S phase, but how cyclin A can accumulate in the presence of active APC has remained unclear. During G1, APC autonomously switches to a state permissive for cyclin A accumulation. Crucial to this transition is the APC(Cdh1)-dependent autoubiquitination and proteasomal degradation of the ubiquitin-conjugating enzyme (E2) UbcH10. Because APC substrates inhibit the autoubiquitination of UbcH10, but not its E2 function, APC activity is maintained as long as G1 substrates are present. Thus, through UbcH10 degradation and cyclin A stabilization, APC autonomously downregulates its activity. This indicates that the core of the metazoan cell cycle could be described as a self-perpetuating but highly regulated oscillator composed of alternating CDK and APC activities (Rape, 2004).

UbcH10 and cancer

Ubiquitin-dependent proteolysis by the 26S proteasome plays a pivotal role in cell cycle progression as well as in tumorigenesis. In this pathway, ubiquitin-conjugating enzyme (E2), together with ubiquitin ligase (E3), transfers ubiquitin to the specific substrate protein(s); however, little is known about the potential contribution of E2 to tumorigenesis. In this study, the expression levels of 17 E2 genes in 25 different human normal tissues and 24 human cancerous cell lines was examined by using a quantitative real-time reverse transcription-PCR. Among the E2 gene family, the expression level of UbcH10 was extremely low in many of the normal tissues but prominent in the majority of cancerous cell lines. Intriguingly, UbcH10 was expressed at high levels in primary tumors derived from the lung, stomach, uterus, and bladder as compared with their corresponding normal tissues, suggesting that UbcH10 is involved in tumorigenesis or progression of the tumor. To further investigate a possible contribution of UbcH10 to malignant transformation and tumor cell proliferation, NIH3T3 cells were transfected with the expression plasmid encoding UbcH10, and stable transfectants were subsequently established. UbcH10-overexpressing cells exhibited an increased incorporation of bromodeoxyuridine, an enhanced growth rate, an increase in saturation density, and a promotion of colony formation in soft agar medium as compared with parental NIH3T3 cells and the control transfectants. Collectively, these results provide the first evidence that UbcH10 is highly expressed in various human primary tumors and that UbcH10 has an ability to promote cell growth and malignant transformation (Okamoto, 2003)

Gene expression profiling of anatomically diverse carcinomas and their corresponding normal tissues was used to identify genes with cancer-associated expression. The ubiquitin conjugase UbcH10 is significantly overexpressed in many different types of cancers and is associated with the degree of tumor differentiation in carcinomas of the breast, lung, ovary and bladder, as well as in glioblastomas. UbcH10 overexpression in gastro-esophageal, and probably other carcinomas may be a direct consequence of chromosomal amplification at the UbcH10 locus, 20q13.1, a region known to be amplified in diverse tumors. To evaluate whether inhibition of UbcH10 function may be therapeutically relevant in cancer, small interfering RNAs (siRNAs) were used to silence UbcH10 transcription selectively. Diminution of UbcH10 expression significantly inhibits both tumor and normal cell proliferation without inducing cell death. However, when combined with agonists of the DR5/TRAIL receptor, siRNAs directed against the UbcH10 transcript dramatically enhances killing of cancer cells, but not of proliferating primary human epithelial cells or fibroblasts. Together, these data demonstrate that UbcH10 plays an important role in tumor development and that its inhibition in combination with agonists of the TRAIL receptor may provide an enhanced therapeutic index (Wagner, 2004).


REFERENCES

Search PubMed for articles about Drosophila vihar

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