InteractiveFly: GeneBrief

Activator of SUMO 1: Biological Overview | References

Gene name - Activator of SUMO 1

Synonyms -

Cytological map position - 87B10-87B10

Function - enzyme

Keywords - regulation of SUMOylation, imaginal disc development, regulation of cell division, tumor suppressor, maternal, hematopoesis, CNS development, activation of Dorsal, protein degradation

Symbol - Aos1

FlyBase ID: FBgn0029512

Genetic map position - chr3R:8258255-8259626

Classification - E1_enzyme_family

Cellular location - cytoplasmic and nuclear

NCBI link: EntrezGene

Aos1 orthologs: Biolitmine

SUMOylation is a highly conserved post-translational modification shown to modulate target protein activity in a wide variety of cellular processes. Although the requirement for SUMO modification (see Drosophila Smt3) of specific substrates has received significant attention in vivo and in vitro, the developmental requirements for SUMOylation at the cell and tissue level remain poorly understood. This study shows that in Drosophila melanogaster, both heterodimeric components of the SUMO E1-activating enzyme are zygotically required for mitotic progression but are dispensable for cell viability, homeostasis and DNA synthesis in non-dividing cells. Explaining the lack of more pleiotropic effects following a global block of SUMO conjugation, it was further demonstrated that low levels of global substrate SUMOylation are detected in mutants lacking either or both E1 subunits. These results not only suggest that minimal SUMOylation persists in the absence of the E1 SUMO-activating complex comprising the Aos1/Uba2 heterodimeric pair (see Schematic representation of the SUMOylation pathway), but also show that the process of cell division is selectively sensitive to reductions in global SUMOylation. Supporting this view, knockdown of SUMO or its E1 and E2 enzymes robustly disrupts proliferating cells in the developing eye, without any detectable effects on the development or differentiation of neighboring post-mitotic cells (Kanakousaki, 2012).

Animal development is achieved through the coordination of cell growth, cell division and cell death. During the early embryogenesis of Drosophila melanogaster, cells divide rapidly, which is facilitated by maternal deposition of components of the cell cycle machinery. After the beginning of zygotic transcription and three rounds of mitosis (14th-16th), most cells enter their final interphase, while cells in the CNS continue to divide. At late embryogenesis, the larval tissues initiate endoreplication cycles and become polyploid. Endoreplication continues throughout the larval stages, supporting cell growth that results in a dramatic increase of larva body size. By contrast, cells of the imaginal discs (appendage primordia destined to form the adult structures at metamorphosis) continue to proliferate throughout larval development (Kanakousaki, 2012).

Although transcriptional regulation is known to play a vital role in controlling patterning and proliferation during Drosophila development, the contribution of post-translational mechanisms, such as SUMOylation, are less well understood. SUMO (Small Ubiquitin-related Modifier) belongs to a family of highly-conserved Ubiquitin-like proteins that can be conjugated to other proteins to alter their properties. SUMO modification is found in all eukaryotic organisms, and the biochemical pathway that regulates SUMO conjugation to target proteins is evolutionarily conserved. SUMO proteins are genetically encoded as precursor molecules that are proteolytically cleaved to expose the glycine-glycine motif used for conjugation. SUMO processing is carried out by specific proteases that can also remove SUMO from substrates. SUMO conjugation to substrates (SUMOylation) starts with the activation of processed SUMO, performed by an E1 SUMO-activating complex comprising the Aos1/Uba2 heterodimeric pair (Johnson, 1997). SUMO is activated by ATP hydrolysis-dependent formation of a thioester bond between the catalytic cysteine of the Uba2 subunit and the C-terminal glycine of SUMO (Bossis, 2006). Activated SUMO is then transferred to the catalytic cysteine of the E2 SUMO-conjugating enzyme Ubc9 (Drosophila Lesswright). Finally, SUMO is transferred to the ε-amino group of a lysine side chain of the target protein with the assistance of E3 SUMO protein ligases, and an isopeptide bond is formed between the lysine of the substrate and the glycine of SUMO (Bossis, 2006). Aos1/Uba2 and Ubc9 are the only E1 and E2 enzymes that facilitate SUMO conjugation in each organism. By contrast, there are several proteins that have been found to have E3 SUMO ligase activity so far, including members of PIAS family, the nuclear pore protein RanBP2 and the Pc2 protein of the polycomb group (Kanakousaki, 2012).

SUMOylation is essential in most organisms and is thought to take place in all tissues and at all developmental stages. Previous investigations have found SUMOylation to be required for numerous cellular processes, including transcription, cell cycle progression, DNA repair, subcellular localization and signal transduction. Although the role of SUMOylation in development, growth and differentiation at the organismal level is not thoroughly studied, there is evidence that ablation of the SUMOylation pathway leads to dramatic developmental defects and early lethality. Ubc9 (SUMO E2) depletion in yeast causes cell cycle arrest at G2/M. Likewise, Aos1 or Uba2 depletion in yeast cells leads to the formation of microcolonies comprising enlarged cells (Johnson, 1997). In mice, a Ubc9 null allele causes early embryonic lethality and apoptosis of the inner cell mass (ICM) with reported mitotic chromosome condensation and segregation defects. Similarly, lack of Ubc9 function causes embryonic lethality in zebrafish. In Drosophila germline clones that remove maternal smt3 (Drosophila SUMO), 70% of embryos fail to hatch and those that survive die during the first larval instar (Nie, 2009). Zygotic mutant animals for the same smt3 mutation die in the early second instar (Nie, 2009). Additionally, P-element induced lesswright alleles (lwr, Drosophila E2) cause late embryonic or first instar larval lethality owing to the inability of maternal Bicoid to enter the nuclei during embryogenesis (Epps, 1998). Apart from these more general defects, SUMO is also reported to have specific roles in individual developmental processes. RNAi knockdown of smt3 (SUMO) in the prothoracic gland results in a prolonged larval life and pupariation is impeded due to inefficient production of Ecdysone (Talamillo, 2008). In addition, transcription factors that are involved in wing disc patterning, such as Vestigial (vg), Spalt (sal) and Spalt-related (salr), are regulated by SUMOylation (Takanaka, 2005; Sanchez, 2010; Kanakousaki, 2012 and references therein).

The present study examined the role of the E1 SUMO-activating enzyme subunits Aos1 and Uba2 during Drosophila development. Surprisingly, it was found that aos1 and uba2 mutants exhibit specific defects in imaginal disc development and lethality at the larval/pupal transition, without any obvious defects in larval cell growth or survival. Aos1 and Uba2 control global SUMOylation, but evidence is also presented that low levels of global SUMOylation can persist in the absence of Aos1/Uba2 activity. Finally, based on zygotic null phenotypes and RNAi knockdowns, it was shown that SUMO E1 activity is crucial for cell division, but may be only minimally required for DNA replication, cell survival and homeostasis in non-dividing cells (Kanakousaki, 2012).

The present study shows that aos1 and uba2 mutant animals exhibit late third instar/early pupal lethality and a severe disruption of imaginal disc and optic lobe development. Mosaic analysis of the aos1 mutation in discs revealed that the mutant cells undergo apoptosis, but when apoptosis is blocked, the aos1 mutant imaginal disc cells arrest in G2/M. The disrupted cell cycle of aos1 mutant disc cells could be the cause of the apoptosis observed in mutant clones. However, it has recently been shown that SUMOylation regulates the JNK pathway as well, through Hipk kinase (Huang, 2011). This suggests that the apoptosis of SUMOylation-deficient cells is probably a synergistic result of many different causes (Kanakousaki, 2012).

The accumulation of SUMOylation-deficient cells in G2/M has been observed both in yeast and in zebrafish upon Ubc9 depletion. However, it is less clear what causes the cell cycle arrest. Activation of Cdk1 was tested as defined by the presence of mitotic cyclins and it was found that both Cyclin A and Cyclin B are present in aos1 mutant cells. Similarly, the mitotic cyclins are stabilized in yeast ubc9 (E2) mutants. The effect of SUMOylation in degradation of mitotic cyclins could be direct, but so far there is no evidence that cyclins are modified by SUMO. By contrast, it has been reported that Pds1 (Securin) is also stabilized in Δsmt3 or Δubc9 mutant yeast cells. Pds1 is an APC (anaphase promoting complex) substrate like the mitotic cyclins, and the same study shows that more APC substrates are stabilized in SUMOylation-deficient cells. Thus, it seems that lack of SUMOylation affects APC-mediated proteolysis. However, it is not known whether any of the APC subunits are modified by SUMO, or if other factors that regulate APC activity require SUMOylation. In addition, no evidence of metaphase arrest was observed in aos1 mutant disc cells, which indicates that there must be earlier defects in these cells apart from the inefficient degradation of substrates by the APC. Another possibility is that SUMOylation regulates activation of Cdk1 for mitotic entry by controlling its phosphorylation through the competitive functions of String phosphatase and Wee1 kinase. In yeast cells, the Wee1 homolog is a target of SUMOylation, and SUMO modification affects its protein stability. In addition, several other proteins that are known to have a role in mitosis are SUMOylation targets in Drosophila (Nie, 2009). Among these, Polo kinase is required for spindle formation, Topoisomerase 2 has a role in chromosome segregation and PP2A functions at multiple stages of the cell cycle (Kanakousaki, 2012).

In zebrafish ubc9 mutants, a small percentage of mutant cells undergo an additional round of DNA replication, resulting in 8N DNA content. In yeast uba2 mutants, it is also reported that about 10% of affected cells have two buds. Hence, upon blockage of progression through mitosis, it seems that some mutant cells can re-enter the cell cycle and become polyploid. These observations indicate that the rest of the cell cycle (but not mitosis) can be completed when SUMOylation is reduced or eliminated. It was also found that endoreplication (S-phase progression) is not affected in larval cells of aos1 mutant animals. Collectively, these observations indicate that SUMOylation is strictly required for G2/M progression in vivo, with lesser or negligible requirements in G1/S (Kanakousaki, 2012).

These findings contrast with the observation that dsRNA knock down of SUMO in Drosophila S2 cells causes a G1/S arrest. Indeed, proteins required for the G1/S transition (Cdc2c/Cdk2) or DNA synthesis (RFC2, PCNA) are SUMOylation targets (Nie, 2009). This may indicate that SUMOylation is required for G1/S progression under certain conditions, although the data indicate that the demand for SUMOylation in G2/M progression is substantially higher in vivo. The fact that mutations in SUMOylation enzymes, such as Ubc9 in zebrafish and yeast, and Aos1 in Drosophila (this work), cause arrest in G2/M could be due to the fact that SUMOylation declines gradually in mutant cells as the enzyme is progressively consumed. A few E1 or E2 enzyme molecules could plausibly catalyze many SUMO conjugation reactions. As a result, cells would arrest when they reached the higher threshold of SUMOylation required for G2/M progression. By contrast, knockdown of SUMO itself in S2 cells (Nie, 2009) may cause a more sudden drop in SUMOylation levels below the threshold required for G1/S progression and thus the cells arrest in G1/S (Kanakousaki, 2012).

A requirement for aos1 in cell cycle progression can explain the developmental defects in the highly proliferative imaginal discs and optic lobes of aos1 mutant larvae. Furthermore, these are tissues where the aos1 gene is most highly expressed, reflecting the need for high levels of SUMOylation in dividing cells. Likewise, early embryonic divisions coincide with high levels of maternal aos1 and the rest of the components of SUMO conjugation pathway (Hashiyama, 2009). Maternal deposition of aos1 can thus explain the survival of zygotic null mutants through embryogenesis. Indeed, upon induction of aos1c06048 germline clones, it was found that embryos generally failed to hatch, similar to the results obtained for smt3 (Nie, 2009). Germ line clones of the aos1N1-null allele failed to produce any eggs, probably owing to very early defects in oogenesis (Kanakousaki, 2012).

One interesting aspect of the aos1 phenotype was developmental delay and the formation of sporadic melanotic tumors. The formation of melanotic tumors has also been observed in lwr mutant larvae, which indicates this may be a common consequence of disrupted SUMOylation. Melanotic tumors in lwr4-3/lwr5 and lwr5/lwr14 mutant larvae are caused by misregulation of the Toll/Dorsal pathway, which results in overproliferation of hemocytes and increased lamellocyte differentiation. However, in lwr mutant animals with a stronger allelic combination (lwr4-3/lwr14), increased lamellocyte differentiation was observed without an increase in the total number of circulating hemocytes. Melanotic tumor formation could still take place in these animals but with lower penetrance. Thus, melanotic tumors are not necessarily the result of hemocyte overproliferation, which is consistent with the findings that disrupted SUMOylation results in defects in cell cycle progression. Instead, an abnormal increase in lamellocyte differentiation due to Toll pathway misregulation could be the cause of the mild melanotic tumor formation observed in aos1c06048 mutant larvae. Indeed, an increase in circulating lamellocytes was recently reported for aos1c06048 mutant larvae (Kalamarz, 2012). Nevertheless, the possibility cannot be excluded that hemocytes in aos1 mutant larvae could still be able to perform a few rounds of division before their maternal Aos1 protein is consumed and mitosis is blocked (Kanakousaki, 2012).

This study found that larval tissues comprising non-diving, endoreplicating cells were not overtly affected by Aos1 depletion. In fact, the aos1 and uba2 mutant phenotypes resembled those of several mutations that inhibit cell cycle progression and cause late third instar larval/early pupal lethality and defects in imaginal disc development. This implies, unexpectedly, that aos1 and uba2 primarily function in cell cycle progression. Therefore global SUMOylation levels were monitored in mutants, and SUMOylation was found to be dramatically reduced in aos1, uba2 and double mutant larvae, but not completely eliminated. The remaining SUMOylation in aos1-null mutants could potentially be attributed to the function of residual maternal Aos1. However, the presence of Aos1 could not be detected by western blot. Another possibility is that the residual SUMOylation is a result of decreased deSUMOylation in aos1 mutant animals in response to disrupted SUMO conjugation, and this leads to some persistently SUMO-modified substrates. An alternative explanation could be that the SUMO-activating function occurred independently of Aos1/Uba2, possibly by an E1 complex primarily assigned to another Ubiquitin-like molecule with low affinity for SUMO. However, there is no evidence so far of an E1 other than Aos1/Uba2 that can activate SUMO. In sum, the current evidence suggests that endoreplicating larval cells have minimal requirements for SUMO conjugation and the residual SUMOylation observed in zygotic E1 mutants is sufficient for their survival and growth. Consistent with these findings, endoreplicating trophoblastic cells in ubc9 mutant mice embryos are viable and morphologically normal (Kanakousaki, 2012).

Supporting the idea of quantitatively lower requirements for SUMOylation outside of cell division, it was found that RNAi-mediated disruption of aos1, uba2, ubc9 and smt3 (SUMO) had no discernible effect on post-mitotic cells in the eye disc. In proliferating cells of the same tissue, however, the same constructs produced severe developmental defects. Similarly, no obvious phenotype was observed when Elav-Gal4 was used to knock down aos1 and uba2 in all differentiated neurons of the entire animal. Thus, as with endoreplicating larval cells, survival and homeostasis of differentiated cells in imaginal discs was not affected by reduction of SUMOylation. Overall these results imply that minimal SUMOylation levels may be sufficient to mediate non-mitotic functions of the pathway (Kanakousaki, 2012).

A reduced requirement for SUMOylation in non-dividing larval cells can explain the late third instar/early pupal lethality of aos1 and uba2 mutants. However, the semi class of lwr (E2) mutants and smt3 mutants both die much earlier. In the case of E2 enzyme, lwr13/lwr13 homozygotes and lwr4-3/lwr13 and lwr5/lwr13 trans-heterozygotes both exhibit lethality at the third instar larval stage. lwr13 is also molecular null as it deletes the entire lwr locus. In the smt3 mutant case, lethality at earlier stages than the aos1 and uba2 mutants could be due to differential perdurance of the respective maternal proteins. An alternative explanation could be that there is an Aos1/Uba2-independent SUMO-activating mechanism that facilitates low level SUMOylation in aos1 and uba2 mutants, but cannot be useful upon elimination of SUMO in smt3 mutants. Combined with previous observations in Drosophila and other organisms, the results suggest that robust SUMOylation is essential for cell division but is required less for the cellular processes that mediate survival and homeostasis. Importantly, the selective requirement for SUMOylation in dividing cells could suggest a potential new avenue for therapeutic intervention in proliferative disease (Kanakousaki, 2012).

Sumoylation is tumor-suppressive and confers proliferative quiescence to hematopoietic progenitors in Drosophila melanogaster larvae

How cell-intrinsic regulation of the cell cycle and the extrinsic influence of the niche converge to provide proliferative quiescence, safeguard tissue integrity, and provide avenues to stop stem cells from giving rise to tumors is a major challenge in gene therapy and tissue engineering. This question was explored in sumoylation-deficient mutants of Drosophila. In wild type third instar larval lymph glands, a group of hematopoietic stem/progenitor cells acquires quiescence; a multicellular niche supports their undifferentiated state. However, how proliferative quiescence is instilled in this population is not understood. This study showed that Ubc9 protein is nuclear in this population. Loss of the SUMO-activating E1 enzyme, Aos1/Uba2, the conjugating E2 enzyme, Ubc9, or the E3 SUMO ligase, PIAS, results in a failure of progenitors to quiesce; progenitors become hyperplastic, misdifferentiate, and develop into microtumors that eventually detach from the dorsal vessel. Significantly, dysplasia and lethality of Ubc9 mutants are rescued when Ubc9(wt) is provided specifically in the progenitor populations, but not when it is provided in the niche or in the differentiated cortex. While normal progenitors express high levels of the Drosophila cyclin-dependent kinase inhibitor p21 homolog, Dacapo, the corresponding overgrown mutant population exhibits a marked reduction in Dacapo. Forced expression of either Dacapo or human p21 in progenitors shrinks this population. The selective expression of either protein in mutant progenitor cells, but not in other hematopoietic populations, limits overgrowth, blocks tumorogenesis, and restores organ integrity. An essential and complex role for sumoylation in preserving the hematopoietic progenitor states for stress response and in the context of normal development of the fly is discussed (Kalamarz, 2012).

In a quest to identify the source of microtumors in Ubc9 mutants, this study discovered that even though Ubc9 protein is ubiquitously expressed, it plays a specific and essential, niche-independent function in maintaining proliferative quiescence within progenitors of the medullary and transition zones. Reduction of sumoylation via knockdown of any of the other core enzymes of the pathway also leads to progenitor dysplasia and tumorogenesis. Once detached from the dorsal vessel, the microtumors float in the hemolymph (Kalamarz, 2012).

The progenitor population that serves as the source of microtumors is heterogeneous with respect to Dome>GFP and ZCL2897 expression. One of the earliest detectable effects of the mutation is on the differential expression of Dome>GFP and ZCL2897 or 76B>GFP in the expanding population. The onset of the effects of Ubc9 mutation coincides with the period when the progenitors in the medulla of the anterior lobes undergoes proliferative restraint. At the same time, cells of the posterior lobes lag behind; they continue to divide and follow a defined heterochronic developmental pattern. It is somewhat surprising that even though the Ubc9 mutation has differential effects on cells of the anterior versus posterior lobes, the overproliferation defects in both are largely rescued by ectopic expression of p21/Dap. This observation suggests a fundamental role for the enzyme in inhibiting cell cycle progression and conferring quiescence to progenitors. Since the decline in Dome>GFP expression precedes overproliferation in mutant lobes and each defect can be rescued by the expression of wild type Ubc9, it is possible that Dome>GFP expression marks the quiescent cell state. The inability of p21 or Dap to restore normal Dome>GFP expression attests to the notion that the sequential series of events, even at the earliest stages of tumorogenesis, can be genetically teased out in vivo (Kalamarz, 2012).

While the changes in cell identities in mutant lobes are complex, the discovery of heterogeneity in the medullary zone populations of anterior and first posterior lobes is consistent with recent reports that this population has distinct fate-restricted cell populations. The current results suggest that lymph gland progenitors are similar to mammalian transit amplifying cells or those in the Drosophila testis, that have limited proliferative capacity and possess a restricted differentiation potential relative to their multipotent stem cells. With an appropriate immune or developmental cue, Drosophila hematopoietic progenitors may re-enter the cell cycle to produce differentiated progeny (Kalamarz, 2012).

What is the physiological significance of retaining some cells in quiescence at this stage in larval life? One possibility is that mitotic exit shelters progenitors from precocious development and provides a mechanism that determines the number of times they must divide before they differentiate. Additionally, a reserve of progenitors, ready to divide and differentiate rapidly guards larvae against natural enemies such as parasitic wasps that attack them at this stage of the life cycle. This tactic parallels mitotic exit of hematopoietic stem cells (HSCs) in mice about three weeks after birth, or in humans, at about four years of age, when they become adult HSCs. The dormant adult HSCs are activated as the organism recovers from injury (Kalamarz, 2012).

This similarity in strategies between flies and humans in normal hematopoiesis is further reinforced even when the process becomes aberrant. Like in dUbc9 mutants, uncontrolled proliferation of progenitors in human leukemias can occur independently of the signals from the niche. It is intriguing that Antp, a niche marker, is also expressed in the dorsal vessel. Furthermore, Dome>GFP expression, undetectable in normal cells, is strongly activated in mutant cells of the dorsal vessel. Thus, it is possible that cues from the cells of the dorsal vessel influence the state of the hematopoietic progenitors and integrity of the lobes. Conversely, the status of the progenitors themselves may determine the association of the lobes to the dorsal vessel. Further analysis of Ubc9 mutants will clarify the role of the microenvironment in supporting progenitor quiescence and maintaining tissue integrity (Kalamarz, 2012).

A key mechanism by which sumoylation maintains proliferative quiescence in larval hematopoiesis is cell cycle regulation through Dacapo/p21. In the embryo, Dap/p21 binds to cyclin E/Cdk2 complexes to block the G1/S transition in cell cycle. Furthermore, the human p21 protein can block mitosis in the Drosophila eye. This function of Dap/p21 in larval hematopoiesis is similar to the roles of p27KIP1 or p21CIP1/WAF1 in enforcing HSC quiescence (Kalamarz, 2012).

Dap is expressed in Dome>GFP progenitors in wild type and mutant glands, and is reduced shortly after Dome>GFP is downregulated in mutant glands. Overexpression of Dap/p21 in these cells leads to decrease in progenitor number. It is noteworthy that dap mutants do not exhibit apparent tumorous overgrowth, a trait that is similar to young p21 null mice. However, with age, or in the presence of other mutations (e.g., oncogenic Ras), p21 null mice are prone to developing tumors. It is therefore very likely that tumorogenesis in Ubc9 mutants is supported not only by loss of Dap/p21 but also by the activation of other oncogenic and pro-inflammatory proteins (Kalamarz, 2012).

The mechanism by which Ubc9 controls Dap protein levels is not known. dap transcription has been studied in embryonic development where it regulates mitotic exit. High dap transcript levels in stage 16 embryonic central and peripheral nervous system, or in differentiating postmitotic cells of a developing eye disc, correlate with exit from mitosis. These observations suggest that regulation of dap transcription is coupled with mitotic exit, and it is therefore possible that its transcription in the lymph gland progenitors is similarly synchronized. Microarray experiments of whole Ubc9 larvae compared to their heterozygous siblings indicate dap transcript downregulation. An intriguing possibility is that Dacapo itself, or another protein in complex with Dap, is a sumoylation target. In high throughput yeast two-hybrid assay, Dap was found to physically interact with Ubc9. Future experiments including biochemical analyses of Dap and interacting proteins are required to test this idea (Kalamarz, 2012).

The causal relationship between cancer and inflammation is now widely accepted, even though the mechanisms that establish and sustain this relationship remain unresolved . Drosophila Toll-Dorsal pathway not only manages immunity, but also governs hematopoietic development. Ubc9 microtumor development requires Rel/NF-kappa B family transcription factors Dorsal and Dif. Aberrant activation of NF-kappa B signaling in Ubc9 mutants resembles hematopoieitic malignancies in vertebrates that arise due to ectopic germline or somatic disruption of the pathway (Kalamarz, 2012).

It has recently been discovered that sumoylation provides a homeostatic mechanism to restrain systemic inflammation in the fly larva, where it keeps the Toll/Dorsal-dependent immune response in check. Ubc9 controls the 'set point' by maintaining normal levels of IkappaB/Cactus protein in immune tissues (Paddibhatla, 2010). The Ubc9 cancer-inflammation model offers novel opportunities to examine the dynamics of tumor growth, its relationship to metastasis, and the links between cancer and inflammation. Ubc9 tumors are sensitive to aspirin. This model is well-suited for identifying and testing drugs that target highly-conserved biochemical mechanisms, such as sumoylation, which oversee self-renewal pathways in progenitor populations (Kalamarz, 2012).

Expression of genes involved in sumoylation in the Drosophila germline

Post-translational modification of proteins by the covalent addition of small ubiquitin-related modifier (SUMO) proteins has been reported to regulate a wide range of cellular processes. However, the spatiotemporal expression pattern of genes encoding the sumoylation machinery during development is largely unknown. This study reports the expression of five sumoylation genes, Uba2, Aos1, smt3, Ulp1, and lesswright (lwr), in the Drosophila germline. Transcripts from all five genes were detected throughout the early embryo by whole-mount in situ hybridization, while they were predominantly expressed in the germline progenitors, or pole cells, in late stage embryos. These genes were also expressed in the germline during oogenesis and spermatogenesis. Finally, it was found that SUMO protein was enriched in the nuclei of pole cells and gametogenic cells. Given that a large fraction of SUMO substrates are nuclear proteins, these data suggest that sumoylation is highly active in the germline. These data provide a basis for understanding how sumoylation regulates germline development (Hashiyama, 2009).

Identification of septin-interacting proteins and characterization of the Smt3/SUMO-conjugation system in Drosophila

The septins are a family of proteins involved in cytokinesis and other aspects of cell-cortex organization. In a two-hybrid screen designed to identify septin-interacting proteins in Drosophila, several genes were isolated, including homologues (Dmuba2 and Dmubc9) of yeast UBA2 and UBC9. Yeast Uba2p and Ubc9p are involved in the activation and conjugation, respectively, of the ubiquitin-like protein Smt3p/SUMO, which becomes conjugated to a variety of proteins through this pathway. Uba2p functions together with a second protein, Aos1p. The Drosophila homologues of AOS1 (Dmaos1) and SMT3 (Dmsmt3) were also cloned and characterized. The biochemical data suggest that DmUba2/DmAos1 and DmUbc9 indeed act as activating and conjugating enzymes for DmSmt3, implying that this protein-conjugation pathway is well conserved in Drosophila. Immunofluorescence studies showed that DmUba2 shuttles between the embryonic cortex and nuclei during the syncytial blastoderm stage. In older embryos, DmUba2 and DmSmt3 are both concentrated in the nuclei during interphase but dispersed throughout the cells during mitosis, with DmSmt3 also enriched on the chromosomes during mitosis. These data suggest that DmSmt3 could modify target proteins both inside and outside the nuclei. No concentration of DmUba2 was observed at sites where the septins are concentrated, and DmSmt3 modification of the three Drosophila septins tested was not detected. However, DmSmt3 localization to the midbody was observed during cytokinesis both in tissue-culture cells and in embryonic mitotic domains, suggesting that DmSmt3 modification of septins and/or other midzone proteins occurs during cytokinesis in Drosophila (Shih, 2002).

To identify proteins interacting with the Drosophila septins, two-hybrid screens were conducted using Pnut, Sep1, and Sep2 as baits. These screens identified 27 positive clones that proved to represent eight genes. Among these were the other septins, as expected from other data indicating that septins interact with one another. In addition, the screens identified Drosophila homologues (Dmuba2 and Dmubc9) of yeast UBA2 and UBC9, whose products are involved in the activation and conjugation of the ubiquitin-like molecule Smt3p/SUMO. These screening results and the recent discovery of Smt3p modification of septins in S. cerevisiae stimulated a study the Smt3p/SUMO conjugation machinery in Drosophila (Shih, 2002).

In further two-hybrid analyses, the C-terminal portion of DmUba2 interacted strongly with full-length Sep1 and Sep2 and with the N-terminal portion of Sep1. Interactions were also detected with the N-terminal portions of Pnut and Sep2 and with the C-terminal portions of Sep1 and Sep2. In contrast, a full-length AD-DmUba2 fusion showed none of these interactions, although other studies indicated that this fusion was functional for other interactions. Interestingly, full-length AD-DmUbc9 showed a pattern of interactions very similar to those seen with the C-terminal portion of DmUba2 (Shih, 2002).

The other genes identified in the screens had not been described previously; they were designated sip1-sip4 (for septin-interacting protein). sip1 (Accession No. AF221101; Drosophila genome annotation No. CG7238) encodes a protein with predicted P-loop and coiled-coil domains; it appeared to interact specifically with the C-terminal portion of Pnut. sip2- sip4 encode proteins without obviously informative motifs. sip2 (CG9188) encodes a protein that interacted with full-length Sep1 and Sep2 but not with Pnut. sip3 (CG1937) encodes a protein that appeared to interact specifically with the C-terminal portion of Sep2. sip4 was identified independently as dip2 (Dorsal interacting protein 2); it encodes a protein that interacts with all of the Sep1 and Sep2 fusions and (weakly) with the N-terminal portion of Pnut (Shih, 2002).

Sequencing of a full-length clone of Dmuba2 showed that the predicted DmUba2 contains 700 amino acids and has 29% sequence identity to yeast Uba2p and 48% identity to human hUba2. Like its homologues and other E1-type enzymes, DmUba2 contains an ATP-binding motif (amino acids 26-31) and the consensus Cys (C175) corresponding to those essential for thiolester bond formation in other E1-type enzymes. The original two-hybrid clone of Dmubc9 appeared to be full length by comparison to yeast UBC9. Antibodies were raised to DmUba2. The affinity-purified antibodies recognized mainly one polypeptide of apparent molecular weight ~97 kDa, which is presumably DmUba2 (predicted molecular weight, 77.5 kDa). Support for this conclusion was obtained by expressing Dmuba2 under GAL-promoter control in yeast. When cells were grown under inducing conditions, the antibodies recognized primarily a polypeptide of apparent molecular weight ~97 kDa that was absent when cells were grown under repressing conditions. Similarly anomalous low mobility on SDS-PAGE has been noted for both Uba2p and hUba2 (Shih, 2002).

In S. cerevisiae, the E1 enzyme for ubiquitin activation is the 1024 amino-acid Uba1p (see Drosophila Uba1). In contrast, Smt3p is activated by a heterodimer of the 636 amino-acid Uba2p, which is related to the C-terminal part of Uba1p, and the 347 amino-acid Aos1p, which is related to the N-terminal part of Uba1p. Similarly, the 700 amino-acid DmUba2 is related in sequence (~40% identity over the ~225 amino acids of the three similarity boxes defined for other Uba1- type and Uba2-type enzymes to the C-terminal part of the putative Drosophila ubiquitin-activating enzyme DmUba1. Therefore, a Drosophila homologue of yeast AOS1 was identified among the ESTs from the BDGP (Shih, 2002).

Sequencing (Accession No. AF193554) showed that Dmaos1 encodes a polypeptide of 337 amino acids that has 28% sequence identity to yeast Aos1p and 40% identity to the human Aos1p homologue Sua1. As expected, DmAos1 is related in sequence to the N-terminal part of DmUba1 (~37% identity over the ~202 amino acids of the similarity boxes as defined previously, suggesting that a heterodimer of DmUba2 and DmAos1 is the Drosophila Smt3/SUMO-activating enzyme. In support of this hypothesis, an interaction was detected between full-length DmUba2 and full-length DmAos1 in the two-hybrid system. An attempt to use the two-hybrid system to delimit the region of DmUba2 responsible for its interaction with DmAos1 was unsuccessful (Shih, 2002).

A homolog of SMT3/SUMO-1 was identified among the BDGP EST clones. The predicted DmSmt3 contains 90 amino acids with 48% identity to yeast Smt3p and 54% identity to human SUMO-1. Polyclonal antibodies were generated and immunoblots were performed on fly extracts, expecting to detect both free DmSmt3 and DmSmt3- modified proteins. Because yeast and mammalian cells contain Smt3p/SUMO-1-specific isopeptidases, which remove Smt3/SUMO from modified proteins, extracts were prepared both with and without N-ethylmaleimide (NEM), an isopeptidase inhibitor. In both extracts, the purified antibodies recognized both a polypeptide of ~16 kDa (presumably free DmSmt3) and many polypeptides of higher molecular weight (presumably DmSmt3-conjugated proteins). As expected, the higher molecular-weight species were both less abundant and of lower average molecular weight when extracts were prepared without NEM (Shih, 2002).

To test the hypothesis that DmUba2/DmAos1 and DmUbc9 are activating and conjugating enzymes for DmSmt3, but not for ubiquitin (DmUb), in vitro protein-binding assays were used to investigate the interactions among these proteins. Because ubiquitin and ubiquitin-like proteins undergo proteolytic cleavage of their C-termini to leave the sequence Gly-Gly, which is essential for both activation and conjugation, DmSmt3 and DmUb were cloned such that they terminated with Gly88 (DmSmt3) or Gly76 (DmUb) and were tagged with His6 at their N-terminal ends. Then incubated purified His6DmSmt3 and purified His6DmUb were incubated with fly extracts. The His6-tagged proteins were isolated using Ni-NTA beads, and the associated proteins were analyzed. As expected, both DmUba2 and DmUbc9 associated only with His6DmSmt3 and not with His6DmUb. The anti-DmUba2 antibodies detected not only the free form of DmUba2 (~97 kDa) but also species whose lower mobilities suggested that they might represent DmUba2 conjugated to one, two, or three molecules of DmSmt3 (and/or some other ubiquitin-like molecule). To ask if the interactions of DmUba2 and DmUbc9 with DmSmt3 involved thiolester bonds, the experiments were repeated but ß-mercaptoethanol (which reduces thiolester bonds) was omitted during sample preparation. As expected, the most abundant species now observed with the anti-DmUba2 antibodies had an apparent molecular weight of ~116 kDa, consistent with its being DmUba2 with a single His6DmSmt3 linked by a thiolester bond. Similarly, the anti-DmUbc9 antibodies now revealed an additional species with an apparent molecular weight of ~30 kDa, presumably representing DmUbc9 with a single His6DmSmt3 linked by a thiolester bond (Shih, 2002).

Immunoprecipitation was used to ask whether DmUba2 and DmUbc9 interact in vivo. When immunoprecipitates were prepared from embryo extracts using antibodies to either protein, the other protein was detected by immunoblotting. Taken together, the results of in vitro binding assays and coimmunoprecipitation suggest that DmUba2/DmAos1 and DmUbc9 are indeed the activating and conjugating enzymes, respectively, for DmSmt3. They may form a complex containing both the E1-type and E2-type enzymes (Shih, 2002).

To begin investigating the roles of the DmSmt3-conjugation pathway in Drosophila, immunofluorescence and confocal microscopy was used to characterize the intracellular localization of DmUba2. Yeast Uba2p is concentrated in the nucleus, but the localization of the homologous enzyme has not been examined in multicellular organisms. Interestingly, DmUba2 is not exclusively nuclear during early embryogenesis. Before migration of the nuclei to the embryo cortex after nuclear division 9, DmUba2 was found largely in the cortex, and its distribution there appeared homogenous. During the interphase preceding nuclear division 10, DmUba2 gradually became organized into a cap corresponding approximately to the cortical actin cap that forms over each nucleus. DmUba2 was also found in the deeper cytoplasm and gradually moved into the nuclei. During mitosis, DmUba2 was dispersed in the cortex and in the cytoplasm near the cortex. During the three subsequent nuclear cycles, the cap-like localization, progressive nuclear accumulation, and dispersion during mitosis of DmUba2 remained evident. However, during the successive cycles, the cap-like cortical localization became more organized and the degree of nuclear enrichment became more pronounced. By cycle 13, although some DmUba2 still localized to the cortex during interphase, it was predominantly nuclear (Shih, 2002).

Because DmUba2 was identified by its two-hybrid interaction with Sep1 and Sep2, whether DmUba2 colocalized with the septins either at the cellularization front or in cleavage furrows in mitotic domains after gastrulation was examined carefully. During cellularization, most DmUba2 localized to nuclei, accumulating preferentially at their apical ends. No DmUba2 was detected at the cellularization front. However, some DmUba2 remained at the cortex, where diffuse septin staining was also observed. During mitosis in older embryos, DmUba2 spread throughout the cell but did not become detectably concentrated in cleavage furrows; it then moved back into the nuclei after mitosis, with no detectable concentration at the midbody. Thus, no substantial colocalization of DmUba2 and septins was detected in early embryogenesis. However, it remains possible that DmUba2 could interact with septins at the cortex in syncytial-blastoderm embryos, during cellularization, or in mitotic cells. DmUba2 was concentrated in nuclei of nondividing cells and dispersed throughout the cell during mitosis throughout embryonic stages 6 to 15. This was particularly striking in the CNS, where the septins, in contrast, are enriched in axons (Shih, 2002).

DmUba2 localization was also examined during oogenesis. DmUba2 localized to the nuclei of both germ cells and somatic follicle cells in the germarium. After encapsulation of the germ-line cells by the follicle cells, DmUba2 remained localized to follicle cell nuclei. DmUba2 localization to nurse cells decreased as egg chamber development progressed, but it remained enriched in the oocyte nucleus. In contrast, Pnut localizes primarily to the basal surface of the follicle cells and is excluded from nuclei (Shih, 2002).

The two-hybrid interactions between the septins and DmUba2 and DmUbc9 might reflect a physiologically significant but transient interaction, such as might occur if Drosophila septins, like yeast septins, are Smt3 modified. To explore this possibility, immunofluorescence was used to examine DmSmt3 localization in cultured cells and in cellularizing and older embryos. DmSmt3 did not colocalize detectably with the septins at the cellularization front. Instead, DmSmt3 localized to nuclei, with a particular enrichment at their apical ends, as did DmUba2. In cultured cells and in cells of postgastrulation embryos, DmSmt3 was concentrated in nuclei throughout interphase. During mitosis, DmSmt3 initially appeared to spread throughout the cell. However, during metaphase, DmSmt3 appeared to concentrate in the region of the chromosomes; this was confirmed by localizing DmSmt3 relative to the mitotic spindle. Strikingly, DmSmt3 was also found concentrated in a spot at cleavage furrows and midbodies both in cultured cells and in dividing embryonic cells; this spot overlapped, but did not appear to coincide fully, with the concentration of the septins in these furrows. DmSmt3 was not enriched at the cleavage furrows early in furrow formation, but only during later stages, and it remained concentrated in the midbody after most DmSmt3, and essentially all of the DmUba2, had reaccumulated in nuclei (Shih, 2002).

DmSmt3 localization was also examined during other stages of embryogenesis. During the syncytial cell cycles, DmSmt3 was concentrated in the nuclei during interphase and appeared to localize to the chromosomes during mitosis. Like DmUba2, DmSmt3 localized primarily to the nuclei of non-mitotic cells throughout the rest of embryogenesis, including in the CNS (where the septins, in contrast, localized to axons). The concentration of DmSmt3 in late cleavage furrows and midbodies suggested that one or more of the Drosophila septins might be modified by DmSmt3. Various experimental approaches were tried to look for DmSmt3-modified septins. None of these approaches detected DmSmt3 modification of Pnut, Sep1, or Sep2. These negative results may mean that the septins are not Smt3 modified. However, immunofluorescence studies detected colocalization of Smt3 with the septins only for a brief period at the end of mitosis, and even at this stage the overlap was not complete. Thus even if septins are Smt3 modified during this period, they would probably comprise only a very small fraction of the total septins in the embryo and thus might have escaped detection (Shih, 2002).

The Drosophila septins appear to be essential for cytokinesis in at least some cell types, and it is likely that they have a variety of non-cytokinesis roles as well. Because studies in yeast suggest that a primary function of the septins is to serve as a matrix or template for the organization of other proteins at the cell surface, the identification of septin-interacting proteins should be critical to the elucidation of septin function in Drosophila. This study began with an attempt to identify such proteins using the yeast two-hybrid system. Of 27 positive clones identified with three septin baits, 17 contained fragments of the septin genes themselves. Because other evidence suggests strongly that the septins interact with each other in vivo, this result suggests that the baits used were good and that the screen was otherwise of high fidelity. Thus, it seems likely that at least some of the other positive clones represent genes whose products really interact with septins in vivo (Shih, 2002).

In conclusion several lines of evidence suggest that the two-hybrid interactions observed between DmUba2, DmUbc9, and the septins reflect physiologically significant interactions. (1) Among the 10 positive clones that did not encode septins, four contained either Dmuba2 or Dmubc9, and Dmuba2 fragments were isolated with two different septin baits. (2) Because DmUba2 and DmUbc9 also interact with each other, the identification of both genes independently in screens using septin baits suggests that the interactions are relevant. (3) While these studies were in progress, it became clear that yeast septins are extensively modified by Smt3p, although the functional significance of that remains uncertain. (4) Several other groups also isolated Uba2 and/or Ubc9 in two-hybrid screens using other protein baits. Several of these interactors, including Drosophila calcium/calmodulin-dependent kinase II (CAM-kinase II; Long, 2000) and Dorsal (Bhaskar, 2000), were subsequently shown to be Smt3 modified (Shih, 2002).

Other genetic data may also reflect an interaction between the septins and the DmSmt3 system. The pnut septin mutation was originally identified as an enhancer of a sina mutation that affects R7 photoreceptor development. Although the significance of this genetic interaction remains unclear, it may reflect the recently discovered crosstalk between Smt3/SUMO modification and ubiquitination. Mammalian SUMO-1 is conjugated to the protein Mdm2, a RING-finger E3 ubiquitin ligase involved in p53 degradation. Modification by SUMO-1 appears to regulate Mdm2 activity and hence the level of p53, probably by regulating the ubiquitination and degradation of Mdm2 itself. Other RING-finger proteins, including Drosophila Sina, interact with Ubc9 family proteins and/or are modified by Smt3/SUMO . Thus, it seems possible that DmSmt3 modification regulates the activities of Sina, such as its role in downregulating the transcriptional repressor Tramtrack (one of whose isoforms is itself DmSmt3 modified), and that Pnut plays a role in mediating the requisite interactions. Finally, in several types of dividing Drosophila cells, it was found that DmSmt3 colocalizes with septins in the cleavage furrows and/or midbodies during cytokinesis. Interestingly, DmSmt3 is not enriched in the furrow during the early stages of furrow formation but only later, at a time when most DmUba2 has moved back into the nuclei. Although DmSmt3 modification of Sep1, Sep2, or Pnut could not be detected (despite considerable effort), it remains possible that one or more of these proteins is modified at low levels or that DmSmt3 is conjugated to Sep4 or Sep5. However, it is also possible that the DmSmt3 in the midzone is conjugated to other proteins, such as the 'chromosomal passenger proteins,' which are associated with the chromosomes and then relocate to the spindle midzone in mitosis. The involvement of such proteins in chromosome segregation as well as in cytokinesis might help to explain the observations suggesting that the Smt3/SUMO system is involved in chromosome segregation (Shih, 2002 and references therein).

It also remains unclear whether the Drosophila septins ever serve as a matrix/template for the localization of the DmSmt3 conjugation system. Although immunofluorescence studies show that DmUba2 and the septins are sometimes in the same compartment, so that interaction would be possible, no persuasive colocalization of the proteins was detected. Thus, elucidation of the possible interactions between the septins and the Smt3 system in Drosophila, and of their functional significance, will need to await further studies using other approaches (Shih, 2002).

Biochemical and two-hybrid studies indicate that there are multiple DmSmt3-modified proteins, that DmSmt3-specific isopeptidases probably exist, that DmUba2 and DmAos1 interact with each other, and that both DmUba2 and DmUbc9 become conjugated to DmSmt3, but not to DmUbiquitin, by thiolester bonds. While these studies were in progress, related findings were also made by other investigators who were led by other routes to the Smt3/SUMO system in Drosophila. In particular, several other proteins were shown to interact with DmUba2 and DmUbc9 using the two-hybrid system, and multiple DmSmt3-modified proteins were observed, supporting the hypothesis that DmSmt3 is indeed conjugated to a variety of proteins in vivo. In addition, the DmUba2/DmAos1 interaction and the conjugation of DmSmt3 to DmUba2 and DmUbc9 by thiolester bonds were also observed using other methods. Finally, it was shown that Dmubc9 can functionally complement a yeast ubc9 mutation. Taken together, these results make clear that the Smt3/SUMO conjugation system is closely conserved between Drosophila, yeast, and mammals. The data also show that DmUba2 and DmUbc9 form a complex in vivo, suggesting that the conjugation machinery may act in a concerted fashion (Shih, 2002).

Studies in other laboratories also provided clues to possible functions of modification by DmSmt3. In particular, the semushi and lesswright mutations are both in Dmubc9, suggesting (from their mutant phenotypes) that DmUbc9 has roles in the nuclear import of the transcription factor Bicoid and in meiotic chromosome segregation. In addition, two other transcriptional regulators, Dorsal and Tramtrack, as well as CAM-kinase II, have also been shown to be modified by DmSmt3. In the case of Dorsal, as with Bicoid, DmSmt3 conjugation appears to promote nuclear localization, whereas Tramtrack modification may help to regulate its activity and/or its degradation by the proteasome, as discussed above. The modification of CAM-kinase II may regulate its activity (Shih, 2002).

Although most suggested functions of the Smt3/SUMO system in Drosophila, as well as in yeast and mammalian cells, center on nuclear proteins, immunofluorescence and two-hybrid data support the hypothesis that there are also cytoplasmic targets. (1) It remains likely that in Drosophila, as in yeast, there is an interaction between the Smt3/SUMO system and the septins, which appear to be exclusively cytoplasmic proteins. (2) Some DmSmt3- modified proteins appear to remain both at the embryo cortex during cellularization and in the midbodies that remain after the nuclear envelopes have reformed at the end of cytokinesis. (3) Although DmUba2 and DmUbc9 are found primarily in nuclei, considerable DmUba2 is also found in the cytoplasm and at the cortex during the syncytial-blastoderm stage, suggesting that DmSmt3 modification of cortical and/or cytoplasmic proteins could occur. (4) The cell-cycle-regulated translocation of DmUba2 between cytoplasm and nucleus both in syncytial blastoderm and in post-cellularization embryos may suggest that the partitioning of the DmSmt3-conjugation system between the cytoplasm and the nucleus is important and thus well regulated during embryonic development (Shih, 2002).

Identification and characterization of a SUMO-1 conjugation system that modifies neuronal calcium/calmodulin-dependent protein kinase II in Drosophila melanogaster

Drosophila Uba2 and Ubc9 SUMO-1 conjugation enzyme homologs (DmUba2 and DmUbc9) were isolated as calcium/calmodulin-dependent kinase II (CaMKII) interacting proteins by yeast two-hybrid screening of an adult head cDNA library. At least one isoform of Drosophila neuronal CaMKII was found to be conjugated to DmSUMO-1 in vivo. The interactions observed in the two-hybrid screen may therefore reflect catalytic events. To understand the role of SUMO conjugation in the brain, a characterization of the system was undertaken. The other required components of the system, Drosophila Aos1 and SUMO-1 (DmAos1 and DmSUMO-1), were identified in expressed sequence tag database searches. Purified recombinant DmUba2/DmAos1 dimer can activate DmSUMO-1 in vitro and transfer DmSUMO-1 to recombinant DmUbc9. DmSUMO-1 conjugation occurs in all developmental stages of Drosophila and in the adult central nervous system. Overexpression of a putative dominant negative DmUba2(C175S) mutant protein in the Drosophila central nervous system resulted in an increase in overall DmSUMO-1 conjugates and a base-sensitive p120 species, which is likely to be DmUba2(C175S) linked to endogenous DmSUMO-1 through an oxygen ester bond. Overexpression of DmUba2(wt) protein in vivo also led to increased levels of DmSUMO-1 conjugates. High level overexpression of either DmUba2(wt) or DmUba2(C175S) in the Drosophila central nervous system caused pupal and earlier stage lethality. Expression in the developing eye led to a rough eye phenotype with retinal degeneration. These results suggest that normal SUMO conjugation is essential in the differentiated nervous system and reveal a potential novel mechanism that regulates neuronal calcium/calmodulin-dependent protein kinase II function (Long, 2000).

A functional interaction between Dorsal and components of the Smt3 conjugation machinery

To identify proteins that regulate the function of Dorsal, a Drosophila Rel family transcription factor, a yeast two-hybrid screen was employed to search for genes encoding Dorsal-interacting proteins. Six genes were identified, including two that encode previously known Dorsal-interacting proteins (Twist and Cactus), three that encode novel proteins, and one that encodes Drosophila Ubc9 (DmUbc9), a protein thought to conjugate the ubiquitin-like polypeptide Smt3 to protein substrates. It was found that DmUbc9 binds and conjugates Drosophila Smt3 (DmSmt3) to Dorsal. In cultured cells, DmUbc9 was found to relieve inhibition of Dorsal nuclear uptake by Cactus, allowing Dorsal to enter the nucleus and activate transcription. The effect of DmUbc9 on Dorsal activity was potentiated by the overexpression of DmSmt3. A DmSmt3-activating enzyme, DmSAE1/DmSAE2, was also identified and it was found to further potentiate Dorsal-mediated activation (Bhaskar, 2000).


Search PubMed for articles about Drosophila Aos1

Bhaskar, V., Valentine, S. A. and Courey, A. J. (2000). A functional interaction between dorsal and components of the Smt3 conjugation machinery. J Biol Chem 275: 4033-4040. PubMed ID: 10660560

Bossis, G. and Melchior, F. (2006). SUMO: regulating the regulator. Cell Div 1: 13. PubMed ID: 16805918

Epps, J. L. and Tanda, S. (1998). The Drosophila semushi mutation blocks nuclear import of bicoid during embryogenesis. Curr. Biol. 8: 1277-1280. PubMed ID: 9822580

Hashiyama, K., Shigenobu, S. and Kobayashi, S. (2009). Expression of genes involved in sumoylation in the Drosophila germline. Gene Expr Patterns 9: 50-53. PubMed ID: 18755298

Huang, H., Du, G., Chen, H., Liang, X., Li, C., Zhu, N., Xue, L., Ma, J. and Jiao, R. (2011). Drosophila Smt3 negatively regulates JNK signaling through sequestering Hipk in the nucleus. Development 138: 2477-2485. PubMed ID: 21561986

Johnson, E. S., Schwienhorst, I., Dohmen, R. J. and Blobel, G. (1997). The ubiquitin-like protein Smt3p is activated for conjugation to other proteins by an Aos1p/Uba2p heterodimer. EMBO J 16: 5509-5519. PubMed ID: 9312010

Kalamarz, M. E., Paddibhatla, I., Nadar, C. and Govind, S. (2012). Sumoylation is tumor-suppressive and confers proliferative quiescence to hematopoietic progenitors in Drosophila melanogaster larvae. Biol Open 1: 161-172. PubMed ID: 23213407

Kanakousaki, K. and Gibson, M. C. (2012). A differential requirement for SUMOylation in proliferating and non-proliferating cells during Drosophila development. Development 139: 2751-2762. PubMed ID: 22745316

Long, X. and Griffith, L. C. (2000). Identification and characterization of a SUMO-1 conjugation system that modifies neuronal calcium/calmodulin-dependent protein kinase II in Drosophila melanogaster. J Biol Chem 275: 40765-40776. PubMed ID: 10995744

Nie, M., Xie, Y., Loo, J. A. and Courey, A. J. (2009). Genetic and proteomic evidence for roles of Drosophila SUMO in cell cycle control, Ras signaling, and early pattern formation. PLoS One 4(6): e5905. PubMed ID: 19529778

Paddibhatla, I., Lee, M. J., Kalamarz, M. E., Ferrarese, R. and Govind, S. (2010). Role for sumoylation in systemic inflammation and immune homeostasis in Drosophila larvae. PLoS Pathog 6: e1001234. PubMed ID: 21203476

Sanchez, J., Talamillo, A., Lopitz-Otsoa, F., Perez, C., Hjerpe, R., Sutherland, J. D., Herboso, L., Rodriguez, M. S. and Barrio, R. (2010). Sumoylation modulates the activity of Spalt-like proteins during wing development in Drosophila. J Biol Chem 285: 25841-25849. PubMed ID: 20562097

Shih, H. P., Hales, K. G., Pringle, J. R. and Peifer, M. (2002). Identification of septin-interacting proteins and characterization of the Smt3/SUMO-conjugation system in Drosophila. J Cell Sci 115: 1259-1271. PubMed ID: 11884525

Takanaka, Y. and Courey, A. J. (2005). SUMO enhances vestigial function during wing morphogenesis. Mech Dev 122: 1130-1137. PubMed ID: 16026969

Talamillo, A., Sanchez, J., Cantera, R., Perez, C., Martin, D., Caminero, E. and Barrio, R. (2008). Smt3 is required for Drosophila melanogaster metamorphosis. Development 135: 1659-1668. PubMed ID: 18367553

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date revised: 15 May 2013

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