InteractiveFly: GeneBrief

Ecdysone-induced protein 63E : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

The puffing response to the late-larval ecdysone pulse consists in the rapid induction of six early puffs in a primary response to the hormone, followed by a secondary response consisting in the regression of these early puffs and the concomitant induction of over 100 late puffs. It is thought that the early puffs represent the expression of genes involved in the regulation of the molting heirarchy, and that the late puffs represent the expression of effector genes. The L63 gene is complex: it codes for Ecdysone-induced protein 63E (Eip63E), which is responsible for the late larval 63E puff. It spans 85 kb of genomic DNA that includes three overlapping transcription units that generate nine different mRNAs encoding five different L63 isoforms. In these regards, L63 more closely resembles the BR-C, E74, and E75 early genes than it does the simple Sgs and L71 secondary-response genes. L63 also differs from these genes in its temporal and spatial expression patterns, again more closely resembling the early genes. Whereas Sgs and L71 gene expression is limited to the terminal stages of the larval salivary gland's existence, one or more of the L63 transcription units are expressed throughout Drosophila development and in many different tissues. These characteristics provide for a redundancy of expression in a gene that mutational analysis shows is required even before metamorphosis occurs. The L63 isoforms contain a common 294-amino-acid region near the carboxy-terminus that shows strong homology to the cyclin-dependent kinase (CDK) protein family. This region provides a CDK function critical to development (Stowers, 2000).

It has been argued that the L63 proteins function as cyclin-dependent kinases, given the strong sequence similarity of L63 to established CDKs, the observation that a mutant which removes a key catalytic site is a likely null, and the results of the rescue experiments with altered L63 proteins generated of in vitro mutagenesis. However, there is no evidence that L63 is involved in the control of cell division as is the case for many CDKs. In fact, several observations suggest otherwise. (1) Qualitative examination of the brains of wild-type and deficiency larvae at pupariation revealed no significant difference in the fraction of cells undergoing mitosis. (2) No differences in the deficiency of polytenization in the salivary gland polytene chromosomes are seen in wandering third-instar mutant larvae, when compared to wild-type animals. (4) Eyes composed entirely of L63 clones are indistinguishable from control mitotic recombinant eyes, indicating that L63 is not required for cell division in the eye. These results suggest that the CDK functions of the L63 proteins are required for functions other than the regulation of cell division. A similar proposal has been made for the closest mammalian relatives of L63 -- namely, the PFTAIRE and PCTAIRE proteins, which may play a role in cell differentiation rather than in cell division (Stowers, 2000).

Given the above observations, the wide spatial and temporal distributions of the L63 proteins, the functional redundancies indicated by the L63 mutants, and the observation that L63 overexpression appears to have no phenotypic consequences, it is proposed that the functional specificity of L63 proteins derives in large measure from associated cyclins and/or other proteins that regulate L63 activity. For instance, an interaction between L63 and one cyclin in an imaginal tissue, like the leg disc (whose metamorphic fate is to form an adult structure) could result in L63 controlling epithelial morphogenesis, while an interaction between L63 and a different cyclin in a strictly larval tissue (such as the salivary gland whose fate during metamorphosis is histolysis) could result in L63 playing a role in programmed cell death. This scenario is consistent with the functions observed for other CDKs. Such a functional specificity has been observed for the mammalian CDK5, a key regulator of neuronal differentiation (see Drosophila CDK5). CDK5 is widely expressed in human and mouse tissues, but its kinase activity is restricted to the brain where its specific activator p35/p25 is expressed. Another example of specific control of CDK activity by different cyclins is that of the S. cerevisiae PHO85 CDK protein. It is essential for the proper regulation of phosphate and glycogen metabolism as well as for cytokinesis. To accomplish these roles it appears that PHO85 may well interact with as many as 10 different cyclins. Mutation of some of these cyclins has shown that they can produce mutant phenotypes that are a subset of those of the PHO85 mutant. Apparently these cyclins exhibit overlapping but unique functions. Using these results as a model, it can be imagined that L63 proteins interact with quite different cyclins or other proteins to provide different developmental functions (Stowers, 2000 and references therein).

Evidence that L63 is required for embryogenesis consists in the observation that L63 mutant embryos that are maternally and zygotically deficient for L63 activity do not complete embryogenesis. Interestingly, recent observations make the possibility plausible that L63 maternal expression may be ecdysone regulated during oogenesis because the same set of early genes induced by ecdysone at the onset of metamorphosis is coordinately regulated by ecdysone during oogenesis. Maternal L63 expression may therefore be part of an ecdysone-regulated hierarchy of gene expression in oogenesis that is used repeatedly during development (Stowers, 2000).

The observation that the majority of zygotically deficient L63 mutants die as larvae indicates that L63 is also required during larval development. From the small pupal phenotype of the few L63 mutants that survive to pupariation it is inferred that L63 may participate in the regulation of organism size. There are numerous ways in which the loss of L63 activity could indirectly reduce organism size; these include, to name a few, a role for L63 proteins in digestion, muscle contraction, or the nervous system. An interesting possibility is that L63 proteins act as direct determinants of organism size. One possible scenario is that these proteins are part of a system that regulates progression from one developmental stage to the next, so that when L63 is absent, precocious developmental transitions occur (Stowers, 2000).

The bent-leg phenotype of rare escaper adult L63 mutants indicates a third function of L63 in development: an involvement in epithelial morphogenesis. Two possibilities were considered for how L63 could be involved in this developmental process: (1) as a regulator of individual epithelial cell shape changes and (2) as a component of a communication system between epithelial cells. One mechanism by which L63 proteins could alter the changes in cell shape due to remodeling of the cytoskeleton is by phosphoregulating critical components of the cytoskeleton, such as actin. Alternatively, if L63 is part of a cell-cell communication system, the bent-leg phenotype of L63 mutants could be explained by a failure of the epithelial cells to communicate to each other their relative spatial positions, thereby altering the evagination process that occurs in the leg disc during pupal development. A role for L63 in the morphogenesis of the leg disc is further supported by recent genetic results that show that L63 mutations genetically interact with genes involved in leg morphogenesis. It should be emphasized that whatever role L63 plays in epithelial morphogenesis, it cannot be generalized to all discs given the finding that eyes composed exclusively of L63 mitotic clones show no morphological defects. The tissue distributions of L63 proteins just before and during metamorphosis also suggest a role for L63 in the cell death of the histolysing larval tissues. In this regard, it is noted that the larval midgut, which expresses little or no L63 protein in mid-third-instar larvae (18 h before pupariation), exhibits strong L63 expression at pupariation while in the midst of the process of cell death and histolysis. In this connection it is also noted that the gastric caeca, which undergo histolysis before the midgut, exhibit strong L63 expression earlier than does the midgut (Stowers, 2000).

REGULATION

Protein Interactions

L63 encodes a CDK-like protein homologous to the mammalian PFTAIRE. It has been shown that L63 provides a CDK-related function critical to development. This study presents the first biochemical characterization of L63 kinase. In addition, two novel Drosophila proteins, PIF-1 and PIF-2 (for PFTAIRE Interacting Factor-1 and -2) are described, identified in a two-hybrid screen for their ability to interact with the amino-terminal region of L63. The full-length PIF-1 cDNA shows an unusual dicistronic organization. PIF-1A and PIF-1B (the L63 interactor) predicted proteins are expressed in vivo, and show a distinct expression profile during development. Interaction between L63 and PIF-1B was confirmed in vitro and in vivo. The role of this interaction remains to be demonstrated, but the data suggest that PIF-1B might serve as a regulator of L63 (Rascle, 2003. Full text of article).

Cyclin Y is a novel conserved cyclin essential for development in Drosophila

The Drosophila gene CG14939 encodes a member of a highly conserved family of cyclins, the Y-type cyclins, which have not been functionally characterized in any organism. This study reports the generation and phenotypic characterization of a null mutant of CG14939, which was renamed Cyclin Y (CycY). The null mutant, CycY^E8, is homozygous lethal with most mutant animals arresting during pupal development. The mutant exhibits delayed larval growth and major developmental defects during metamorphosis, including impaired gas bubble translocation, head eversion, leg elongation, and adult tissue growth. Heat-shock-induced expression of CycY at different times during development resulted in variable levels of rescue, the timing of which suggests a key function for zygotic CycY during the transition from third instar larvae to prepupae. CycY also plays an essential role during embryogenesis since zygotic null embryos from null mothers fail to hatch into first instar larvae. Evidence is provided that the CycY protein (CycY) interacts with Eip63E, a cyclin-dependent kinase (Cdk) for which no cyclin partner had previously been identified. Like CycY, the Eip63E gene has essential functions during embryogenesis, larval development, and metamorphosis. The data suggest that CycY/Eip63E form a cyclin/Cdk complex that is essential for several developmental processes (Liu, 2010).

Cyclins are a family of highly conserved proteins that activate cyclin-dependent kinases (Cdks) to regulate the cell cycle, transcription, and other cellular processes. The founding members of the family, cyclins A and B, were first discovered as proteins that oscillated throughout the cell cycle, peaking in late G2 and M phases. These proteins were later shown to be required to activate the serine/threonine protein kinase, Cdc2 (also known as Cdk1), which is required for entry into M phase in most eukaryotes. Other cyclins with sequence similarity to cyclins A and B were subsequently identified and shown to be required at other points during the cell cycle. The best characterized of these in metazoans include D-type cyclins, which partner with Cdk4 to control G1 phase events, and E-type cyclins, which partner with Cdk2 to control the transition from G1 to S phase. Several other members of the cyclin family do not show cell-cycle-dependent degradation or synthesis and some have been shown to play roles in cellular processes that are not directly related to cell cycle regulation. One group of cyclins, for example, regulates transcription by activating Cdks that can phosphorylate the carboxy-terminal tail of the large subunit of RNA polymerase II. Several additional members of the cyclin family remain uncharacterized. This study describes the initial characterization of one such novel cyclin encoded by the Drosophila gene CG14939, which has been renamed Cyclin Y (CycY). Although this cyclin is highly conserved through evolution, no member of its family has been functionally characterized in any organism (Liu, 2010).

The defining feature of the cyclin family is a homologous region called the cyclin domain, which includes the region responsible for interaction with a Cdk. Detailed studies on specific examples of Cdk/cyclin complexes have shown that the cyclin domain is essential and sufficient for interaction with and activation of the Cdk partner. Thus, while specific Cdk partners have not been identified for every cyclin, all are thought to play the role of activating one or more Cdks. In addition to activating kinase activity, the cyclin may influence the substrate specificity or determine the subcellular localization of the active complex (Liu, 2010).

This study presents data suggesting that one Cdk partner for Drosophila Cyclin Y is Ecdysone-induced protein 63E (Eip63E). The Eip63E gene encodes five highly related and apparently functionally redundant protein isoforms, all of which have homology to cyclin-dependent kinases. The proteins are most similar to the poorly characterized mammalian Cdks called PFTAIRE, so named because of the amino acid sequence in the conserved helix that binds to cyclins. Although a cyclin partner for Eip63E has not been identified, rescue experiments using mutant variants of the protein have suggested that its activity depends on cyclin binding. In those experiments, mutation of a conserved glycine adjacent to the PFTAIRE (G243), which in other Cdks is required for cyclin binding, abolished the ability of an Eip63E transgene to rescue null Drosophila embryos to adulthood. Similarly, mutation of a conserved isoleucine (I249), which is also required for cyclin binding in other Cdks, diminished the ability of Eip63E to promote development. A directed yeast two-hybrid screen by Rascle (2003) identified two potential regulators of Eip63E, Pif-1 and Pif-2, but neither of these proteins has any similarity to cyclins. In a high throughput yeast two-hybrid screen, however, one Eip63E-interacting protein that was identified was the CG14939 protein (Liu, 2010 and references therein).

The name of Eip63E derives from the fact that one of the three transcription units of the Eip63E gene is induced in response to pulses of the steroid hormone ecdysone, which triggers crucial developmental transitions including metamorphosis. Phenotypic characterization of Eip63E loss-of-function mutants has shown that it has essential roles in several developmental processes. The majority of zygotic null mutants die during larval development, while only a small percentage survive to pupation. The mutants that survive take 2-3 days longer to pupariate than their heterozygous siblings and are generally smaller than wild-type pupae. These phenotypes point to a role for Eip63E in larval development and metamorphosis and further suggest that this Cdk may be involved in growth control. Mutant eye clones, however, show no morphological or cell cycle defects, leading Stowers to conclude that Eip63E does not regulate the cell cycle. Eip63E proteins have also been shown to be important for embryogenesis since zygotic null embryos from null mothers fail to hatch into first instar larvae. Interestingly, this maternal effect can be complemented by zygotic expression. Thus, while it is clear that this ecdysone-inducible gene is important for metamorphosis and other developmental events, the molecular partners for Eip63E and the pathways in which it may function have yet to be discovered (Liu, 2010).

This study reports the generation of a null mutant allele of CycY and shows that its phenotype is similar to that of Eip63E mutants. CycY plays major essential roles during metamorphosis, especially during pupariation. CycY is essential for embryogenesis and that this requirement could be partially rescued by zygotic expression. Finally, it was confirmed that CycY and Eip63E specifically interact in Drosophila cells and it was showm that the interaction depends on a conserved phosphorylation target on CycY, Ser389 (Liu, 2010).

Drosophila CG14939 has a single predicted transcript that encodes a protein with 406 residues. Between amino acids 205 and 328 lies a cyclin domain, a conserved region that defines the cyclin family of proteins. The closest human homolog of CG14939 is a poorly characterized gene called Cyclin Y (CCNY). Genes in a number of other species have also been named Cyclin Y on the basis of their sequence similarity to human CCNY. CG14939 is more similar to the Y cyclins from other species than it is to any other Drosophila melanogaster gene, indicating that it belongs to this orthologous family of proteins. Outside of the cyclin domain the protein has virtually no sequence similarity to other cyclins. However, CycY has been highly conserved through evolution. Clear CycY orthologs are found in all metazoans with fully sequenced genomes, including bilaterians (e.g., insects, nematodes, vertebrates), cnidarians (e.g., the sea anenome, Nematostella vectensis), and the placozoan, Trichoplax adhaerens. Cyclin Y is also found in the choanoflagellate, Monosiga brevicollis, the closest known unicellular relative of metazoans, suggesting that the Y-type cyclins originated prior to the first multicellular species. Cyclin Y proteins from all of these species share substantial sequence similarity over most of their length, including regions outside of the cyclin domain. In contrast, plants, fungi, and other nonmetazoan species do not have proteins with extensive sequence similarity to CycY, though they do contain the CycY-specific cyclin domain; this cyclin domain is distinct from other cyclin domains and appears to be conserved throughout the eukaryotic kingdom. In metazoan species the level of CycY conservation is particularly high. For example, the Drosophila protein shares 52% identity with the human CCNY protein. This level of conservation is much higher than that observed for the cell cycle cyclins (e.g., cyclins A, B, D, and E), which share between 20% and 41% identity between human and Drosophila. This suggests that CycY has an important and potentially conserved function. Surprisingly, the function of Cyclin Y has not been studied and CycY mutants have not been reported for any model organism (Liu, 2010).

To determine the function of Drosophila CycY, a loss-of-function mutant allele was generated. Advantage was taken of the availability of a strain, d03228 bearing a P-element inserted 1958 bp downstream of the CycY stop codon and 5723 bp upstream of the start codon of the neighboring gene, crol. This insertion itself has no visible effect on the function of any genes in this region since the homozygous d03228 adults are completely viable and normal. Imprecise excision was used to generate a small deletion around the original P-element. The deletion, E8, completely removed the CycY coding region while leaving the coding regions of the neighboring genes intact. Expression of the neighboring genes, crol and Pde1c, was confirmed using RNA extracted from homozygous and heterozygous E8 second instar larvae. In contrast, CycY transcription was undetectable in homozygous E8 larvae. The E8 deletion is referred to as CycY^E8 (Liu, 2010).

Two additional lines of evidence indicate that CycY is the only gene affected in strain CycY^E8. First, CycY^E8 fully complemented crol⁰⁴⁴¹⁸, a lethal null allele of the neighboring gene; crol⁰⁴⁴¹⁸ also complemented the mutant phenotype of CycY^E8. Thus, although CycY^E8 lacks the first noncoding exon of crol, a crol transcript is expressed and appears to be fully functional. Second, all of the abnormalities that were observed in homozygous CycY^E8 mutants can be rescued either by a CycY genomic transgene or by ubiquitous expression of a CycY cDNA using heat-shock induction. Combined these results indicate that the CycY^E8 mutant strain is a null mutant for CycY (Liu, 2010).

Homozygous CycY^E8 mutants or CycY^E8 over a deficiency that removes CycY (Df(2L)Exel6030) produce no viable adults, indicating that CycY is an essential gene. To analyze the lethal phase, eggs from a self-cross of CycY^E8/CyO flies were collected for 12 hr and aged for another 30 hr. Of 366 embryos examined, 89 (24.3%) remained unhatched while 277 (75.7%) hatched to first instar larvae. Since ~25% of the embryos from this cross should be homozygous CyO, which is lethal during embryogenesis, one-third of the embryos that hatched should be homozygous CycY^E8, indicating that zygotic expression of CycY is not essential for embryogenesis (Liu, 2010).

To evaluate whether CycY is required during larval and pupal development, 180-200 first instar larvae of CycY null mutants (homozygous CycY^E8 or CycY^E8/Df(2L)Exel6030) or their siblings were picked and their morphology and development were followed for 15 days, after which no additional adults eclosed. CycY null mutants did not show obvious larval lethality since the majority (90% or 93%) of first instar larvae developed into pupae, which is a rate similar to their heterozygous siblings (84% or 94%, respectively). However, delayed growth during larval development was observed. By the time third instar larvae in the heterozygous group started to wander, CycY null mutant larvae were still at the feeding stage and exhibited dramatically smaller body sizes. The CycY^E8 homozygotes eventually grew to sizes that were 80%-90% of the heterozygotes before pupariation. The delay in larval growth could be rescued with a genomic CycY transgene. The delay was also evident in the timing of pupariation. The first pupa of CycY^E8 heterozygotes was observed at 6 days after egg deposition (AED), while the first pupa of CycY^E8 homozygotes was observed at 7 days AED. On the basis of the number of pupae that formed each day in the two strains, it was estimated that puparium formation of CycY^E8 homozygous mutants was delayed for ~13 hr relative to that of the heterozygous controls. The genomic CycY transgene shortened this delay to ~5 hr. Similar results were obtained with the CycY^E8/Df(2L)Exel6030 mutant (Liu, 2010). CycY null mutants were arrested predominately during pupal stages, but with variable expressivity. The final developmental stages of animals from each genotype were scored on the basis of the presence of defined morphological markers. Two major lethal phases were observed. The early lethal phase was between pupal stages P3 and P5; for example, all 162 CycY^E8 mutants that pupated developed to stage P3 but only 61% reached stage P5. In contrast, all of the 152 heterozygotes that pupated reached stage P5, and all but two eventually emerged as adults. The CycY null pupae that were arrested at stage P3 or P4 showed a variety of developmental defects, including defects in gas bubble translocation, head eversion, leg elongation, and adult tissue growth. Many mutant individuals stopped further development with the newly formed gas bubble still in the middle of the abdomen. In others the gas bubble translocated to the posterior portion of the puparium as in wild type, but then failed to completely relocate to the anterior, which may hinder head eversion. Many of the mutant pupae showed different amounts of empty space inside the pupal case, which was probably due either to the failure of gas bubble translocation or to insufficient adult tissue growth. A defect in leg elongation was also prevalent. Some mutant individuals had partially elongated legs that were either shorter than normal and did not reach the bottom of the abdomen or were bent. More severe cases showed no sign of leg elongation. Wings also did not achieve full extension. The CycY^E8/Df(2L)Exel6030 mutant had the same range of phenotypes as homozygous CycY^E8 (Liu, 2010).

The late lethal phase of the CycY null was between stages P14 and P15, almost at the end of pupal development. For example, while 41% of the CycY^E8 homozygous pupae reached stage P14, only 13% reached stage P15. The P14-arrested mutants exhibited the prominent malformed leg phenotype that was also observed during earlier pupal stages. In addition to the morphological defects, CycY null pupae were generally shorter and much lighter than wild-type pupae (Liu, 2010).

Among the small fraction of CycY^E8 pupae that reached stage P15, 8 out of 23 (35%) arrested during the process of eclosion. The remainder eclosed into adults, but the majority (13 out of 15) died very quickly with their wings still folded. Most of these adults displayed short bent legs. Only two animals successfully eclosed into adults that looked normal, though they were smaller than newly emerged heterozygous control adults and they survived for <2 days. When the mutants that were arrested during eclosion were manually dissected from the pupal case, a layer of white tissue could be seen, which seemed to adhere adult structures to the inside wall of the pupal case. All of the CycY null mutant defects described above could be rescued by introduction of a CycY genomic transgene (Liu, 2010).

The null mutant phenotype of CycY suggested an important function during metamorphosis. To determine the developmental time point at which CycY expression is required, transgenic flies were generated that expressed myc-tagged CycY from a heat-shock promoter. A series of different heat-shock regimes was performed to compare their ability to rescue the lethality of homozygous CycY^E8. Heat shock on the first 3 days after egg laying failed to rescue the viability of homozygous CycY^E8 mutants. However, when heat shock was extended for 1 or 2 more days, which included late third instar larvae, the rescue ability was dramatically increased to 30%-35%. If CycY was also provided during early pupal stages, the rescue ability increased further to 50%-60%. If CycY expression was withheld until 4 days after egg laying, a 50% rescue rate could still be achieved. However, if heat shock was delayed for 1 more day, the rescue ability decreased to only 13%. Combined, these data suggest that the most important period for zygotic CycY expression is from the late larvae to the early stages of pupal development, consistent with the first major lethal phase of the CycY^E8 mutant (Liu, 2010).

To see whether CycY is expressed at the developmental times when it appears to be needed, quantitative real-time PCR was used to determine the CycY mRNA levels. It was found that the relative abundance of CycY mRNA fluctuated over a narrow range during development. The highest mRNA level was observed in 0- to 1-hr embryos, most likely due to maternal deposition. CycY message levels then decreased from later embryogenesis through the first and second instar larval stages but increased again in third instar larvae and peaked at pupal stages. The transcription variation of CycY is thus consistent with its essential requirement for pupariation (Liu, 2010).

The mutant phenotypes described above were based on zygotic null mutants, which showed normal embryogenesis and slow but otherwise normal larval development. To test whether maternally expressed CycY contributes to early development, maternal null mutants were generated using the ovo^D1 dominant female sterile technique. Hs-FLP/w*; CycY^E8 FRT40A/ovo^D1 FRT40A females were heat shocked for 2 hr during larval development to express FLP recombinase and promote homologous recombination between the CycY^E8 FRT40A and ovo^D1 FRT40A chromosomes. Since ovo^D1 is dominant female sterile, mothers will lay eggs only if homozygous CycY^E8 FRT40A germline cells are generated and CycY is not essential for oogenesis. Mothers that received heat-shock treatment during larval development were crossed with w¹¹¹⁸ males and the number and development of the eggs laid were monitored. It was observed that heat-shock treated CycY^E8 FRT40A/ovo^D1 FRT40A females could lay similar numbers of eggs as heat-shock treated FRT40A/ovo^D1 FRT40A females, indicating that CycY is not essential for at least some of the major processes of oogenesis. However, ~40% of the eggs from CycY^E8 mothers had fused dorsal appendages, suggesting that CycY may play a role in axis specification (Liu, 2010).

To test for a maternal contribution to embryogenesis, females with homozygous CycY^E8 germline cells were generated using the ovo^D1 dominant female sterile technique, and were crossed with CycY^E8/CyO, Act5C-GFP males. Zygotic null progeny were identified by absence of the GFP balancer. Interestingly, the majority (99.6%) of zygotic null embryos from null mothers failed to hatch, suggesting that maternal expression of CycY is essential for embryogenesis. Surprisingly, when females with homozygous CycY^E8 germline cells were crossed with w¹¹¹⁸ males, 7.3% of the embryos hatched into first instar larvae and 73% of these larvae developed into normal adults. Taken together, these data suggest that maternally provided CycY plays an important role during embryogenesis, but that this role can be accomplished at least to a limited extent by zygotic expression (Liu, 2010).

Cyclin proteins generally serve as regulatory subunits for Cdks. In a previous high throughput yeast two-hybrid screen an interaction was detected between CycY and Eip63E, a Cdk with no known cyclin partner. To test specificity, additional two-hybrid assays were conducted using additional Cdks and cyclins. It was found that CycY interacted only weakly or not at all with other Cdks, including Cdk1, Cdk2, Cdk4, Cdk5, Cdk7, Cdc2rk, and CG7597. Likewise, Eip63E interacted with CycY and CycC, a protein known to be promiscuous in two-hybrid assays, but only weakly or not at all with CycA, CycB, CycB3, CycD, CycE, CycG, CycH, CycJ, CycK, CycT, Koko, and CG16903. To further confirm and test the specificity of the Eip63E-CycY interaction, tagged versions of Cdks and cyclins were expressed in cultured Drosophila cells and interaction was tested by co-AP followed by immunoblotting. In the co-AP assay, CycY interacted strongly with Eip63E but only weakly or not at all with Cdk2, Cdk4, or Cdc2rk. Eip63E, on the other hand, interacted much more strongly with CycY than with other cyclins tested, including CycK, CycD, and CG31232 (Koko). As expected, Glycine 243 (G243) of Eip63E, which is essential for its function in vivo, is required for binding to CycY. In further support of the interaction between these proteins, a recent study demonstrated an interaction between the human homolog of Eip63E, PFTK1, and human CycY using yeast two-hybrid and co-AP assays from human cells (Jiang, 2009). Taken together, these data and the studies with the human orthologs support the notion that CycY and Eip63E constitute a conserved cyclin-Cdk pair (Liu, 2010).

A recent large-scale phosphoproteome study in Drosophila embryos identified several phosphorylated peptides from the CycY protein. A number of the phosphorylation sites are in highly conserved serine residues, suggesting that they may affect CycY function. One of these residues, S389, has also been found to be phosphorylated in human CycY, both in nuclear and cytoplasmic fractions. Position Ser389 in the Drosophila protein is conserved in every species examined. Moreover in one of the two preceding positions of every CycY there is another serine (S388 in Drosophila), which was also identified as a phosphorylated residue in the human protein. As a first test of the potential importance of these residues a Drosophila CycY S389A mutant and S388A/S389A double mutant was generated, and its Cdk-binding ability was measured. The Ser389A mutant had a dramatically decreased ability to bind Eip63E. The double mutant did not further diminish Cdk binding, indicating that S388 does not contribute to the interaction. While these results point to a role for S389 in Cdk interaction, it could not be shown that phosphorylation is important, since a S389E mutant also failed to interact with the Cdk (data not shown) (Liu, 2010).

If Eip63E and CycY form a functional Cdk/cyclin complex in vivo, their mutant phenotypes might be expected to be similar. Previous studies have shown that Eip63E is important for embryogenesis, larval development, and morphogenesis. Those studies demonstrated that the majority of Eip63E null mutants die during larval development, while a small percentage survive to pupal stages with an occasional adult escaper. It was also shown that puparium formation in Eip63E mutants is delayed by 2-3 days, pupae are small, and the rare adult escapers have a bent-leg phenotype and short life spans. All of these phenotypes are similar to those observed for CycY^E8. To further compare the Eip63E and CycY loss-of-function phenotypes, a detailed side-by-side phenotypic characterization was performed. A transheterozygous null mutant, Eip63E⁸¹/Eip63E^GN50 was used and its phenotype was compared with that of CycY^E8. It was found that CycY and Eip63E null mutants showed similar developmental defects, though the Eip63E null mutant phenotype was generally more severe. Both mutants displayed a major lethal phase during metamorphosis. While CycY mutants showed lethality during early or late pupal stages, the majority of Eip63E mutants died at earlier pupal stages. Both mutants also showed similar metamorphosis defects, including gas bubble translocation defects, failed head eversion, and leg elongation defects. In addition, pupae of both mutants were similarly small in weight and length. Finally, both mutants exhibited delayed puparium formation, for 13 hr in the case of CycY, and 37 hr for Eip63E. It has been shown that Eip63E has a zygotically rescuable maternal contribution to embryogenesis, similar to observation for CycY. The striking similarity between the mutant phenotypes of Eip63E and CycY, combined with the specific physical interaction between the proteins in yeast two-hybrid and co-AP assays, supports the idea that CycY and Eip63E may function together in vivo. The possibility, however, cannot be excluded that one or both proteins have additional partners. For example, one potential explanation for the earlier lethality and more severe phenotype of Eip63E mutants relative to the CycY null is that Eip63E may have functions independent of CycY and these may involve other cyclin partners. Alternatively, the subtle differences in CycY and Eip63E mutant phenotypes may be due to differences in the levels of perdurance of their maternal components. Further in vivo analysis of the interaction will be needed to distinguish these possibilities (Liu, 2010).

It is concluded that Cyclin Y is a highly conserved protein. Only minimal information is available for the human ortholog, CCNY. The gene is broadly expressed in human tissues, with particularly high levels in testis. Localization studies with GFP fusions in cell lines have shown that one isoform of human CycY, which has also been called Cyclin X, is nuclear while another isoform may be anchored to the cell membrane via a conserved myristoylation signal. Recently, CCNY was identified as a potential susceptibility factor for inflammatory bowel disease (IBD), a complicated genetic disorder affecting the intestinal mucosa. A single nucleotide polymorphism (SNP) located in an intron of CCNY was found to be strongly associated with the two IBD subphenotypes, Crohn's disease and ulcerative colitis, though it is not yet clear whether CCNY plays a direct role in these diseases. Another study found that human CycY is among a number of proteins that are significantly upregulated in metastatic colorectal cancer cells, though again it is not clear whether this cyclin contributes to the phenotype of these cells. The establishment of a CycY-deficient animal model could provide a system for studying conserved functions of Cyclin Y and for understanding its potential role in human diseases (Liu, 2010 and references therein).

Cell cycle control of Wnt receptor activation

Low-density lipoprotein receptor related proteins 5 and 6 (LRP5/6; Drosophila Arrow) are transmembrane receptors that initiate Wnt/β-catenin signaling. Phosphorylation of PPPSP motifs in the LRP6 cytoplasmic domain is crucial for signal transduction. Using a kinome-wide RNAi screen, it was shown that PPPSP phosphorylation requires the Drosophila Cyclin-dependent kinase (CDK) L63. L63 and its vertebrate homolog PFTK are regulated by the membrane tethered G2/M Cyclin, Cyclin Y, which mediates binding to and phosphorylation of LRP6. As a consequence, LRP6 phosphorylation and Wnt/β-catenin signaling are under cell cycle control and peak at G2/M phase; knockdown of the mitotic regulator CDC25/string, which results in G2/M arrest, enhances Wnt signaling in a Cyclin Y-dependent manner. In Xenopus embryos, Cyclin Y is required in vivo for LRP6 phosphorylation, maternal Wnt signaling, and Wnt-dependent anteroposterior embryonic patterning. G2/M priming of LRP6 by a Cyclin/CDK complex introduces an unexpected new layer of regulation of Wnt signaling (Davidson, 2009).

Wnt/β-catenin signaling regulates patterning and cell proliferation throughout embryonic development and is widely implicated in human disease, notably cancer. Two principal classes of transmembrane (TM) receptors function to transduce Wnt/β-catenin signaling; the seven pass TM Frizzled (Fz) proteins and the single pass TM low density lipoprotein receptor-related proteins 5 and 6 (LRP5/6; Drosophila Arrow). Frizzled receptors activate β-catenin-dependent (canonical) as well as β-catenin-independent (noncanonical, such as planar cell polarity) pathways, while LRP5/6 function more specifically in the Wnt/β-catenin pathway (Davidson, 2009).

LRP6 signaling requires Ser/Thr phosphorylation of its intracellular domain (ICD), which contains five PPPSPXS dual phosphorylation motifs comprising Pro-Pro-Pro-Ser-Pro (PPPSP) and directly adjacent casein kinase 1 (CK1) sites. Phosphorylation of the most N-terminal PPPSP (S1490) involves glycogen synthase kinase 3 (GSK3), while CK1g phosphorylates two Ser/Thr clusters near S1490. Phosphorylation of CK1 sites is downstream of, and requires, PPPSP phosphorylation; however, alternative epistasis models have also been proposed. Both PPPSP and CK1 site phosphorylation is necessary for Axin binding to LRP6 and Wnt/β-catenin pathway activation. Phosphorylated PPPSPXS motifs directly inhibit the ability of GSK3 to phosphorylate β-catenin, providing a potential mechanism linking LRP6 activation to β-catenin stabilization. Investigating how LRP6 phosphorylation is regulated is thus crucial for understanding Wnt receptor activation and downstream signaling. Constitutive, non-Wnt-induced S1490 phosphorylation has been observed, suggesting that additional proline-directed kinases may be involved, such as the ERK or Cyclin-dependent kinase (CDK) subgroups (Davidson, 2009).

CDKs are regulators of the cell cycle and require Cyclin partners, whose levels are precisely controlled during the cell cycle, endowing CDKs with both temporal activity and substrate specificity. Several less well-characterized CDK-like proteins exist, including the PFTAIRE kinase subfamily. This study reports on the identification of a Cyclin/PFTAIRE-CDK complex that phosphorylates LRP6 S1490 in a cell cycle-dependent manner, which brings Wnt/β-catenin signaling under G2/M control and introduces a surprising new principle in Wnt regulation (Davidson, 2009).

An important issue in the field of Wnt/β-catenin signaling concerns the regulation of LRP5/6/Arrow function via phosphorylation. This study has identified the unusual plasma membrane tethered Cyclin Y/PFTAIRE complex which functions predominantly at the G2/M phase of the cell cycle to phosphorylate the PPPSP motifs of LRP6. The results suggest a G2/M priming model of LRP5/6/Arrow phosphorylation, where the Cyclin Y/CDK complex phosphorylates LRP6 at PPPSP motifs, which then primes adjacent phosphorylation by CK1. However, PPPSP priming alone is not sufficient for phosphorylation by CK1, as Wnt-induced LRP6 aggregation is also required. Combined phosphorylation at PPPSP and CK1 sites then promotes Gsk3-Axin binding to LRP6 and signalosome formation. Since GSK3 and Cyclin Y/CDK are both essential for LRP6 priming they apparently act nonredundantly. So why is there a dual kinase input to PPPSP phosphorylation? The phosphorylation of LRP6 by GSK3 occurs in acute response to Wnt signaling and it was suggested that it serves to amplify receptor activation. Cyclin Y/CDK phosphorylates Wnt independently at G2/M, thereby gating signal transduction in proliferating cells. One possibility is that individually both kinases prime LRP6 substoichiometrically at the five PPPSP sites and that only their combined action is sufficient for full LRP6 signaling competence (Davidson, 2009).

These findings have important implications for the link between proliferation and Wnt signaling. It has been long known that there is cross talk between mitogenic growth factors and Wnt signaling. The current results may explain why mitogenic growth factors synergize with Wnt/β-catenin signaling, namely by G2/M priming of LRP6 through enhanced cell proliferation, which sensitizes LRP6 for incoming Wnt signals. Moreover, not only extracellular but also intracellular cell cycle check point regulators controlling G2/M entry are likely to affect Wnt signaling (Davidson, 2009).

Wnt/β-catenin signaling itself promotes G1 progression by inducing c-myc and cyclin D1. This suggests that Wnt/β-catenin signaling can entrain a positive feedback loop in proliferating cells by promoting cell cycle progression, which triggers LRP6 phosphorylation at G2/M. Simultaneous stimulation by Wnt and mitogenic growth factors could initiate such a loop. Indeed, the results may explain the previously noted G2/M enrichment of β-catenin and Wnt signaling. Likewise, protein levels of the direct Wnt target gene Axin2, considered a marker gene for Wnt/β-catenin signaling, also peak during mitosis (Davidson, 2009).

What may be the function of a Wnt positive feedback loop during the cell cycle? One of the many roles of Wnt/beta-catenin signaling is to promote cell proliferation and the positive feedback loop suggested by this study may enhance the systems' levels properties of the cell cycle. Specifically, the loop may promote synchrony of cell cycle regulated events or constitute a bistable switch between cell proliferation and cell cycle exit (Davidson, 2009).

One interesting question raised by this study concerns preferential transcription of Wnt target genes around G2/M. Most genes are transcriptionally silenced between late prophase and early telophase, yet TOPFLASH reporter and AXIN2 peak around G2/M. It will therefore be interesting to investigate whether Wnt target genes are transcribed during the more permissive stages G2, early prophase, or late telophase (Davidson, 2009).

Another important question raised by this study is whether G2/M priming is essential or only modulatory for Wnt/β-catenin signaling in general, in particular in light of Wnt signaling in nondividing cells. The fact that LRP6 signaling is promoted by G2/M phase does not exclude Wnt/β-catenin signaling in other cell cycle phases or in nondividing cells. Even though during interphase the levels of LRP6 signalosomes, Sp1490, β-catenin, and reporter activation are lower compared to G2/M, such Wnt/β-catenin signaling is likely physiologically relevant and may involve additional PPPSP kinases, such as GSK3. Surprisingly little is known about Wnt/β-catenin signaling in nondividing cells. In transgenic Wnt-reporter mice, Wnt activity is detected in apparently postmitotic cells in the adult brain, retina, and certain liver cells. In the adult liver, Wnt/β-catenin signaling controls perivenous gene expression. Furthermore, Wnts play a role in axon remodeling in postmitotic neurons and at least one study suggests that this can involve the β-catenin pathway. In light of the current results it will be interesting to examine more systematically Wnt/β-catenin signaling and in particular the LRP6 kinases involved in postmitotic cells (Davidson, 2009).

Traditionally it is thought that Wnt/β-catenin signaling acts to regulate gene expression of downstream targets. Why then should Wnt/β-catenin signaling peak at G2/M? One likely answer is that components of the Wnt/β-catenin pathway play a crucial role during mitosis beyond transcriptional activation. In C. elegans, Wnt signaling regulates the orientation of the mitotic spindle in early development. In mammalian cells, phosphorylated β-catenin itself binds to centrosomes and is involved in spindle separation during mitosis. Likewise, GSK3, Adenomatous polyposis coli protein (APC) and Axin2, which are components of the β-catenin destruction complex, also have direct functions in mitosis. Taken together these data suggest that Cyclin Y/CDK phosphorylates LRP6 at G2/M to induce Wnt/β-catenin signaling for orchestrating a mitotic program (Davidson, 2009).

DEVELOPMENTAL BIOLOGY

Deletion mapping of sequences required for the ecdysone induction of the 63E late puff suggests that L63B transcription, and possibly that of L63C, is ecdysone responsive. Northern analyses were used to show that the temporal profile of L63B (Ecdysone-induced protein 63E) transcription mimics that of known ecdysone-responsive genes and that the L63A profile is more complex, while L63C transcription is too weak to be detected by these analyses. While sequence overlap exists between beta3 and the gamma1 exon of L63C, a probe specific for gamma1 sequences fails to detect any mRNAs on an identical Northern blot, even though it contains more L63C nucleotides than are present in the beta3 exon. The temporal profile of the L63B1/B2 mRNAs is like that of several ecdysone-responsive genes. The L63B1/B2 midembryonic expression is like that observed for the ecdysone-inducible early-late DHR3 gene and the EcR-A and EcR-B1 transcription units that encode respective ecdysone receptor isoforms. The embryonic ecdysone pulse presumed to induce these genes peaks at 10 h and is well suited to generating the L63B embryonic expression pattern. Similar arguments apply to the strong L63B1/B2 expression that extends from late third instar (L3) to the middle of pupal development (108-168 h) when DHR3 and EcR-A are also strongly expressed in conjunction with the late-larval, prepupal, and pupal ecdysone pulses that peak at 120, 130, and 150 h, respectively. The same temporal pattern of transcription is seen for the band of RNA for which the probe consists of sequences in the common 8-13 exons. This band was assigned to the 2.6-kb L63B3 mRNA because the match of lengths and pattern are unique (Stowers, 2000).

In contrast to L63B transcription, the observed temporal profile for the 3.8-kb L63A1 and L63A2 mRNAs indicates that, with one exception, the ecdysone pulses do not influence the abundance of these mRNAs in whole animals. That exception is an increase in transcript abundance during the 120- to 168-h interval that could result from the prepupal and/or pupal ecdysone pulses. It should, however, also be noted that this increase in the L63A mRNA might also result from the same regulatory mechanisms that cause a reappearance of the 63E puff during the middle of the prepupal period when the ecdysone levels are very low. Given that the 3.1-kb L63A3 mRNA is represented by the 3.0-kb band of the middle blot, then its expression is largely confined to embryogenesis. The large abundance of all of the L63A mRNAs during the first few hours of embryogenesis are probably of maternal origin, particularly because the abortion of transcription during the short nuclear division cycles of this period makes it highly unlikely that the long L63A primary transcript could be zygotically produced (Stowers, 2000).

Monoclonal anti-L63 antibodies divide into two classes, one class against epitopes in the N-terminal tail and the other class against epitopes in the C-terminal tail, with all epitopes present in all five L63 isoforms. Specificity criteria for L63 in the tissue staining are that the pattern obtained with an antibody from one class (A1-29; N-terminal epitope) is the same as that obtained with antibody from the other class (E3-53; C-terminal epitope). L63 is expressed throughout development and in many tissues. However, some tissues show different L63 expression levels at different developmental stages, especially at metamorphosis. Comparison of the larval midgut shows that the L63 protein increases from little or nothing in feeding mid-third-instar larvae (i.e., 18 h before pupariation) to high levels in white prepupae (i.e., at pupariation), implying that one or more of the L63 transcription units is activated in response to the late-larval pulse of ecdysone. The same developmental correlation of L63 with ecdysone titer is observed in ovaries that show no staining in mid-third-instar larvae; rather, by pupariation, they contain high levels of L63. The gastric caeca, which exhibit high levels of L63 at mid-third instar, show signs of programmed cell death by pupariation and disappear a few hours later. Another pattern of L63 expression is seen in both the larval salivary gland and the imaginal discs. In mid-third instar, both exhibit significant levels of L63 that increase severalfold by pupariation, as if L63 is induced in two phases. Interestingly, a similar developmental expression profile for L63 is observed in the ring gland, the site of ecdysone biosynthesis, with moderate expression levels at mid-third instar and high expression levels at white prepupae. L63 is primarily localized to the cytoplasm. A similar conclusion can also be drawn from close examination of the salivary gland staining. The observation that L63 protein is present in some tissues before the later larval ecdysone pulse is consistent with temporal patterns of L63A and total L63 mRNA expression and the predominantly premetamorphic lethal phase of L63 mutants (Stowers, 2000).

EFFECTS OF MUTATION

Evidence that L63 is an essential gene was obtained from the analysis of seven lethal EMS mutations in the 63E region. Molecular analysis of the 81 mutation shows that it is a mutant allele of the L63 gene, hereafter denoted L63⁸¹, consisting of an in-frame deletion of the DNA sequences encoding residues 226-241 within the conserved CDK region of the L63 isoforms. Western analyses of proteins in the L63⁸¹ mutants reveal the expected shift in mobility resulting from the deletion of 16 amino acids. One of these is the highly conserved lysine (K234) that forms an essential part of the ATP-binding catalytic triad in the human CDK2 protein. Hence, it is likely that the L63⁸¹ mutation is null for L63. The Df E1/Df GN50 transheterozygote is an even better candidate for an L63 null because Df E1 eliminates all three of the L63 transcription initiation sites along with adjacent, presumably regulatory DNA, while Df GN50 eliminates virtually all L63 coding sequences. The supposition that the Df E1/Df GN50 heterozygote is an L63 null is further confirmed by the inability to detect any L63 protein by Western analysis of this mutant genotype at metamorphosis. As expected, the Df E1/Df GN50 transheterozygote exhibits a lethal phase much like that of the L63⁸¹/Df GN50. Both mutant combinations show a heterogeneous lethal phase with the majority of mutant animals dying during larval development and a small percentage living to pupation with an occasional adult escaper. Further evidence that the lethality of these heterozygotes results from alteration of L63 is that the lethal L63⁸¹/DfGN50 and DfE1/DfGN50 heterozygotes can be rescued to adulthood by heat-shock-induced expression of the L63B1 isoform. This result provides strong evidence that defective L63 genes cause the above lethalities. No aberrant phenotypic consequences of the ectopic L63B1 expression were observed in these rescue experiments (Stowers, 2000).

The redundancy of the L63 isoform functions is indicated not only by the ability of a single isoform, L63B1, to rescue Df E1/Df GN50 embryos to adulthood, but also by the viability of deletion mutants in which the production of a particular isoform is eliminated. The most striking illustration of this redundancy is the finding that Df 1-11/Df GN50 animals are viable and can be perpetuated as a stock. Df 1-11 removes all of the exons specific to the L63A proteins (exons a1-3), as well as potential regulatory regions both upstream and downstream of the L63A transcription initiation site. Given that the Df GN50 is null for L63, the viability of Df1-11/Df GN50 animals indicates that the L63A1 and 2 proteins are not required when the L63B proteins are present. The reduction of L63 protein levels in Df1-11/Df GN50 late larvae, compared to the Canton-S wildtype, may result not only from the Df GN50 deletion, but also from the absence of L63A proteins due to the Df 1-11 deletion. If this is the case, then the protein band seen above the Df FF/Df GN50 main band, but not seen in Df 1-11/Df GN50, may represent one of the L63A isoforms (Stowers, 2000).

The Df FF and Df CC deficiencies eliminate the B1 exon and hence the L63B transcription initiation site. The observation that Df CC can be maintained indefinitely as a homozygote indicates that the L63B transcription initiation site and neighboring sequences are not required for viability. One cannot, however, conclude that the L63B1, B2, and B3 proteins are not required in the presence of the L63A1 and A2 proteins because the L63B1 protein is encoded by the L63C1 and 2 mRNAs as well as the L63B1 and B4 mRNAs. Furthermore, there is no obvious reason why an additional L63C mRNA encoding the L63B2 protein might not be made by the same exon 5/7 splice used for the L63A2 and L63B2 mRNAs. Indeed, the fact that such an L63C cDNA was not detected may well result from the low abundance of the L63C mRNAs in the wildtype (Stowers, 2000).

The rescue of Df E1/Df GN50 animals to adulthood provided an opportunity for carrying out a self-cross of w; HSL63/1; DfE1/DfGN50 animals. The observation that none of the embryos from this cross were observed to hatch demonstrates an embryonic requirement for the L63 protein. Moreover, this observation raises the possibility of a maternal effect because Df E1/Df GN50 mutants derived from females not maternally deficient for L63 die later in development. The viability of Df 1-11/Df GN50 animals (maternally deficient for L63A) allowed this possibility to be confirmed by showing that the direction of the cross Df 1-11/Df GN50 3 Df E1/TM6b affects the lethal phase of the L63 mutant genotype. When female Df 1-11/Df GN50 are crossed to male Df E1/TM6b, all Df E1/Df GN50 L63 mutant progeny die prior to the third-instar developmental stage, while if the reciprocal cross is performed a significant percentage of Df E1/Df GN50 mutant progeny survive to third instar or further, exhibiting a lethal phase similar to that of Df E1/Df GN50 animals from the cross Df E1/TM6b 3 Df GN50/TM6b. Notably, when L63 maternally deficient w; HSL63/1; DfE1/DfGN50 mothers are crossed to Df E1/TM6b males, TM6b progeny survive to adulthood. This result indicates that the embryonic requirement for L63 that is absent in the L63 maternal deficient females is zygotically rescuable by the wild-type L63 function present on the TM6b chromosome (Stowers, 2000).

Phenotypic analysis of rare L63 null mutant animals that survive past the third instar stage reveal developmental roles for L63. One such role is a general effect on the overall size of the animal. L63 mutant pupae range in size from approximately one-third to two-thirds that of wildtype under uncrowded conditions and take 2-3 days longer than their heterozygous siblings to reach pupariation. A second and more specific developmental role for L63 is an involvement in epithelial morphogenesis. This is evidenced by the bent-leg phenotype in rare escaper adult L63 mutant animals. While such L63 mutant animals do manage to eclose they almost immediately fall into the food and die soon thereafter. Both the small pupal size and the bent-leg phenotypes are rescued along with viability in the heat-shock-induced L63 cDNA rescue experiments. In contrast to the morphogenetic defect observed in L63 mutant legs, no morphogenetic defects are observed in L63⁸¹ mutant eyes composed exclusively of mitotic clones of L63 cells. Furthermore, analysis of the electrophysiological properties of L63 mutant eyes by electroretinogram reveals no defects in phototransduction or synaptic transmission (Stowers, 2000).

The transgene rescue technique was adapted to test for CDK function by attempting to rescue Df E1/Df GN50 animals with heat-shock-induced expression of mutant L63 proteins in place of wild-type L63B1. Thus, in these experiments a mutant version of the L63B1 cDNA was linked to the hsp70 promoter in the context of a P-element vector that was germ-line transformed to generate a mutant HS L63 chromosome. 32°C as well as 37°C heat shocks were used, thereby testing for rescue at different in vivo concentrations of the mutant L63 protein. Residues known to be important for cyclin binding, kinase activity, and phosphorylation in CDK proteins were chosen for site-directed mutagenesis. If these functional hallmarks of the CDK family contribute to the in vivo function of L63, it would be predicted that mutating these residues would adversely affect the ability of the altered L63 protein to rescue the Df E1/Df GN50 mutant. The conserved glycine (G243) and isoleucine (I249) residues of L63B1 correspond to human CDK2 residues known to be crucial for binding cyclin A. In the human CDK2/CYCA cocrystal structure, the glycine corresponding to the L63 G243 is adjacent to the cyclin-interacting PSTAIRE domain (PFTAIRE in L63). It is thought to be essential at that position because it is the only amino acid that will allow the two residues directly adjacent to it to form hydrogen bonds with the human cyclin A. The isoleucine residue in human CDK2 that corresponds to the L63 I249 is crucial because it fits tightly into a hydrophobic pocket of the human cyclin A. G243A L63 mutant protein fails to rescue the Df E1/Df GN50 animals exposed to either the 32°C or the 37°C heat shocks and the I249L mutant provides only partial rescue compared to the L63B1 wild-type (17% with 32°C heat shock and 68% with 37°C heat shock). These differences cannot be attributed to differences in the heat-shock response because the wild-type and mutant protein levels were similar for a given temperature. These results are taken as strongly supportive of the proposition that L63 protein interacts with cyclins (Stowers, 2000).

The glutamic acid residue at 251 in L63B1 (E251) corresponds to a highly conserved residue in both cyclin-dependent kinases and other, more distantly related, protein kinases. In the human CDK2, this residue assists the highly conserved lysine and aspartic acid residues (the other two members of the catalytic triad -- K234 and D344 in L63B1) in correctly positioning the gamma phosphate of ATP for nucleophilic attack by serine and threonine substrates. Mutation of the glutamic acid to glutamine results in complete loss of the rescue function at both temperatures -- a result in strong support of the minimal proposition that L63 is a protein kinase. The serine residue S359 in L63B1 corresponds to a conserved threonine phosphorylation site in human CDK2 (T160) and in other CDKs, including the T169 site in the yeast CDC28. Phosphorylation of this residue is critical for full CDK activity. The L63B1 mutant in which this serine is replaced by alanine (S359A) cannot rescue Df E1/Df GN50 whether induced at 32°C or 37°C (Stowers, 2000).

An attempt was made to see if the N-terminal tail plays an essential role in L63 function by constructing a mutant, delta202 L63, in which the N-terminal 202 residues of L63B1 were eliminated. No rescue by delta202 L63 was observed when the heat shocks were carried out at 32°C, but 31% rescue occurs with 37°C heat shocks. This result cannot, however, be directly compared with those of the other L63 rescue constructs because the amount of delta202 L63 stably produced by the 37°C heat shocks is only 15% of that obtained for the other L63 constructs. What can be said about the nonconserved N-terminal tail is that it is not necessary for L63 function, although it may enhance that function or protect L63 from denaturation and/or destruction. An ancillary experiment was carried out in which it was asked whether either of the bona fide Drosophila CDKs (cdc2 and cdc2c) could rescue the Df E1/Df GN50 null. The answer was a clear no, even though both proteins were produced at high levels by the 37°C heat shocks (Stowers, 2000).

EVOLUTIONARY HOMOLOGS

Cloning and expression of genes coding for PFTAIRE and PCTAIRE proteins

It has been suggested that cell-type determination in Dictyostelium discoideum is dependent on the position of a cell in the cell cycle at the time of starvation. In order to understand the molecular basis of this phenomenon, studies on the cell cycle were initiated and the isolation of a Dictyostelium gene encoding a homolog of the Cdc2 kinase has been described. Additional cdc2 genes could not be isolated from Dictyostelium using polymerase chain reaction technology, but a gene has been isolated that is highly related to cdc2. The encoded product is a protein of 33 kDa that shares over 60% identity to the cell-cycle-dependent Cdc2 kinases. However, despite this high level of identity, the gene is not capable of complementing the temperature-sensitive cdc28 mutant of Saccharomyces cerevisiae. Furthermore, the gene product shares some characteristics with the recently described PCTAIRE proteins; it contains a PCTAIRE motif instead of the Cdc2 kinase conserved PSTAIRE sequence; it does not possess the conserved GDSEID sequence that is involved in the activation of the enzyme and it has a Ser in the position equivalent to Thr-161. However, the Dictyostelium protein exhibits a slightly higher level of identity to the Cdc2 kinases than to the PCTAIRE proteins and is smaller than any of the PCTAIRE proteins thus far identified. Since the gene product has characteristics of both Cdc2 kinases and PCTAIRE proteins the gene product has been designated Crp (Cdc2-Related PCTAIRE) kinase. The gene is expressed as two transcripts of 1.5 and 1.8 kb and the expression is developmentally regulated with low levels of mRNA in vegetative cells and significantly higher levels throughout the remainder of the differentiation process. These results suggest the possibility that the gene product is involved in Dictyostelium differentiation rather than growth. This report is the first evidence for a highly-related cdc2 gene in unicellular eukaryotes. It also demonstrates for the first time that a unicellular eukaryote expresses a protein containing the PCTAIRE sequence (Michaelis, 1993).

Recent studies on the molecular mechanisms controlling the mammalian cell cycle have disclosed a large family of cdc2-related serine/threonine kinases. Among this gene family, the PCTAIRE protein kinases comprise a distinct subfamily of unknown cellular function. To analyze the genomic structure and chromosomal location of the PCTAIRE-1 and -3 genes, human cosmid clones for each gene were isolated by screening a human genomic library with murine PCTAIRE cDNA probes. Overlapping clones encompassing approximately 60 kb of genomic DNA were obtained for both PCTAIRE-1 and -3. These clones have abeen confirmed to encode authentic PCTAIRE genes by the detection of exon-intron structures and the coincidence of the nucleotide sequence of exons to that of the published human cDNAs. Using these cosmid clones as probes for FISH analyses, the chromosomal loci for PCTAIRE-1 and PCTAIRE-3 were assigned to bands Xp11 and 1q31-q32, respectively (Okuda, 1994).

The roles of the cyclin dependent kinase (Cdk) family in murine germ cell development have been examined by studying the expression of five Cdk family genes (Cdc2, Cdk2, Cdk4, Pctaire-1, and Pctaire-3) in mouse reproductive organs. Northern blot and in situ hybridization analyses revealed distinctive expression patterns for these genes, with striking cellular, lineage, and developmental stage specificity. Cdk expression is observed in cell types with proliferative activity: Cdc2 and Cdk2 expression in premeiotic spermatocytes in the testis, and Cdc2, Cdk2, and Cdk4 expression in granulosa cells of ovarian follicles. Cdc2 transcripts are most abundant in late pachytene to diplotene spermatocytes, which are soon to undergo meiosis. Surprisingly, expression of Cdk family genes is also observed in non-proliferating cell types. All five Cdk family genes examined are expressed in Sertoli cells of the adult testis; these cells are no longer mitotically active. With regard to Pctaire-1 and Pctaire-3, the highest levels of expression are observed in postmeiotic spermatids. Immunoblot analysis also reveals the presence of high levels of Pctaire-1 in postmeiotic germ cells. These results suggest that Cdk family kinases may exhibit various functions in germinal and somatic cells during gametogenesis, not only in the cell cycle but also in other regulatory processes, including differentiation (Rhee, 1995).

A rat PCTAIRE-1 cDNA clone was isolated by immunoscreening of a PC12 cDNA library, followed by 5' RACE (rapid amplification of cDNA ends) to determine the 5' end. The rat PCTAIRE-1 cDNA sequence is 96% identical to mouse PCTAIRE-1 and contains an alternatively spliced exon of 131 bp near the 5' end. Although a mouse cDNA containing this exon has been reported, examination of several mouse cell lines has provided no evidence for expression of the corresponding mRNA. In contrast, reverse transcription and polymerase chain reaction (RT/PCR) across this region using RNA from proliferating, differentiated, and apoptotic PC12 cells demonstrates that alternatively spliced forms of PCTAIRE-1 mRNA, with and without this exon, are expressed. Both forms of PCTAIRE-1 mRNA are also expressed in vivo in neonatal rat brain, although other tissues examined contained only the form lacking the alternatively spliced exon. In the absence of the alternatively spliced exon PCTAIRE-1 mRNA contains an open reading frame of 1488 bp, corresponding to a 55-kDa protein that is 97% identical to mouse PCTAIRE-1 protein. When the alternatively spliced exon is present, this open reading frame is terminated by a stop codon and a second open reading frame is initiated, predicting a second PCTAIRE-1 protein of 52 kDa. The two predicted PCTAIRE-1 proteins are identical downstream of the splice site, but share no homology at their N-terminal ends (Gao. 1996).

PCTAIRE are members of a subfamily of Cdc2-related kinases that have been shown to be preferentially expressed in post-mitotic cells. To examine the neural functions of PCTAIRE, rat cDNA clones encoding PCTAIRE 1, 2, and 3 were isolated, and their expression patterns in the brain were analyzed. Among the three rat PCTAIREs, only PCTAIRE 2 was found to be specifically expressed in the brain. Furthermore, its expression is transiently increased during brain development, peaking 7-15 days after birth. Within the brain, PCTAIRE 2 is concentrated in the neuronal layers of the hippocampus and olfactory bulb, which mostly consist of post-mitotic neurons. In an immunocytochemical experiment, immunoreactivity for PCTAIRE 2 was detected in the cell bodies and extended neurites of neurons, but not in astrocytes. The PCTAIRE 2 protein was recovered in the particulate fraction and resistant to solubilization with non-ionic detergent, suggesting that PCTAIRE 2 might be present as a component of a large protein complex. An immunoprecipitation assay revealed that the PCTAIRE 2 is associated with Ser/Thr-phosphorylating activity for histone H1, and that its activity depends on association with a regulatory partner that can be released under high-salt conditions. These findings suggest that PCTAIRE 2 is a Ser/Thr kinase that might play a unique role in terminally differentiated neurons (Hirose, 1997).

A cDNA encoding a cdc2-related protein kinase, named PFTAIRE, has been cloned and characterized. It is expressed primarily in the postnatal and adult nervous system. Several populations of terminally differentiated neurons and some neuroglia express PFTAIRE mRNA and protein. In neurons, PFTAIRE protein is localized in the nucleus and cytoplasm of cell bodies. The anatomical, cellular, and ontogenic patterns of PFTAIRE expression in the nervous system differ from those of p34cdc2 and cdk5, which are expressed in brain and several other mitotic tissues. Proteins of approximately 58-60 kDa coprecipitate specifically with PFTAIRE from cytosolic protein preparations of adult mouse brain and transfected cells. These proteins appear to be the major endogenous substrates associated with this kinase activity. The temporal and spatial expression patterns of PFTAIRE in the postnatal and adult nervous system suggest that PFTAIRE kinase activity may be associated with the postmitotic and differentiated state of cells in the nervous system and that its function may be distinct from those of p34cdc2 and cdk5 (Lazzaro, 1997).

A murine cDNA encoding for a novel putative Cdk-related protein kinase, which has been named Pftaire-1, has been isolated by screening a testis cDNA library for new serine/threonine kinases. Pftaire-1 shows 50% and 49% amino acid identity with Cdk5 and Pctaire-3, respectively, and contains the eleven subdomains characteristic of the protein kinases. By Northern blot analysis two transcripts of approximately 5.5 and 4.9 kb in size have been detected. These transcripts are expressed at low levels in all murine tissues tested, except in the brain, testis and embryo, where high expression is detected. Cellular localization of the mRNAs by in situ hybridization analysis shows that Pftaire-1 is expressed in late pachytene spermatocytes in the testis and in post mitotic neuronal cells both in the brain and the embryo, suggesting a role of Pftaire-1 both in the process of meiosis as well as neuron differentiation and/or function (Besset, 1998).

PCTAIRE-1 is a member of the cyclin-dependent kinase (cdk) family whose function is unknown. The pattern of PCTAIRE-1 protein expression was examined in a number of normal and transformed cell lines of various origins and it was found that the kinase is ubiquitous. PCTAIRE-1 exhibits cytoplasmic distribution throughout the cell cycle. Confocal microscopy shows that PCTAIRE-1 does not colocalize with components of the cytoskeleton or with the endoplasmic reticulum. Endogenous PCTAIRE-1 and ectopically expressed PCTAIRE-1 display kinase activity when myelin basic protein is used as an acceptor substrate. Similar to other members of the cyclin-dependent kinase family, PCTAIRE-1 seems to require binding to a regulatory subunit to display kinase activity. PCTAIRE-1 activity is cell cycle dependent and displays a peak in the S and G2 phases. The low level of kinase activity observed until the onset of S phase correlates with elevated tyrosine phosphorylation of the molecule (Charrasse, 1999).

A selective antibody to a synthetic peptide corresponding to an N-terminal sequence of the PCTAIRE-1 protein has been developed. In rodent brain extracts it recognizes only the protein doublet characteristic of PCTAIRE-1, and this signal is completely abolished by preincubation of the antibody with the immunopeptide. Immunolabeling experiments done with this PCTAIRE-1-specific antibody reveal that the protein is widely distributed in the rodent brain as are the mRNAs visualized using an antisense riboprobe corresponding to the entire PCTAIRE-1 open reading frame. Two types of PCTAIRE-1 protein localizations were observed: (1) a diffuse labeling of almost all brain regions, particularly intense in the molecular layer of the cerebellum and the mossy fiber region of the hippocampus, and (2) a spot-like localization in the nuclei of large neurons, such as cerebellar Purkinje cells and pyramidal cells of the hippocampus. Colocalization with the B23 protein allows the identification of these compartments as nucleoli. These results suggest a nucleolar function of PCTAIRE-1 in large neurons and a role in regions containing important granule cell projections (Le Bouffant, 2000).

Protein interactions of Pftaire and Pctaire proteins

PCTAIRE-1 is a member of the cyclin-dependent kinase (cdk)-like class of proteins, and is localized mainly in the mammalian brain. Using the yeast two-hybrid system a mouse brain cDNA library was screened with PCTAIRE-1 as bait, and several clones were isolated coding for the mouse homologs of the following proteins: p11 (also known as calpactin I light chain) and the eta, theta (also known as tau) and zeta isoforms of 14-3-3 proteins. That these four proteins interact with PCTAIRE-1 was confirmed by demonstrating the biochemical interactions using the pure recombinant proteins. The fact that 14-3-3 proteins are known to interact with many other intracellular proteins (such as C-kinase, Raf, Bcr, P13-kinase) and p11 with annexin II (a major pp60[v-src] and C-kinase substrate) suggests that PCTAIRE-1 might be part of multiple signal transduction cascades and cellular protein networks (Sladeczek. 1997).

An antibody directed against the C-terminal part of PCTAIRE-1 recognizes three proteins in rodent brain. The high-molecular-mass band is most abundant in the cerebellum, hippocampus and cortex. It migrates at the same apparent molecular mass as recombinant PCTAIRE-1 and interacts, like recombinant PCTAIRE-1, with p11 (also known as calpactin I light chain) and 14-3-3 proteins. Combination of p11 or 14-3-3 affinity resins with immunoprecipitation and peptide elution allow for a purification of a full-length PCTAIRE-1 preparation having significant kinase activity. These results suggest that PCTAIRE-1 is an active kinase in brain. The catalytic core region of PCTAIRE-1, which is common for all cyclin-dependent kinases, does not interact with p11 and 14-3-3 proteins in the two-hybrid assay. Full interaction with p11 and 14-3-3 proteins requires both, the N-terminal and C-terminal ends of PCTAIRE-1, suggesting that complex three-dimensional arrangements are responsible for these interactions. A low-molecular-mass protein (migrating at about 30 kDa) that was also recognized by the antibody directed against the carboxy-terminal part of PCTAIRE-1, is abundant and almost homogeneously distributed in all brain areas investigated. Database searches starting with the amino acid sequences of two peptides obtained by tryptic digestion of this protein have yielded cDNA and genomic sequences, allowing for the composition of a DNA sequence coding for a putative 26 kDa protein containing both peptides. This protein has no important sequence similarity with any other known protein. But many DNA sequences are found in databases with an almost 100% identity with parts of the 26 kDa protein coding sequence. These results allow these widely distributed cDNA sequences to be attributed to an existing 26-kDa protein and the gene has been localized within two recently published genomic sequences (Le Bouffant, 1998).

PCTAIRE 2 is a Cdc2-related kinase that is predominantly expressed in the terminally differentiated neuron. To elucidate the function of PCTAIRE 2, proteins that associate with PCTAIRE 2 were screened by the yeast two-hybrid system. A positive clone was found to encode a novel protein that could bind to PCTAIRE 2 in vitro as well as in vivo, and was designated as Trap (tudor repeat associator with PCTAIRE 2). The overall structure of Trap shows no significant homology to any proteins, but contains five repeated domains (the tudor-like domain), conserved in Drosophila Tudor protein. Trap associates with the N-terminal domain of PCTAIRE 2 through its C-terminal domain, which contains two tudor-like domains. PCTAIRE 1, but not PCTAIRE 3, can also associate with Trap. Trap is predominantly expressed in brain and testis, and gradually increases during brain development throughout life, consistent with the expression pattern of PCTAIRE 2. Immunoreactivities for PCTAIRE 2 and Trap were colocalized to the mitochondria in COS 7 cells. Immunohistochemical analyses shows that PCTAIRE 2 and Trap are distributed in the same cell layer of the cerebral cortex and cerebellum. These findings suggest that Trap is a physiological partner of PCTAIRE 2 in terminally differentiated neurons (Hirose, 2000).

PCTAIRE protein kinases interact directly with the COPII complex and modulate secretory cargo transport

The export of secretory cargo from the endoplasmic reticulum is mediated by the COPII complex. In common with other aspects of intracellular transport, this step is regulated by protein kinase signalling. Recruitment of the COPII complex to the membrane is known to require ATP and to be blocked by the protein kinase inhibitor H-89. The identity of the specific protein kinase or kinases involved remains equivocal. This study shows that the Sec23p subunit of COPII interacts with PCTAIRE protein kinases. This interaction is shown using two-hybrid screening, direct binding and immunoprecipitation. Inhibition of PCTAIRE kinase activity by expression of a kinase-dead mutant, or specific depletion of PCTAIRE using RNAi, leads to defects in early secretory pathway function including cargo transport, as well as vesicular-tubular transport carrier (VTC) and Golgi localization. These data show a role for PCTAIRE protein kinase function in membrane traffic through the early secretory pathway (Palmer, 2005).

Pctaire1 phosphorylates N-ethylmaleimide-sensitive fusion protein: implications in the regulation of its hexamerization and exocytosis

Pctaire1, a member of the cyclin-dependent kinase (Cdk)-related family, has recently been shown to be phosphorylated and regulated by Cdk5/p35. Although Pctaire1 is expressed in both neuronal and non-neuronal cells, its precise functions remain elusive. A yeast two-hybrid screen was performed to identify proteins that interact with Pctaire1. N-Ethylmaleimide-sensitive fusion protein (NSF), a crucial factor in vesicular transport and membrane fusion, was identified as one of the Pctaire1 interacting proteins. The D2 domain of NSF, which is required for the oligomerization of NSF subunits, binds directly to and is phosphorylated by Pctaire1 on serine 569. Mutation of this phosphorylation site on NSF (S569A) augments its ability to oligomerize. Moreover, inhibition of Pctaire1 activity by transfecting its kinase-dead (KD) mutant into COS-7 cells enhances the self-association of NSF. Interestingly, Pctaire1 associates with NSF and synaptic vesicle-associated proteins in adult rat brain. To investigate whether Pctaire1 phosphorylation of NSF is involved in regulation of Ca(2+)-dependent exocytosis, the effect was examined of expressing Pctaire1 or NSF phosphorylation mutants on the regulated secretion of growth hormone from PC12 cells. Interestingly, expression of either Pctaire1-KD or NSF-S569A in PC12 cells significantly increases high K(+)-stimulated growth hormone release. Taken together, these findings provide the first demonstration that Pctaire1 phosphorylation of NSF regulates the ability of NSF to oligomerize, implicating an unexpected role of this kinase in modulating exocytosis. These findings open a new avenue of research in studying the functional roles of Pctaire1 in the nervous system (Liu, 2006).

Regulation of the CDK-related protein kinase PCTAIRE-1 and its possible role in neurite outgrowth in Neuro-2A cells

PCTAIRE-1 is a CDK-related protein kinase found in terminally differentiated cells in brain and testis, and in many immortalised and transformed cell lines. Bacterially expressed PCTAIRE is completely inactive as a protein kinase, but is a very good substrate for protein kinase A (PKA), which phosphorylates a total of four sites in the N-terminus of PCTAIRE-1. Phosphorylation of one of these sites, Ser119, generates a 14-3-3 binding site, which is functional in vitro as well as in vivo. Mutation of another PKA site, Ser153, to an alanine residue generated an activated kinase in transfected mammalian cells. This activity was comparable to that of CDK5 activated by a bacterially expressed, truncated version of p35(nck), p21. Gel filtration analysis of a brain extract suggested that monomeric PCTAIRE-1 was the active species, implying that PCTAIRE-1 may not be a true CDK, in that it does not require a partner (cyclin-like) subunit for kinase activity. Finally, it was found that various forms of PCTAIRE-1 transfected into neuroblastoma cell lines could either promote or inhibit neurite outgrowth, suggesting a potential role for the PCTAIRE-1 gene product in the control of neurite outgrowth (Graeser, 2002).

Myocardin-related transcription factors regulate the Cdk5/Pctaire1 kinase cascade to control neurite outgrowth, neuronal migration and brain development

Numerous motile cell functions depend on signaling from the cytoskeleton to the nucleus. Myocardin-related transcription factors (MRTFs) translocate to the nucleus in response to actin polymerization and cooperate with serum response factor (Srf) to regulate the expression of genes encoding actin and other components of the cytoskeleton. This study shows that MRTF-A (Mkl1) and MRTF-B (Mkl2) redundantly control neuronal migration and neurite outgrowth during mouse brain development. Conditional deletion of the genes encoding these Srf coactivators disrupts the formation of multiple brain structures, reflecting a failure in neuronal actin polymerization and cytoskeletal assembly. These abnormalities were accompanied by dysregulation of the actin-severing protein gelsolin and Pctaire1 (Cdk16) kinase, which cooperates with Cdk5 to initiate a kinase cascade that governs cytoskeletal rearrangements essential for neuron migration and neurite outgrowth. Thus, the MRTF/Srf partnership interlinks two key signaling pathways that control actin treadmilling and neuronal maturation, thereby fulfilling a regulatory loop that couples cytoskeletal dynamics to nuclear gene transcription during brain development (Mokalled, 2010).

Two cyclin-dependent kinase pathways are essential for polarized trafficking of presynaptic components in C. elegans

Polarized trafficking of synaptic proteins to axons and dendrites is crucial to neuronal function. Through forward genetic analysis in C. elegans, a cyclin (CYY-1) and a cyclin-dependent Pctaire kinase (PCT-1) necessary for targeting presynaptic components to the axon was identified. Another cyclin-dependent kinase, CDK-5, and its activator p35, act in parallel to and partially redundantly with the CYY-1/PCT-1 pathway. Synaptic vesicles and active zone proteins mostly mislocalize to dendrites in animals defective for both PCT-1 and CDK-5 pathways. Unlike the kinesin-3 motor, unc-104/Kif1a mutant, cyy-1 cdk-5 double mutants have no reduction in anterogradely moving synaptic vesicle precursors (SVPs) as observed by dynamic imaging. Instead, the number of retrogradely moving SVPs is dramatically increased. Furthermore, this mislocalization defect is suppressed by disrupting the retrograde motor, the cytoplasmic dynein complex. Thus, PCT-1 and CDK-5 pathways direct polarized trafficking of presynaptic components by inhibiting dynein-mediated retrograde transport and setting the balance between anterograde and retrograde motors (Ou, 2010).

Pctaire and X chromosome inactivation

Previously reported data on the X inactivation status of the ubiquitin activating enzyme E1 (UBE1) gene have been contradictory, and the issue has remained unsettled. Three lines of evidence are presented that UBE1 is expressed from the inactive X chromosome and therefore escapes X inactivation. (1) By RNA in situ hybridization, UBE1 RNA is detected from both the active and inactive X chromosomes in human female fibroblasts. (2) UBE1 is expressed in a large panel of somatic cell hybrids retaining inactive human X chromosomes, including two independent hybrids that do not require UBE1 expression for survival. (3) Sites at the 5' end of UBE1 are unmethylated on both active and inactive X chromosomes, consistent with the gene escaping inactivation. In order to address whether other genes that escape inactivation map to the same region of the X chromosome, the expression of genes mapping adjacent to UBE1 was also examined. The gene for PCTAIRE-1 (PCTK1) maps within 5 kb of UBE1 and similarly escapes X inactivation by the somatic cell hybrid assay, whereas six other genes that are within 1 Mb of UBE1 in Xp11.23 are silenced on the inactive X chromosome. Comparative mapping studies of the homologous loci in mouse establish that Ube1-x and Pctk1 are also within close physical proximity on the murine X chromosome, and expression studies of the Pctk1 gene determine that, similar to Ube1-x, it is subject to X inactivation in mouse. Methylation of CpG residues at restriction sites at the 5' end of both genes on the murine inactive X chromosome are consistent with both genes being subject to X inactivation in mouse, in contrast to their expression status in humans (Carrel, 1996).

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Search PubMed for articles about Drosophila Ecdysone-induced protein 63E

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