InteractiveFly: GeneBrief

Cyclin Y: Biological Overview | References

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 (Stowers, 2000). 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 (Stowers, 2000). 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 (Stowers, 2000). 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 (Rascle, 2003; Stowers, 2000). 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 (Stowers, 2000), 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 (Zhai, 2008). 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 (Beausoleil, 2004; Olsen, 2006). 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 (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 (Stowers, 2000). 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 is also noted that Stowers (2000) showed that Eip63E has a zygotically rescuable maternal contribution to embryogenesis, similar to the 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 (Jiang, 2009; Li, 2009). 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 (Jiang, 2009). 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) (Zeng, 2005), while CK1g phosphorylates two Ser/Thr clusters near S1490 (see Davidson, 2005). Phosphorylation of CK1 sites is downstream of, and requires, PPPSP phosphorylation (Davidson, 2005); however, alternative epistasis models have also been proposed (Yum, 2009). Both PPPSP and CK1 site phosphorylation is necessary for Axin binding to LRP6 and Wnt/β-catenin pathway activation (Davidson, 2005; Tamai, 2004; Zeng, 2005). Phosphorylated PPPSPXS motifs directly inhibit the ability of GSK3 to phosphorylate β-catenin, providing a potential mechanism linking LRP6 activation to β-catenin stabilization (Cselenyi, 2008; Piao, 2008; Wu, 2009). 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 (Bilic, 2007). 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 (Macdonald, 2008; Wolf, 2008; Zeng, 2005; Zeng, 2008). 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).

Search PubMed for articles about Drosophila Cyclin Y

Beausoleil, S. A., et al. (2004). Large-scale characterization of HeLa cell nuclear phosphoproteins. Proc. Natl. Acad. Sci. 101: 12130-12135. PubMed ID: 15302935

Bilic, J., et al. (2007). Wnt induces LRP6 signalosomes and promotes dishevelled-dependent LRP6 phosphorylation. Science 316: 1619-1622. PubMed ID: 17569865

Cselenyi, C. S., et al. (2008). LRP6 transduces a canonical Wnt signal independently of Axin degradation by inhibiting GSK3's phosphorylation of beta-catenin, Proc. Natl. Acad. Sci. 105: 8032-8037. PubMed ID: 18509060

Davidson, G., et al. (2005). Casein kinase 1 gamma couples Wnt receptor activation to cytoplasmic signal transduction. Nature 438: 867-872. PubMed ID: 16341016

Davidson, G., et al. (2009). Cell cycle control of wnt receptor activation. Dev. Cell 17(6): 788-99. PubMed ID: 20059949

Jiang, M., et al. (2009). Cyclin Y, a novel membrane-associated cyclin, interacts with PFTK1. FEBS Lett. 583: 2171-2178. PubMed ID: 19524571

Li, X., et al. (2009). Identification and characterization of cyclin X which activates transcriptional activities of c-Myc. Mol. Biol. Rep. 36: 97-103. PubMed ID: 18060517

Liu, D. and Finley, R. L. (2010). Cyclin Y is a novel conserved cyclin essential for development in Drosophila. Genetics 184(4): 1025-35. PubMed ID: 20100936

Macdonald, B. T., et al. (2008). Wnt signal amplification: activity, cooperativity and regulation of multiple intracellular PPPSP motifs in the Wnt coreceptor LRP6. J. Biol. Chem. 283: 16115-16123. PubMed ID: 18362152

Olsen, J. V., et al. (2006). Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. Cell 127: 635-648. PubMed ID: 17081983

Piao, S., et al. (2008). Direct inhibition of GSK3beta by the phosphorylated cytoplasmic domain of LRP6 in Wnt/beta-catenin signaling. PLoS ONE 3: e4046. PubMed ID: 19107203

Rascle, A., Stowers, R. S., Garza, D., Lepesant, J. A. and Hogness, D. S. (2003). L63, the Drosophila PFTAIRE, interacts with two novel proteins unrelated to cyclins. Mech. Dev. 120(5): 617-28. PubMed ID: 12782278

Stowers, R. S., et al. (2000). The L63 gene is necessary for the ecdysone-induced 63E late puff and encodes CDK proteins required for Drosophila development. Dev. Biol. 221(1): 23-40. PubMed ID: 10772789

Tamai, K., et al. (2004). A mechanism for Wnt coreceptor activation. Mol. Cell 13: 149-156. PubMed ID: 14731402

Wolf, J., et al. (2008). T.R. Palmby, J. Gavard, B.O. Williams and J.S. Gutkind, Multiple PPPS/TP motifs act in a combinatorial fashion to transduce Wnt signaling through LRP6. FEBS Lett. 582: 255-261. PubMed ID: 18083125

Wu, G., et al. (2009). Inhibition of GSK3 phosphorylation of beta-catenin via phosphorylated PPPSPXS motifs of Wnt coreceptor LRP6. PLoS ONE 4: e4926. PubMed ID: 19293931

Yum, S., et al. (2009). The role of the Ser/Thr cluster in the phosphorylation of PPPSP motifs in Wnt coreceptors. Biochem. Biophys. Res. Commun. 381: 345-349. PubMed ID: 19309792

Zeng, X., et al. (2005). A dual-kinase mechanism for Wnt co-receptor phosphorylation and activation. Nature 438: 873-877. PubMed ID: 16341017

Zeng, X., et al. (2008). Initiation of Wnt signaling: control of Wnt coreceptor Lrp6 phosphorylation/activation via frizzled, dishevelled and axin functions. Development 135: 367-375. PubMed ID: 18077588

Zhai, B., et al. (2008). Phosphoproteome analysis of Drosophila melanogaster embryos. J. Proteome Res. 7: 1675-1682. PubMed ID: 18327897

date revised: 11 September 2010

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