Cyclin D: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | References

Gene name - Cyclin D

Synonyms - Cdi3: Cyclin-dependent kinase interactor 3

Cytological map position -

Function - Regulatory subunit of cyclin dependent kinase

Keywords - cell cycle

Symbol - CycD

FlyBase ID:FBgn0010315

Genetic map position -

Classification - G1 cyclin

Cellular location - nuclear



NCBI links: Precomputed BLAST | Entrez Gene
BIOLOGICAL OVERVIEW

The morphogenetic furrow of the developing Drosophila eye provides a model system for studying the role of cell cycle genes and their control of cell proliferation and differentiation. A depression in the apical surface of the eye imaginal disc epithelium of the third instar larva, the morphogenetic furrow moves from the posterior region of the disc to the anterior. The passing of the furrow results in cell differentiation and in the induction of synchronous cell proliferation marked by the orderly expression of cell cycle genes.

Prior to the passing of the furrow there are many S phase and mitotic cells expressing Cyclin A and Cyclin B. Initially, a domain of string expression occurs, prior to the passing of the furrow, found in a narrow band of cell roughly 5- to 6-cell diameters wide. Cyclin D expression reaches high levels immediately after the induction of string. The string domain represents a transition point: S phase ceases at its anterior boundary, expression of Cyclin A and Cyclin B terminates, and an increase in cells in mitosis is seen within the string domain (Thomas 1994). Significantly, the band of Cyclin D expression occurs before cells reenter into the cell cycle. Cyclin E is expressed posterior to the band of Cyclin D expression, in a region partially overlapping the synchronous band of S-phase cells (Thomas 1994).

What is the function of string and Cyclin D expression ahead of the developing furrow? The expression of string is accompanied by an increase in the number of cells in mitosis as would be expected if the function of string were to drive cells in G2 into a round of mitosis. This increase in density of mitotic activity represents a two fold increase over the number seen in the unpatterned region anterior to the furrow (Thomas, 1994).

Cells in G1 are inhibited from entering S phase. Thus some aspect of cell cycle regulation inhibits passage into S for a certain period of time. The occasional S phase cells seen overlapping the anterior edge of STG mRNA suggests that cells already in S phase complete it before progressing into G2. One gene involved in regulation of the G1 phase is roughex. Mutations in roughex cause cells to circumvent G1, and all cells enter S phase, including cells that would normally differentiate. This leads to defects in early steps of pattern formation and cell fate determination.

The G1 period is accompanied by cell shape changes that define the furrow and marks the beginning of cluster differentiation. In the midst of the furrow, cellular interactions play a role in restricting the number of R8 cells that form within the morphogenetic furrow to one per cluster. At the posterior edge of the morphogenetic furrow, cells are formed into regularly spaced preclusters that contain postmitotic precursors serving as the first five photoreceptor cells to differentiate, R8 and R2-5. Following an extended G1 period, marked by Cyclin D and Cyclin E expression and the passing of the furrow, the remaining cells pass synchronously into S phase (following the passage of the furrow) and then into G2 and M, accompanied by the expression and degradation of Cyclins A and B. The expression of Cyclin D and E prior to the passing of the furrow set the stage for the cycle of synchronous division in the cells that do not immediately differentiate. Paradoxically, although all cells passing through the furrow express Cyclin D and Cyclin E, only a fraction of those cells undergo the synchronized division that follows the passing of the furrow (Thomas, 1994 and Finley, 1996).

Cyclin D does not provide essential Cdk4-independent functions in Drosophila

The three mammalian D-type cyclins are thought to promote progression through the G1 phase of the cell cycle as regulatory subunits of cyclin-dependent kinase 4 and 6. In addition, they have been proposed to control the activity of various transcription factors without a partner kinase. This study describes phenotypic consequences of null mutations in Cyclin D, the single D-type cyclin gene in Drosophila. As previously observed with null mutations in the single Drosophila Cdk4 gene, these mutations do not primarily affect progression through the G1 phase. Moreover, the apparently indistinguishable phenotypes of double (CycD and Cdk4) and single mutants (CycD or Cdk4) argue against major independent functions of Cyclin D and Cdk4. The reduced cellular and organismal growth rates observed in both mutants indicate that Cyclin D-Cdk4 acts as a growth driver (Emmerich, 2004).

D-type cyclin-cdk complexes are of crucial importance in human tumorigenesis. Since these complexes have been conserved in evolution, it is readily possible to use model organisms like D. melanogaster for functional characterizations. This study extends previous characterization of Drosophila Cdk4 mutants by phenotypic comparisons with CycD mutants. As observed for Cdk4, Cyclin D is not required for progression through the G1 phase of the cell cycle. Some escapers develop to the adult stage even when both maternal and zygotic Cdk4+ or CycD+ function is abolished. Moreover, FACS analyses demonstrate that the cell-cycle profile of wing-imaginal disc cells homozygous for null mutations in Cdk4 or CycD is essentially indistinguishable from that of wild type. The evidence therefore is not consistent with the prevailing idea that D-type cyclin-cdk complexes primarily regulate progression through the G1 phase. In cultured mammalian cells, where the most support for this suggestion has accumulated, D-type cyclin-cdk complexes have been shown to act in part by titrating CIP/KIP inhibitors away from CycE/Cdk2 complexes, which are thus freed to stimulate cell-cycle progression. In contrast, binding of Dacapo, the single known Drosophila CIP/KIP family member, to Drosophila Cyclin D-Cdk4 has not been detectable. This provides a potential explanation for the apparent discrepancy. It should be noted, however, that the strong genetic interactions reported among CycD, CycE, and Cdk2, previous interaction tests performed with Rbf indicate that Drosophila CyclinD-Cdk4 complexes do play a significant, if redundant, activating role in the E2F/RBF network, just as described in mammals. In the CycD and Cdk4 mutants, CycE/Cdk2 complexes are presumably sufficient to perform this function (Emmerich, 2004).

While not revealing a specific role during G1, the Drosophila mutant phenotypes provide compelling evidence that Cyclin D-Cdk4 promotes cellular growth and thereby accelerates progression through all the cell-cycle phases proportionally. CycD and Cdk4 mutants develop into small but normally proportioned flies with an average weight of ~20% less than that of wild-type siblings. Conversely, overexpression of Cyclin D and Cdk4 has the opposite effect, causing increased growth in organs such as the eye, wing, and salivary glands. Moreover, growth regulation by Cyclin D and Cdk4 is also clearly apparent at the cellular level. Clones of wing-imaginal disc cells either lacking one of the Cyclin D-Cdk4 complex partners or overexpressing the complex grow slower or faster, respectively, than wild-type clones (Emmerich, 2004).

In Drosophila, the growth-promoting function of Cyclin D-Cdk4 can be interrogated using genetic approaches. Initial results have so far argued that Cyclin D-Cdk4 is not part of one of the other pathways (insulin/TOR, ras, myc, bantam), which are known to control cellular and organismal growth rates. However, the Hif-1 prolyl hydroxylase has been identified as a key growth stimulator downstream of Cyclin D-Cdk4. This raises the possibility that Cyclin D-Cdk4 is interconnected with metabolic pathways sensitive to oxygen levels. Future analyses might therefore reveal whether an involvement in oxygen-related metabolism represents the evolutionary conserved role of Cyclin D-Cdk4 in multicellular eukaryotes and throw a new light on its significance in human tumors, where oxygen limitation is a known and crucial challenge. Cyclin D-Cdk4 has also recently been implicated in the JAK-STAT pathway by an independent genetic approach in Drosophila (Emmerich, 2004 and references therein).

This comparison of CycD and Cdk4 mutant phenotypes is also of interest with regard to functions provided by these proteins independently. In particular, D-type cyclins have been proposed to regulate a number of transcription factors without a partner kinase. Moreover, overexpression of UAS-CycD alone or UAS-Cdk4 alone does often have phenotypic consequences that vary in extent with different GAL4 driver lines. For instance, ey-GAL4-driven UAS-CycD expression suppresses the inhibitory effects of simultaneous UAS-RBF1 expression dramatically. Similarly, da-GAL4-driven UAS-CycD expression during development of otherwise wild-type flies results in an increased adult fly weight. However, in these experiments in Drosophila, the overexpressed Cyclin D might execute its effect in combination with excess Cdk4 expressed from the endogenous Cdk4 gene, as suggested by the finding that da-GAL4-driven UAS-CycD expression in Cdk4 mutants increases adult fly weight at most marginally. The findings that loss of CycD+ or Cdk4+ function, as well as simultaneous loss of both CycD+ and Cdk4+ function, results in essentially indistinguishable phenotypes and does not necessarily prevent development to the adult stage, demonstrate that neither Cyclin D nor Cdk4 provides essential functions in Drosophila independently of each other (Emmerich, 2004).

CycD/Cdk4 and discontinuities in Dpp signaling activate TORC1 in the Drosophila wing disc

The molecular mechanisms regulating animal tissue size during development are unclear. This question has been extensively studied in the Drosophila wing disc. Although cell growth is regulated by the kinase TORC1, no readout exists to visualize TORC1 activity in situ in Drosophila. Both the cell cycle and the morphogen Dpp are linked to tissue growth, but whether they regulate TORC1 activity is not known. This study developed an anti-phospho-dRpS6 antibody that detects TORC1 activity in situ. Unexpectedly, it was found that TORC1 activity in the wing disc is patchy. This is caused by elevated TORC1 activity at the cell cycle G1/S transition due to CycD/Cdk4 phosphorylating TSC1/2.TORC1 is also activated independently of CycD/Cdk4 when cells with different levels of Dpp signaling or Brinker protein are juxtaposed. This study has thereby characterize the spatial distribution of TORC1 activity in a developing organ (Romero-Pozuelo, 2017).

During animal development, tissues increase tremendously in mass, yet stop growing at very stereotyped sizes in a robust manner. For instance, the Drosophila wing is specified as a cluster of circa 50 cells, which increases in mass ~500-fold before terminating growth. Once growth has ceased, the left and right wings of an individual fly are virtually identical in size, to within 1%, illustrating the robustness of this process. How animal tissue size is regulated is a fundamental open question in developmental biology (Romero-Pozuelo, 2017).

As mitotically growing tissues develop, two independent cellular processes occur in a coordinated manner: proliferation and cell growth. By itself, proliferation -- the division of cells -- does not lead to mass accumulation. This was nicely shown in the Drosophila wing where overexpression of E2F speeds up the cell cycle, but leads to a normally sized tissue containing more, smaller cells. For a tissue to grow, cells need to accumulate biomass. The mechanisms interconnecting cell proliferation and cell growth are not completely understood. In organisms from yeast to humans, growth is in large part regulated by the target of rapamycin complex 1 (TORC1) kinase. TORC1 promotes biomass accumulation by promoting anabolic metabolic pathways such as protein, lipid, and nucleotide biosynthesis, while repressing catabolic processes such as autophagy. Hence, to understand tissue growth it would be of interest to study the spatial distribution of TORC1 activity in a developing tissue. This line of investigation has been hampered, however, by the lack of readouts for TORC1 activity that can be used in situ (Romero-Pozuelo, 2017).

One signaling pathway that strongly affects tissue size is the Dpp pathway. Dpp is expressed and secreted by a stripe of cells in the medial region of the wing imaginal disc, and forms an extracellular morphogen gradient that both helps to pattern the wing and affects its size. In the absence of Dpp signaling during development, only small rudimentary wings are formed. In contrast, overexpression of Dpp leads to strong tissue overgrowth, in particular along the axis of the morphogen gradient. Several models have been proposed for how Dpp signaling regulates wing size. The exact mechanism by which Dpp regulates tissue size, however, is an unresolved issue. Dpp signaling acts to repress expression of a transcription factor called Brinker. Brinker appears to mediate most of the size effects of Dpp signaling. When Brinker is genetically removed, Dpp signaling becomes dispensable for wing growth. Given that Dpp signaling promotes tissue growth, an open question is whether Dpp signaling promotes TORC1 activity (Romero-Pozuelo, 2017).

Thia study examined whether Dpp signaling promotes TORC1 activity in the Drosophila wing disc. To this end, a phospho-RpS6 (pS6) antibody was developed that allows TORC1 activity to be assayed in situ in tissue. This reagent reveals unexpectedly that TORC1 activity in the growing wing disc is neither uniform nor graded, but is instead patchy. This patchiness is mediated via CycD/Cdk4 and the tuberous sclerosis 1 (TSC1)-TSC2 complex in response to cell cycle stage. Using this pS6 antibody, this study found that TORC1 activity is also induced by discontinuities in Dpp signaling or discontinuities in Brinker levels. It is proposed that these discontinuous conditions may be analogous to regenerative conditions that happen in the wing disc in response to tissue damage. In sum, this work reveals the pattern of TORC1 activity in the context of a developing organ (Romero-Pozuelo, 2017).

TORC1 activity in the wing disc is modulated by the cell cycle, with cells in early S phase showing the highest TORC1 activity. Interestingly, an accompanying paper finds similar results in the Drosophila eye disc (Kim, 2017). This might reflect a metabolic requirement by early S-phase cells for large amounts of nucleotide biosynthesis, an anabolic process promoted by TORC1. Indeed, in various contexts S6K and TORC1 activity were found to be required for the transition from G1 to S. Connections between mechanistic TOR (mTOR) and the cell cycle have previously been found in cultured cells. In human fibroblasts, mTOR shuttles in and out of the nucleus in a cell cycle-dependent manner, peaking in the nucleus shortly before S phase. The relevance of this subcellular relocalization to what is observe in this study, however, is unclear. In fibroblasts, S6K1 activity was found to be highest during early G1, whereas in HeLa cells it was found to be highest during M phase. In sum, it is unclear to what extent cells in culture recapitulate endogenous development, or whether the influence of the cell cycle on TORC1 activity is very context dependent. The TSC1/2 complex has been reported to be phosphorylated by cell cycle-dependent kinases.TSC1 is phosphorylated on Thr417 by Cdk1 during the G2/M transition. This inhibitory phosphorylation would lead to elevated TORC1 activity during G2/M, which does not fit with what was observe here, and thus might be relevant in a different developmental context. Instead, this study found that TSC2 can be phosphorylated by the CycD/Cdk4 complex on Ser1046, and possibly other sites as well, and that this leads to activation of TORC1. This fits with several observations in the literature. Firstly, in U2OS cells the TSC complex was also found to bind cyclin D, leading to its phosphorylation at unknown sites. In U2OS cells, this causes destabilization of the Tsc1 and Tsc2 proteins, which was not observed in this study. Secondly, Tsc1/2 and CycD/Cdk4 were previously found to interact genetically in Drosophila: The reduced tissue growth caused by Tsc1 + Tsc2 overexpression was found to be fully suppressed by expression of CycD + Cdk4. This fits well with the current data suggesting that CycD/Cdk4 directly inhibits the TSC complex via phosphorylation. Thirdly, Cyclin D and Cdk4 were previously reported in Drosophila to promote cell and tissue growth, fitting with activation of the TORC1 complex by CycD/Cdk4. It is worth noting that some patchy TORC1 activity is still seen in CycD- or Cdk4-null discs and in discs with the single phospho-site mutations in TSC2. Hence it is possible that Cdk4 may not be the only factor regulating TORC1 activity in response to the cell cycle, and that Cdk4 might phosphorylate TSC2 on additional sites (Romero-Pozuelo, 2017).

What are the roles of CycD/Cdk4 in cell cycle progression and cell growth? Whereas mammals have three cyclin D genes, CycD1-3, and two CycD binding kinases, Cdk4 and Cdk6, Drosophila has a single CycD, a single Cdk4, and no Cdk6. Hence Drosophila provides an opportunity to elucidate the function of the CycD/Cdk4 complex without difficulties arising from redundancy. Indeed, results in Drosophila clearly show that CycD/Cdk4 promotes cell growth and not cell cycle progression. Both CycD- and Cdk4-null animals are viable, and fluorescence-activated cell sorting (FACS) analysis of null cells revealed that they have a normal cell cycle profile, indicating that they are dispensable for normal cell cycle progression. Instead, Cdk4- and CycD-null animals are 10%-20% smaller than controls, indicating that they promote cell growth. The finding that CycD/Cdk4 activates TORC1 during the G1/S transition can provide one mechanism by which the CycD/Cdk4 complex promotes growth. Hence, from these data it is proposed that in Drosophila the CycD/Cdk4 complex is not part of the core machinery required for cell cycling, but is rather an effector 'side branch' activated at G1/S to promote cell growth. Data from the mouse suggest something similar. CycD1, CycD2, and CycD3 knockout mice are all viable. One could imagine this to be due to redundancy between these three genes, but actually CycD1, CycD2, CycD3 triple-knockout mice survive to mid-gestation, and the triple-knockout mouse embryonic fibroblasts proliferate relatively normally. The mid-gestation lethality of the triple knockouts appears to be due to specific effects in hematopoietic and myocardial cells. Hence, cyclins D1-D3 are also dispensable for cell cycle progression in mice. Interestingly, CycD1 knockout mice and CycD1, CycD2 double-knockout mice are viable but have reduced body size, reminiscent of the size phenotype observed in CycD knockout flies. In sum, despite CycD/Cdk4 being claimed in most reviews on the cell cycle as playing an important role in G1/S progression, it appears that this complex may function rather to promote cell growth in a cell cycle-dependent manner (Romero-Pozuelo, 2017).

Does Dpp control growth in the wing? When discontinuities in Dpp activity or in Brinker levels were genetically induce, activation was observed of TORC1 at the site of discontinuity. Hence, Dpp signaling per se does not appear to activate TORC1; rather, the comparison between high Dpp signaling and low Dpp signaling cells does. In an unperturbed disc, no pattern of pS6 staining was observed that correlates with the Dpp activity gradient, which is highest medially and drops toward the anterior and posterior extremities. This might be due to the fact that in an unperturbed disc the Dpp and Brinker gradients are smooth and do not have such discontinuities. A similar effect of Dpp was previously observed on cell prolife ration, except that in this case the effect of the Dpp discontinuity was very transient, lasting only a few hours after clone induction, whereas the effect seen on growth is sustained. Dpp signaling is, nonetheless, required for growth, because in the absence of Dpp, small vestigial wings are formed. Hence one interpretation might be that low levels of Dpp signaling are continuously required for growth, but that Dpp signaling becomes instructive for tissue growth only when discontinuities in the gradient arise, perhaps as a result of tissue damage or cell delamination, to initiate a regenerative response (Romero-Pozuelo, 2017).

One additional interesting non-autonomous phenomenon observed is that sometimes when a region of the wing disc has high pS6 levels, the rest of the disc loses its typically patchy pS6 pattern and becomes pS6 negative. This phenomenon is not understood, and future work will be necessary to understand it molecularly (Romero-Pozuelo, 2017).


PROTEIN STRUCTURE

Amino Acids - 452

Structural Domains

Drosophila Cyclin D shows strongest similarity to cyclins in a 135 amino acid region, extending from residue 157 to residue 292. This region contains matches to the 20 most conserved residues of most cyclins. The first 97 amino acids contain PEST sequences involved in rapid protein turnover. Drosophila Cyclin D is 39% identical to human Cyclin D2 but is only 18 to 24% identical to the other classes of cyclins identified either in humans or Drosophila. The consensus Cyclin D sequence consists of 65 residues, of which 24 are unique to D cyclins (Finley, 1996).


Cyclin D: Evolutionary Homologs | Regulation | Developmental Biology | References

date revised: 6 Jan 96 

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