Cyclin-dependent kinase 4: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - Cyclin-dependent kinase 4

Synonyms -

Cytological map position - 53C7--8

Function - signaling

Keywords - growth, cell cycle

Symbol - Cdk4

FlyBase ID: FBgn0016131

Genetic map position - 2-

Classification - cyclin-dependent protein kinase

Cellular location - nuclear



NCBI links: Precomputed BLAST | Entrez Gene
BIOLOGICAL OVERVIEW

Recent literature
Icreverzi, A., de la Cruz, A. F., Walker, D. W. and Edgar, B. A. (2015). Changes in neuronal CycD/Cdk4 activity affect aging, neurodegeneration, and oxidative stress. Aging Cell [Epub ahead of print]. PubMed ID: 26219626
Summary:
Mitochondrial dysfunction has been implicated in human diseases, including cancer, and proposed to accelerate aging. The Drosophila Cyclin-dependent protein kinase complex cyclin D/cyclin-dependent kinase 4 (CycD/Cdk4) promotes cellular growth by stimulating mitochondrial biogenesis. This study examine the neurodegenerative and aging consequences of altering CycD/Cdk4 function in Drosophila. Pan-neuronal loss or gain of CycD/Cdk4 was found to increase mitochondrial superoxide, oxidative stress markers, and neurodegeneration and decreases lifespan. RNAi-mediated depletion of the mitochondrial transcription factor, Tfam, can abrogate CycD/Cdk4's detrimental effects on both lifespan and neurodegeneration. This indicates that CycD/Cdk4's pathological consequences are mediated through altered mitochondrial function and a concomitant increase in reactive oxygen species. In support of this, CycD/Cdk4 activity levels in the brain were shown to affect the expression of a set of 'oxidative stress' genes. These results indicate that the precise regulation of neuronal CycD/Cdk4 activity is important to limit mitochondrial reactive oxygen species production and prevent neurodegeneration.

Adlesic, M., Frei, C. and Frew, I.J. (2016). Cdk4 functions in multiple cell types to control Drosophila intestinal stem cell proliferation and differentiation. Biol Open [Epub ahead of print]. PubMed ID: 26879465
Summary:
The proliferation of intestinal stem cells (ISCs) and differentiation of enteroblasts to form mature enteroendocrine cells and enterocytes in the Drosophila intestinal epithelium must be tightly regulated to maintain homeostasis. This study shows that genetic modulation of CyclinD/Cdk4 activity or mTOR-dependent signalling cell-autonomously regulates enterocyte growth, which influences ISC proliferation and enteroblast differentiation. Increased enterocyte growth results in higher numbers of ISCs and defective enterocyte growth reduces ISC abundance and proliferation in the midgut. Adult midguts deficient for Cdk4 show severe disruption of intestinal homeostasis characterised by decreased ISC self-renewal, enteroblast differentiation defects and low enteroendocrine cell and enterocyte numbers. The ISC/enteroblast phenotypes result from a combination of cell autonomous and non-autonomous requirements for Cdk4 function. One non-autonomous consequence of Cdk4-dependent deficient enterocyte growth is high expression of Delta in ISCs and Delta retention in enteroblasts. The study postulates that aberrant activation of the Delta-Notch pathway is a possible partial cause of lost ISC stemness. These results support the idea that enterocytes contribute to a putative stem cell niche that maintains intestinal homeostasis in the Drosophila anterior midgut.

Adlesic, M., Frei, C. and Frew, I. J. (2016). Cdk4 functions in multiple cell types to control Drosophila intestinal stem cell proliferation and differentiation. Biol Open [Epub ahead of print]. PubMed ID: 26879465
Summary:
The proliferation of intestinal stem cells (ISCs) and differentiation of enteroblasts to form mature enteroendocrine cells and enterocytes in the Drosophila intestinal epithelium must be tightly regulated to maintain homeostasis. This study shows that genetic modulation of CyclinD/Cdk4 activity or mTOR-dependent signalling cell-autonomously regulates enterocyte growth, which influences ISC proliferation and enteroblast differentiation. Increased enterocyte growth results in higher numbers of ISCs and defective enterocyte growth reduces ISC abundance and proliferation in the midgut. Adult midguts deficient for Cdk4 show severe disruption of intestinal homeostasis characterised by decreased ISC self-renewal, enteroblast differentiation defects and low enteroendocrine cell and enterocyte numbers. The ISC/enteroblast phenotypes result from a combination of cell autonomous and non-autonomous requirements for Cdk4 function. One non-autonomous consequence of Cdk4-dependent deficient enterocyte growth is high expression of Delta in ISCs and Delta retention in enteroblasts. It is postulated that aberrant activation of the Delta-Notch pathway is a possible partial cause of lost ISC stemness. These results support the idea that enterocytes contribute to a putative stem cell niche that maintains intestinal homeostasis in the Drosophila anterior midgut.

Complexes of D-type cyclins and cdk4 or 6 are thought to govern progression through the G1 phase of the cell cycle. In Drosophila, single genes for Cyclin D and Cdk4 have been identified, simplifying genetic analysis. Drosophila Cdk4 has been shown to interact with Cyclin D and Retinoblastoma-family protein as expected, but is not absolutely essential for cell cycle progression or vitality. Flies homozygous for null mutations develop to the adult stage and are fertile, although only to a very limited degree. It is concluded that progression into and through the cell cycle can occur in the absence of Cdk4 (Meyer, 2000). However, the growth of cells and of the organism is reduced in Cdk4 mutants, indicating a role for D-type cyclin-dependent protein kinases in the modulation of growth rates. The cellular response to CycD-Cdk4-driven growth varies according to cell type. In undifferentiated proliferating wing imaginal cells, CycD-Cdk4 causes accelerated cell division (hyperplasia) without affecting cell cycle phasing or cell size. In endoreplicating salivary gland cells, CycD-Cdk4 causes excessive DNA replication and cell enlargement (hypertrophy). In differentiating eyes, CycD-Cdk4 causes hypertrophy in post-mitotic cells (Datar, 2000).

D-type cyclins, their kinase partners cdk4 and 6, the INK inhibitors and the kinase substrate retinoblastoma protein (Rb) are all known for their crucial importance in human tumorigenesis. At the cellular level, Rb has been shown to regulate progression through the G1 phase of the mammalian cell cycle, predominantly by binding to E2F transcription factors, which control a large number of genes involved in cell proliferation and DNA replication. Rb represses expression of E2F target genes by recruiting histone deacetylase activity and by inhibiting the E2F transcriptional activation domain. The ability of Rb to block progression through G1 is abolished by Rb hyperphosphorylation, which is initiated by D-type cyclin-dependent kinases during G1. In mammalian cells, the synthesis of D-type cyclins is controlled by extracellular signals. Mitogens induce a rapid accumulation of D-type cyclins. Conversely, antimitogens or withdrawal of mitogens result in a rapid decline. D-type cyclins are therefore thought to function as a functional link between extracellular signals and the cell cycle machinery. Accordingly, D-type cyclin-cdk complexes might be predicted to play an important role in the regulation of cell proliferation during development (Meyer, 2000 and references).

Given that Drosophila CycD-Cdk4 is not essential for cell cycle progression, how can these results be reconciled with the standard view that CycD-Cdk4 acts through Rb to control E2F activity? It is suggested that Cyclin D-Cdk4 and Cyclin E-Cdk2 might act as independent RBF kinases in Drosophila. Overexpression of either cyclin D-Cdk4 or Cyclin E results in an increase of hyperphosphorylated Rb. The specific Cyclin E-Cdk2 inhibitor Dacapo severely inhibits Rb hyperphosphorylation. Thus, Drosophila Cyclin E-Cdk2, which is known to phosphorylate RBF efficiently in vitro, might also phosphorylate Rb in Cdk4 mutants, thereby explaining why a decrease in Rb phosphorylation cannot be observed in Cdk4 mutants. The suggestion that Cyclin D-Cdk4 and Cyclin E-Cdk2 might act as functionally overlapping RBF kinases must remain tentative. The finding that Cdk4 mutants are extremely sensitive to a reduction of the Cyclin E+ and Cdk2+ gene dose further stresses the intimate functional relationship between Cyclin D-Cdk4 and Cyclin E-Cdk2 but does not address their interaction hierarchy. Even though the idea that Cyclin D-Cdk4 and Cyclin E-Cdk2 might act as overlapping RBF kinases in Drosophila is favored, it is emphasized that a number of phenotypic differences resulting from both loss- and gain-of-function mutations in the genes encoding the subunits of these different kinase complexes clearly demonstrate that they must also have distinct functions (Meyer, 2000 and references).

Drosophila Cdk4 is clearly required for normal growth of cells and the organism. Size regulation at the level of cells, as well as at the level of organs and organism is an important but poorly understood process. A number of observations in yeast and mammalian cells have indicated that cell cycle progression is dependent on a critical cell size. Many cell types delay the G1-S transition (at a point called START in yeast and at restriction point in mammalian cells) until a critical size has been reached. Slow growth due to nutrient or growth factor limitations therefore results primarily in an extension of the G1 phase. In budding yeast, the Cln cyclins, and in particular Cln3p, have been implicated in this coupling of cell growth and cell cycle progression. Cln3p is a very unstable protein which might only accumulate to an effective concentration that triggers progression through START when ribosome numbers have increased sufficiently. Experimental uncoupling of growth rates and the rates of Cln3p accumulation affects primarily cell division size without a comparable change of cell cycle duration. D-type cyclin-cdk complexes have been proposed to regulate progression through the restriction point in response to growth factors and mitogens in a comparable manner. However, proliferating Cdk4 mutant cells in wing imaginal discs are of wild-type size and progress more slowly through the cell cycle with a proportionate extension of G1, S and G2 phases. This phenotype is incompatible with an exclusive role of Cdk4 in the coupling of cell size and progression through the restriction point. Cdk4 appears to regulate, rather than simply sense growth (Meyer, 2000).

Reduced growth has also been reported to result from inactivation of mouse cdk4 function (Rane, 1999; Tsutsui, 1999). Intriguingly, the phenotypic similarities observed in cdk4 mutant mice and flies extend further. Mutant mice are also infertile, particularly when female. In these females, infertility has been shown to be caused by deficits in the hypothalamic pituitary axis rather than by developmental abnormalities in reproductive organs (Rane, 1999). In addition, cdk4 mutant mice developed diabetes within 2 months after birth due to a loss of the insulin-expressing ß-islet cells (Rane, 1999). In Drosophila, a striking similarity of the phenotypic consequences of mutations in Cdk4 and in genes of the insulin signaling pathway is noted. Therefore, a future analysis of the relationship of Cdk4 and insulin signaling in Drosophila might reveal a striking evolutionary conservation (Meyer, 2000).

Apart from emphasizing functional homology, this analysis also reveals an apparent clear difference between the regulatory role of cyclin D-Cdk4 in Drosophila and in mammalian cells. In mammalian cells, D-type cyclin complexes do not only function catalytically as protein kinases. They also act stoichiometrically by titrating the cdk inhibitors p21CIP1 and p27KIP1. Thereby, they contribute to the activation of cyclin E-Cdk2 complexes. In contrast, the Drosophila cdk inhibitor Dacapo (the only detectable CIP/KIP family member in the known genome sequence) could not be detected in Cyclin D-Cdk4 complexes. This apparent inability of Drosophila Cyclin D-Cdk4 to titrate Dacapo, might explain why, to some extent, mammalian D-type cyclin complexes behave in a functionally distinct manner (Meyer, 2000).

In summary, these observations clearly implicate Cdk4 in the control of growth rates at the cellular and organismal level. Moreover, while these results confirm an involvement of Cdk4 in the regulation of cell proliferation, they also emphasize the importance of alternative pathways. In Drosophila, the developmental program of cell proliferation is surprisingly normal in the absence of Cdk4. The fact that the cyclin D-Cdk4 pathway is of paramount importance for tumorigenesis, therefore, should probably not lead to the view that it is the only pathway controlling entry into and exit from the cell cycle (Meyer, 2000).

The question arises: how does cyclin D/Cdk4 promote cellular growth in addition to cell cycle progression? To assess the function of Drosophila Cyclin D, the effects of overexpressing it were studied in actively proliferating wing imaginal disc cells. Cell clones overexpressing CycD and/or Cdk4 and marked with co-expressed green fluorescent protein (GFP) were induced in developing wings using the flip-out Gal4 system. Tissues were dissociated and analyzed by fluorescence activated cell sorting (FACS) 48 h post-induction (h.p.i.), to determine the proportion of cells in each phase of the cell cycle, and to assess effects on cell size. Expressing either CycD or Cdk4 alone has no detectable effects. Hence, both CycD and Cdk4 were overexpressed. CycD-Cdk4-overexpressing cells were compared with wild-type, non-expressing cells from the same discs. In parallel experiments, overexpressed Drosophila E2F/DP or cyclin E truncates the G1 phase and decreases the cell size. However, no changes in cell cycle phasing or cell size were found in CycD-Cdk4-overexpressing cells. These data differ markedly from the effects of cyclin D1 or D2 overexpression demonstrated in cultured vertebrate cells (Jiang, 1993; Quelle, 1993; Resnitzky, 1994 and 1995) and suggest that the Drosophila CycD-Cdk4 complex does not promote G1-S transitions directly in vivo (Datar, 2000).

To assess the effect of CycD-Cdk4 on rates of cell growth and proliferation, CycD-Cdk4-expressing cell clones were evaluated in situ in wing imaginal discs. Although overexpressed CycD-Cdk4 does not alter cell cycle phasing, it does affect the proliferative rate of cells in the developing wing. At 44 h.p.i. a significant increase in the number of cells in CycD-Cdk4-expressing clones, which had a median of 16 cells, compared with 12 cells in controls, was noted. This corresponds to a 10% decrease in the cell doubling time, from 12 h in controls to 10.8 h in cells overexpressing CycD-Cdk4. Since cells overexpressing CycD-Cdk4 are not reduced in size, it follows that they must acquire more mass than control cells over the same developmental period. Consistent with this it was observed that CycD-Cdk4-overexpressing clones encompass 150% of the area of controls at 44 h.p.i., and 180% of the area of controls at 67 h.p.i. It is concluded that CycD-Cdk4 can stimulate cellular and clonal growth. In the context of the developing wing this increased growth results in faster cell division without detectable changes in cell size (Datar, 2000).

It was next asked what effect CycD-Cdk4 overexpression would have on the development of adult tissues. Adult wings containing CycD-Cdk4-expressing clones show no gross abnormalities in overall shape, veination, bristle or trichome patterning. Optical sections through pupal wings failed to reveal significant changes in cell morphology or cell density within clones overexpressing CycD-Cdk4. However, a striking enlargement of ommatidiae was observed in adult eyes that clonally overexpress CycD-Cdk4. This phenotype requires expression of both CycD and Cdk4. Optical sections of pupal eyes confirmed that overexpressed CycD-Cdk4 leads to increased cell size (hypertrophy) and that this effect is cell autonomous. A mild disorganization in the regular arrangement of ommatidia was observed, but no specific defects in cell differentiation or overall patterning. Hypertrophy occurs in several cell types and structures, including primary pigment and cone cells, photoreceptors and interommatidial bristles. In ommatidiae overexpressing CycD-Cdk4, five cone cells were often observed instead of the normal four, suggesting that an extra cell division occurred (Datar, 2000).

CycD-Cdk4 was also induced in the eye using the GMR-Gal4 and sev-Gal4 drivers, which are expressed starting late in eye development posterior to the morphogenetic furrow (MF), as cells enter their ultimate or penultimate cell cycle and begin to differentiate. Using these drivers to induce CycD-Cdk4 expression it was found that all ommatidiae are enlarged, as is the entire eye, which bulges out of the head in an ominous fashion. Light and transmission electron microscopy of pupal and adult eyes from GMR-Gal4, UAS-CycD, UAS-Cdk4 flies reveal enlarged photoreceptor cell bodies and rhabdomeres, and excessive accumulations of actin, suggesting that CycD-Cdk4 increases cellular protein content as well as cell volume. Driving expression of two copies of CycD-Cdk4 with a single copy of GMR-Gal4 led to even larger ommatidiae and eyes, indicating dose dependence. No significant eye overgrowth was observed when CycD was expressed alone or when it was co-expressed with a kinase-impaired mutant form of Cdk4 (Cdk4D175N). Thus, the bulging eye phenotype appears to require phosphorylation of CycD-Cdk4 substrates. In comparison, CycE or E2F does not cause eye overgrowth when expressed under GMR-Gal4 control, even though both factors promote ectopic DNA replication and cell proliferation in the eye (Datar, 2000).

To assess these phenotypes further FACS was performed on eye discs in which UAS-GFP was co-expressed with either UAS-CycD-Cdk4 or UAS-CycE under GMR-Gal4 control. FACS analysis shows that overexpression of either CycD-Cdk4 or CycE significantly increases the fraction of S- and G2-phase cells posterior to the MF (GFP+ cells). Normally these cells are nearly all arrested in G1, and thus both cyclins appear to perturb the normal program of cell cycle exit at differentiation. The effects of CycD-Cdk4 and CycE on cell size, however, were dramatically different. CycD-Cdk4 greatly increases the size of posterior eye cells, whereas CycE caused a slight decrease in cell size. This cell size effect is not phase specific: even G1 cells overexpressing CycD-Cdk4 are much larger than controls. These results are consistent with findings in wing cells in that CycD-Cdk4 appears to promote both cell cycle progression and cellular growth, whereas CycE affects only cell cycle progression. In addition, neither CycE nor CycD-Cdk4 completely prevents the G1 arrest that accompanies cell differentiation, but only CycD-Cdk4 appears to continue promoting growth in post-mitotic cells, leading to cellular hypertrophy (Datar, 2000).

Many studies have indicated that the major substrates for vertebrate D-type cyclin-Cdk complexes are the Rb family proteins. A Drosophila homolog of Rb, RBF, inhibits cell division when overexpressed in the wing imaginal disc. RBF does not immediately block cell growth, and as a result there is a large increase in cell size. To test interactions between CycD-Cdk4 and RBF, all three genes were co-expressed together in wings, salivary glands or eyes. Using the flip-out Gal4 method in the wing, it was found that cells co-expressing CycD-Cdk4 and RBF cycle more rapidly than cells expressing RBF alone, and thus that CycD-Cdk4 attenuates the inhibitory effects of RBF on cell cycle progression. Interestingly, although cell clones co-expressing CycD-Cdk4 and RBF have fewer cells than wild-type controls, they encompassed substantially more area. Flow cytometry and in situ cell size measurements confirm that the increased mass of these clones is due to increased cell size. The large size of cells co-expressing CycD-Cdk4 and RBF, despite a nearly normal division rate, suggests that CycD-Cdk4 promotes extra growth even while RBF slows cell cycle progression. A logical inference is that CycD-Cdk4 promotes growth via targets other than RBF. This effect is less evident at a later time-point (67 h.p.i.), perhaps because cell cycle suppression by RBF eventually throttles even the growth of CycD-Cdk4-expressing clones (Datar, 2000).

RBF-CycD interaction tests were performed in the larval salivary gland, a differentiated tissue in which cell growth is accomplished by cycles of DNA endoreplication. To express UAS-linked target genes, the F4-Gal4 driver was used, that commences its expression late in embryogenesis after cell proliferation in the salivary primordium is complete and stays active throughout the larval stages. Results obtained in the salivary glands are consistent with those described above for the wing. F4-Gal4-driven expression of CycD-Cdk4 results in oversized salivary glands with nuclei containing excessively endoreduplicated polytene chromosomes. Co-expression of cyclin D with the kinase impaired variant Cdk4D175N or Cdk2 does not induce this size increase, suggesting that the protein kinase activity of cyclin D-Cdk4 complexes is required for the stimulation of salivary gland DNA endoreplication and growth. Although expression of Cdk4D175N alone has little effect on salivary growth, overexpressed RBF strongly inhibits both growth and DNA endoreplication. Even stronger growth suppression is observed when RBF is co-expressed with Cdk4D175N. As in wings and eyes, the growth-inhibitory effects of RBF are attenuated by simultaneous co-expression of CycD-Cdk4 (Datar, 2000).

Similar results are obtained when RBF and CycD-Cdk4 are co-expressed in the eye using ey-Gal4, which is expressed throughout the eye primordium beginning very early in eye development. Expression of RBF under ey-Gal4 control causes a dramatic reduction of the adult eye. This loss of eye tissue is suppressed virtually completely when CycD-Cdk4 is co-expressed with RBF, providing further evidence that CycD-Cdk4 can functionally inactivate RBF. Interestingly, suppression of RBF is also observed when CycD is expressed without the Cdk4 kinase subunit or to a lesser extent when CycD is co-expressed with the kinase-impaired variant Cdk4D175N. This suggests that CycD may suppress RBF function by utilizing an endogenous Cdk, or in a kinase-independent fashion (Datar, 2000).

Although ectopic RBF suppresses growth in developing wings, salivary glands and eyes, growth inhibition by RBF is secondary to its effects on cell cycle progression (Neufeld, 1998). The tests described above are consistent with this interpretation, since in all cases both cell cycle progression (DNA replication) and growth are affected coincidentally. However, these tests cannot rule out the possibility that RBF inhibits cellular growth directly. Therefore the late acting eye specific drivers, GMR-Gal4 and sev-Gal4, were used to test whether RBF could suppress growth in non-cycling cells. Expression of RBF alone using these drivers has little effect on eye size, suggesting that RBF cannot suppress the post-mitotic growth that normally occurs in the eye. Moreover, co-expressed RBF does not substantially suppress eye overgrowth caused by GMR-Gal4- or sev-Gal4-driven CycD-Cdk4. Given this, a parsimonious interpretation of all these interaction tests is that whereas CycD-Cdk4 can promote cellular growth in both proliferating and non-cycling cells, RBF can suppress growth only in cells that are undergoing cycles of DNA replication. In this case, growth suppression by ectopic RBF most likely stems from its ability to inhibit cell cycle progression (Datar, 2000).

To further test RBF function, FLP/FRT-mediated mitotic recombination was used to generate cell clones homozygous for a null allele of rbf (rbf14). Areas of rbf14/14 cell clones were measured in wing discs, and these were compared with the areas of their rbf+/+ sister clones ('twin spots'). Cell clones mutant for rbf are slightly smaller than their wild-type twin spots, though the difference is not statistically significant. Thus, loss of RBF does not confer a growth advantage. FACS analysis of rbf14/14 cells shows a reduced cell size and, surprisingly, an increased G1 population. Microscopic examination reveals many pyknotic nuclei associated with rbf14/14 mutant clones, suggesting elevated levels of apoptosis. Reduced cell size and poor viability have also been observed in hyperproliferative cell clones overexpressing E2F/DP, and similar characteristics have been described for mouse embryo fibroblasts lacking pRB, p107 or p130. One explanation for these phenotypes is that they arise when cell division rates outpace rates of cell growth (Neufeld, 1998). This may also be the case for rbf14/14 cells (Datar, 2000).

Finally, large rbf14/14 clones were generated in the eye using the Minute technique. Although these clones populate large fractions of the eye they do not exhibit the hypertrophic characteristics noted when CycD-Cdk4 is overexpressed. Instead, rbf14/14 clones in the adult eye exhibit slight to moderate hypoplasia and mild defects such as missing or duplicated inter-ommatidial bristles and fused ommatidiae. Similar phenotypes have been noted in eyes overexpressing E2F or cyclin E, both of which promote extra cell division in the eye without increasing growth. These results are consistent with the interpretation that RBF functions to inhibit cell cycle progression, rather than to inhibit cellular growth directly. If this is the case, stimulation of cellular growth by CycD-Cdk4 must be, at least in part, independent of RBF (Datar, 2000).

Thus Drosophila CycD-Cdk4 does not have a specialized function in promoting G1 progression, but instead promotes accumulation of cellular mass, i.e. growth. The effects of CycD-Cdk4 differ profoundly from those of its presumed targets, RBF, E2F and CycE. In numerous assays, RBF, E2F and CycE behave as specific cell cycle regulators that affect cellular growth only indirectly, via their effects on the cell cycle. While these findings differ from those in cultured mammalian cells, they are remarkably consistent with data from in vivo studies of the murine D-type cyclins. Just as overexpressing CycD-Cdk4 in the fly wing or eye causes hyperplasia, targeted overexpression of CycD1 in mice can promote epidermal, mammary and thymic hyperplasia, and mutational activation of Cdk4 can cause pancreatic hyperplasia (Rane, 1999). Likewise, overexpressed cyclin D3 can promote endomitosis in platelets, much as overexpressed Drosophila CycD-Cdk4 drives extra DNA endoreplication in salivary gland cells. CycD1 knockout mice are smaller than their littermates and display hypoplasia of the retina and mammary epithelium, tissues that normally express CycD1 at high levels. Mice lacking CycD2 display hypoplastic growth phenotypes. Cdk4 mutant mice, like Cdk4 mutant Drosophila, are also small (Datar, 2000 and references therein).

Although it is not yet clear whether these growth effects in other systems are cell autonomous, studies of mouse embryo fibroblasts (MEFs) suggest that some are. Proliferating MEFs lacking CycD1 or Cdk4 have normal G1/S/G2 profiles and so, like studies in flies, cast doubt upon a direct role for CycD-Cdk4 in G1-S progression. However, CycD1 or Cdk4 mutant MEFs are delayed in activating DNA replication upon serum stimulation, an effect that could be attributed to impaired growth. MEFs from mice lacking p16INK4A, a specific inhibitor of CycD-dependent kinases, also exhibit accelerated growth. Taken together, all of these observations fall into place to support the view that one cellular function of cyclin D-Cdk complexes is to stimulate cellular growth (Datar, 2000 and references therein).

D-type cyclins are believed to function primarily by suppressing the function of the 'pocket' proteins: pRb, p107 and p130. Numerous genetic interactions have been described between Drosophila CycD-Cdk4 and RBF (Datar, 2000) that indicate in vivo cross-regulation of these gene products. CycD-Cdk4 counteracts the growth-suppressive effects of RBF in wing and eye imaginal discs, and also in the endoreplicating salivary gland. As in vertebrates, these interactions might reflect direct phosphorylation and neutralization of RBF by CycD-Cdk4 kinase. However, these interactions could also be indirect consequences of the effects of CycD-Cdk4 on cellular growth. For instance, growth stimulation by CycD-Cdk4 may increase the activity of CycE-Cdk2, which is also a potent suppressor of RBF activity (Datar, 2000 and references therein).

Although the CycD-RBF interactions describe for Drosophila confirm current paradigms to some extent, they also highlight the differences between CycD-Cdk4 and RBF function. For instance, RBF’s ability to suppress growth is limited to cells actively undergoing DNA replication, such as proliferating cells in the imaginal discs and endoreplicating cells in the salivary gland. In the post-mitotic eye, where CycD-Cdk4 is a potent stimulator of growth, RBF is virtually inert. Moreover, loss of RBF does not enhance growth in either proliferating or post-mitotic cells, and thus does not phenocopy gain of CycD-Cdk4 function. Indeed, all the data on RBF suggest that it functions specifically to suppress cell cycle progression, and that its growth-suppressive effects are secondary consequences of this function. CycD-Cdk4, in contrast, promotes growth in both proliferating and post-mitotic cells. An important implication of this analysis is, therefore, that Drosophila CycD-Cdk4 must promote growth via targets other than RBF. These growth-regulatory targets remain unknown, but the requirement for a catalytically active Cdk4 subunit suggests that they are phosphorylation substrates. Potential candidates include pocket proteins other than RBF, or factors that affect biosynthesis directly, such as regulators of protein synthesis or turnover. Consistently, reports describe non-Rb targets for mammalian CycD1, and indicate that the transforming activity of CycD1 does not require Rb binding (Datar, 2000 and references therein).

How CycD-Cdk4 fits into the hierarchy of genes that regulate growth in Drosophila is unclear, and will remain obscure until mutations in the cycD gene are identified. It is tempting to speculate that CycD-Cdk4 might mediate the growth effects of the Wnt, Bmp and Egf signaling pathways, which orchestrate patterned growth in Drosophila and throughout the animal kingdom. However, there are presently scant data to support this hypothesis in flies. Recent work has revealed that insulin receptor/Pi3K/AKT signaling is also an important growth control pathway in Drosophila, and may respond to nutritional conditions. Like CycD-Cdk4, genes in this pathway appear to be growth specific and to have little role in tissue patterning or differentiation. But there are clear distinctions between CycD-Cdk4-driven growth and Pi3K-driven growth. In the wing for instance, CycD-Cdk4 accelerates growth in a balanced fashion such that cell cycle phasing and cell size remain normal, whereas Pi3K-driven growth is characterized by a truncated G1 phase and increased cell size. The origin of these differences is presently unclear. One plausible explanation is that CycD-Cdk4 can promote G2-M progression, perhaps by repressing RBF and activating Cdc25/Stg, whereas Pi3K cannot. In support of this idea it has recently been shown (Gabrielli, 1999) that human Cdk4 activity is required for the G2-M transition in HeLa cells(Datar, 2000 and references therein).

There is an abundance of correlative evidence associating cyclin D with diverse cancers. It is telling that nearly all cases are associated with elevated levels of cyclin D function as a result of amplification or translocation of the cyclin D locus, or inactivation of the cyclin D-specific inhibitor, p16INK4A. When cyclin D-Cdk4 activity is increased in Drosophila, overgrowth is induced as well. In thinking about how the human D-type cyclins are involved in the progression of cancer, it may prove fruitful to consider them, not as regulators of G1 progression, but as promoters of cellular growth (Datar, 2000 and references therein).

Nutrition controls mitochondrial biogenesis in the Drosophila adipose tissue through Delg and Cyclin D/Cdk4

Mitochondria are cellular organelles that perform critical metabolic functions: they generate energy from nutrients but also provide metabolites for de novo synthesis of fatty acids and several amino acids. Thus mitochondrial mass and activity must be coordinated with nutrient availability, yet this remains poorly understood. This study demonstrated that Drosophila larvae grown in low yeast food have strong defects in mitochondrial abundance and respiration activity in the larval fat body. This correlates with reduced expression of genes encoding mitochondrial proteins, particularly genes involved in oxidative phosphorylation. Second, genes involved in glutamine metabolism are also expressed in a nutrient-dependent manner, suggesting a coordination of amino acid synthesis with mitochondrial abundance and activity. Moreover, this study shows that Delg (Ets97D, CG6338, Delg), the Drosophila homologue to the alpha subunit of mammalian transcription factor NRF-2/GABP, is required for proper expression of most genes encoding mitochondrial proteins. The data demonstrate that Delg is critical to adjust mitochondrial abundance in respect to Cyclin D/Cdk4, a growth-promoting complex and glutamine metabolism according to nutrient availability. However, in contrast to nutrients, Delg is not involved in the regulation of mitochondrial activity in the fat body. These findings are the first genetic evidence that the regulation of mitochondrial mass can be uncoupled from mitochondrial activity (Baltzer, 2009).

In eukaryotes, cellular organelles are separated from the cytoplasm through lipid membranes, creating compartments with unique biological properties. Rather than static, organelle size and function are often dynamic, and tightly regulated in response to various stimuli. One of the best-studied organelles are mitochondria, which show large cell-type specific variations in morphology and abundance, demonstrating that mitochondria are highly regulated. Mitochondrial dysfunction is linked to various diseases, including metabolic disorders and cellular aging, therefore these organelles are critical for cellular homeostasis. Mitochondria perform multiple metabolic functions, most notably the generation of energy from carbohydrates, fatty acids and amino acids. Equally important, mitochondria also provide metabolites for anabolic processes such as de novo synthesis of fatty acids and amino acids. Although the metabolic biochemical reactions are well established, how these processes are coordinated in vivo is just beginning to be understood. One interesting question is how nutrients control mitochondrial mass and activity, and how this regulation affects cellular metabolism (Baltzer, 2009).

During cellular growth, amino acids are used for protein synthesis. In higher eukaryotes, essential amino acids are taken up through the diet, whereas nonessential amino acids are synthesized de novo. For the latter, mitochondria are critical, since they provide oxaloacetate for aspartate and asparagine, as well as 2-oxoglutarate (α-ketoglutarate) for glutamate, glutamine, arginine and proline biosynthesis. Of these amino acids, glutamine is particularly interesting: First, many cell types take up large amounts of glutamine, which can be used to produce cytoplasmic NAD+, NAPDH and lactate in a process called glutaminolysis. Second, since the TCA cycle intermediate citrate can be used as a substrate for fatty acid synthesis, glutamine can be converted into 2-oxoglutarate, thus replenishing the TCA cycle. Third, efflux of cytoplasmic glutamine, either taken up or synthesized de novo, is directly linked to the uptake of essential amino acids, both in mammals and Drosophila. Interestingly, all three processes are highly active in cancer cells, under conditions of high metabolic activity. One would therefore expect tight coordination between nutrients, mitochondrial activity and amino acid synthesis, in particular glutamine, yet factors mediating such links have not been described (Baltzer, 2009).

Mitochondria contain their own genome (mtDNA), encoding a small number of proteins required for oxidative phosphorylation (OXPHOS), as well as tRNAs and rRNAs for mitochondrial translation. The majority of mitochondrial proteins are encoded by the nuclear genome, including factors for mitochondrial transcription and translation. These proteins are translated in the cytoplasm and imported into mitochondria. Accordingly, the transcription of these nuclear genes is believed to be rate limiting for mitochondrial mass and activity. To understand the nutrient-specific regulation of mitochondria, one has to characterize how these nuclear transcription factors are regulated in response to nutrients. In Drosophila, genes encoding mitochondrial proteins are highly expressed during the larval growth and feeding period. Subsequently, as the larvae stop feeding at the end of the last larval instar and prepare for metamorphosis, expression of these genes is strongly downregulated. Thus Drosophila larval growth is an ideal system to study how mitochondria are regulated in response nutrients in vivo. This has been exploited in recent microarray studies, where expression profiles of normal fed and starved larvae were compared: Indeed, starvation led to a strong downregulation of genes involved in mitochondrial translation, respiration, TCA cycle, fatty acid oxidation and mitochondrial transport. Similar findings have been published using microarrays from fed or starved adult flies. Comparing larval fat body and muscle tissues, Teleman et al. discovered that many of these genes respond in a cell-type specific manner. Therefore, factors must exist that mediate a tissue-specific transcriptional control in response to nutrients. One candidate for such a factor is dFoxo, the fly homologue to mammalian forkhead O-type transcription factors (FoxO family). Importantly, dFoxo does only mediate the nutrient responsiveness for a subset of genes encoding mitochondrial proteins. This implies that other transcription factors must exist, yet they have not been described in Drosophila (Baltzer, 2009 and references there).

This study used Drosophila to characterize mitochondria in a developing organism in vivo. Focus was placed on the larval fat body, the fly adipose/liver tissue. Fat body cells are specified during embryogenesis, and show an enormous increase in cell size during larval stages that is accompanied by endoreduplication of mitochondrial DNA to a C-value of ~256. Growth of these cells is directly regulated by nutrient uptake, making the larval fat body an ideal system to study mitochondria in response to nutrition and nutrient-sensitive growth-promoting pathways. It was show that low-yeast food conditions, and thus amino acid starvation, leads to strongly reduced mitochondrial abundance and respiration activity. This correlates with reduced expression of genes encoding mitochondrial proteins, including enzymes involved in glutamine metabolism. Moreover, Delg (Ets at 97D), the fly homologue to the alpha subunit of mammalian transcription factor NRF-2/GABP, functions as a key regulator for mitochondrial mass. Surprisingly, reduced mitochondrial mass in delg mutants does not translate into reduced OXPHOS activity. Rather, residual mitochondria compensate by being more active. More importantly, the data show that Delg is critical to adjust mitochondrial abundance and expression levels of enzymes required for glutamine metabolism in response to nutrient availability. Finally, it was observed that the nutrient-sensitive growth-promoting complex Cyclin D/Cdk4 requires Delg for its effect on mitochondria. Thus the data demonstrate how Cyclin D/Cdk4 and Delg coordinate mitochondrial abundance and glutamine metabolism with nutrient availability in vivo (Baltzer, 2009).

Drosophila Delg (Drosophila Ets like gene) is a close homologue to mammalian NRF-2α, being 39% identical in amino acids sequence. In particular, the ETS domain is highly conserved, and 12 of the 13 residues that bind NRF-2α are also conserved. Delg was first identified as one of several Drosophila proteins containing an ETS domain. Specific mutants have developmental defects, particularly during oogenesis. A null mutant background (delg613/Df(3R)ro80b) is lethal during pupal stages, whereas a hypomorphic allele (delgtne) gives raise to viable but sterile adults (Schulz, 1993). To stain for mitochondria, MitoTracker, which gave an abundant staining in the cytoplasm of wild type fat body cells, was first used. In contrast, delg null mutant cells showed a strong decrease in staining. Although reduced, mutant cells still retained staining that localized in a perinuclear manner. When quantified, a 30% reduction in MitoTracker staining was observed in delg mutant cells. Since these stainings were done on fixed tissues, they reflect mitochondrial abundance, but not mitochondrial activity. To further assay mitochondrial mass, NAO was used; this specifically labels the mitochondrial phospholipid cardiolipin, and is commonly used as a good readout to estimate mitochondrial mass. delg mutant cells showed a strong reduction in NAO. Again, residual mitochondria were concentrated around the nucleus. Finally, using electron microscopy, it was noticed that delg mutants had similar numbers of mitochondria, but mitochondria were strongly reduced in size, being on average 50% smaller in area. To test whether this effect is cell-autonomous, delg homozygous mutant clones were induced using the Flp/FRT system. Mutant cells, recognized by the absence of GFP, showed a strong reduction in MitoTracker. Taken together, these data demonstrate that mitochondrial mass is reduced in delg mutant fat body cells in a cell-autonomous manner. In contrast, ectopic expression of Delg did not result in an increase in mitochondrial abundance, demonstrating that Delg is required but not sufficient to control mitochondrial mass in vivo (Baltzer, 2009).

In Drosophila as well as other insects, the fat body is the major organ for de novo biosynthesis of fatty acids, leading to the storage of lipids as triacylglycerols. Equally important, the fat body is known to release amino acids, such a glutamine and proline, which are synthesized from the mitochondrial metabolite 2-oxoglutarate. Therefore, one would expect that mitochondrial mass and activity are regulated in response to nutrients in the larval fat body. Indeed, this study shows that strong decreases in mitochondrial abundance, respiration activity as well as expression levels of enzymes involved in glutamine and proline metabolism occur under low-yeast food. Under these feeding conditions, amino acids and fatty acids, which are both provided by yeast, become limited. Delg mutants show very similar phenotypes compared to normal fed controls, and do not show additive phenotypes in respect to mitochondrial abundance and amino acid metabolism upon low-yeast food. It is therefore proposed that Delg functions as a transcription factor to coordinate mitochondrial functions according to nutrient availability. One of these aspects is to adjust the synthesis of non-essential amino acids to the uptake of essential amino acids. In this respect, de novo synthesis of L-glutamine is particularly interesting, as the efflux of its cytoplasmic pool is used, both in mammals and Drosophila, for import of essential amino acids. It is proposed that Delg either directly senses nutrients, most likely amino acids, or is controlled by upstream sensors. Since the nutrient-sensitive Cyclin D/Cdk4 pathway functions through Delg, the latter seems more likely. Given the key role of the fat body in metabolic homeostasis of the whole animal, one might expect that fat body mitochondria be regulated differently from mitochondria in other tissues. Indeed, the phenotypes were specific to the fat body, demonstrating that Delg functions primarily in this tissue to coordinate the different anabolic and catabolic functions of mitochondria (Baltzer, 2009).

Mammalian NRF-2 was identified through its binding to the promoters of cytochrome c oxidase (COX) subunits (Virbasius, 1993; Gugneja, 1995), and has been purified as GABP (LaMarco, 1989). Active NRF-2 is a heterotetramer consisting of two alpha and two beta subunits. The alpha subunits mediate DNA binding, which requires direct GGAA/T repeats in the promoters. Accordingly, electromobility assays as well as luciferase reporter assays have shown that these motifs are functionally important. This is particular well understood for genes encoding electron transport proteins, as well as for mitochondrial protein import. Furthermore, direct NRF-2 binding to several promoters was shown by chromatin immunoprecipitation. Thus biochemical evidence links NRF-2/GABP to the transcriptional control of nuclear genes encoding mitochondrial proteins. Accordingly, RNAi studies found reduced expression of several COX subunits in cells having reduced NRF-2α levels, leading to reduced COX activity. Surprisingly, genetic data have not supported the biochemical data: MEFs lacking NRF-2α/GABPα do not have reduced mRNA or protein levels of several putative NRF-2 targets, and mitochondrial phenotypes were not reported. Drosophila Delg is the closest fly homologue to mammalian NRF-2α. Two-hybrid data show that Delg can bind to the Drosophila NRF-2β homologue CG32343, and preliminary data show that CG32343 mutants have mitochondrial defects very similar to delg mutants. Taken together, these data show that Delg functions analogues to mammalian NRF-2α, and the data are the first genetic evidence that links any member of the NRF-2α family to mitochondrial biogenesis (Baltzer, 2009 and references therein).

Of particular interest is the strong reduction in mitochondrial size in the delg mutants. This implies that mitochondrial fusion might be defective, and/or that fission occurs at an increased rate. Indeed, based on microarray data, expression of Opa1-like (CG8479), the fly homologue to mammalian fusion protein OPA1, showed a significant, 2-fold reduction in expression in delg mutant fat body samples. In contrast, fly homologues to mammalian Mitofusins, which are well-established fusion factors (fly homologues Marf/CG3869 and Fuzzy onions/CG4568), were not differently expressed. More work is required to test whether delg mutant have defects in mitochondrial fusion. In addition, when delg homozygous mutant clones were induced, a strong reduction was noted in cell size, yet the nuclear size, shown by the DAPI staining, was not changed, demonstrating growth defects, This is surprising, since endoreplication, and thus nuclear size, normally correlates with cell size in this tissue. Since mitochondrial biogenesis has been shown to correlate with nuclear DNA synthesis, the data suggest that Delg might be involved to link S-phase and potentially cell size to mitochondrial mass. Future work will be required to address this hypothesis (Baltzer, 2009).

Genes involved in mitochondrial OXPHOS activity, including RFeSP and Blw, showed similar reduced expression in the delg mutant or under low yeast nutrition. Importantly, additive defects were detected when delg mutant were grown under low yeast. Moreover, when oxygen consumption was measured in permeabilized fat body tissues, state 3 respirations were strongly affected by low yeast nutrition, yet this was independent of Delg. This demonstrates that factors other than Delg must regulate mitochondrial OXPHOS activity in response to nutrients. One candidate is Spargel/CG9809, the fly homologue to mammalian PGC-1 proteins, which are transcriptional coactivators that control mitochondrial mass and activity in response to external stimuli. Indeed, Spargel functions in parallel to Delg, and mediates a link between insulin-signalling and the expression of genes encoding mitochondrial proteins. Therefore, Delg and Spargel mediate two parallel pathways that control mitochondrial mass and OXPHOS activity in response to nutrients (Baltzer, 2009).

Drosophila Cyclin D/Cdk4 is a cyclin-dependent protein kinase complex, and controls cellular growth levels in addition to regulating cell cycle progression. Importantly for this study, overgrowth induced by ectopic expression of Cyclin D/Cdk4 is insensitive to nutrient conditions, demonstrating that the Cyclin D/Cdk4 pathway is nutrient-responsive. This study shows that ectopic expression of Cyclin D/Cdk4 in the larval fat body is sufficient to drive mitochondrial abundance in a Delg-dependent manner, suggesting a mechanism where Cyclin D/Cdk4 coordinate growth levels and mitochondrial mass. Furthermore, one would expect that the transcriptional activity associated with Delg is regulated in response to Cyclin D/Cdk4. Indeed, using lacZ insertions into the loci of several genes encoding mitochondrial proteins, including Blw, clonal expression of Cyclin D/Cdk4 was sufficient to stimulate expression of these genes. However, this effect was restricted to wandering third instar larvae, but was not seen in feeding L1, L2 or L3 larvae. Moreover, when larvae were grown under low-yeast conditions, ectopic expression of Cyclin D/Cdk4 led to reduced expression of these genes, again based on lacZ insertions. It is concluded that Cyclin D/Cdk4 is not a general activator of Delg function, but might mediate a nutrient and/or developmental-dependent control (Baltzer, 2009).

In mammals, D-type cyclins bound to Cdk4 or Cdk6 are best characterized for their functions during cell cycle progression, but several reports have shown additional roles, in particular the regulation of multiple transcription factors by direct binding. Importantly for this study, mitochondrial size and activity are regulated in response to mammalian Cyclin D1: Knockdown or knockout of Cyclin D1 leads to larger mitochondria that are more active. Conversely, ectopic expression of Cyclin D1 inhibits mitochondrial activity, a function that requires binding to Cdk4. Moreover, Cyclin D1 and NRF-1 are functional linked: Cyclin D1 stimulates NRF-1-dependent transcription, the two proteins interact, and a protein kinase associated to immunoprecipitated vCyclin D1 can phosphorylate NRF-1. This suggests a mechanism where Cyclin D1/Cdk4 inhibit mitochondrial mass and activity through inhibition of NRF-1 function. Thus D-type cyclins bound to Cdk4 have opposite effects on mitochondrial mass in mammals compared to flies, suggesting that different mechanisms have been adopted during evolution to control mitochondria (Baltzer, 2009).


PROTEIN STRUCTURE

Amino Acids - 317

Structural Domains

Analysis of full-length cDNAs indicates the presence of the characteristic pRb-binding motif LXCXL (Meyer, 2000)

In addition to the previously identified Drosophila cdc2 and cdc2c genes, four additional cdc2-related genes have been identified with low stringency and polymerase chain reaction approaches. Sequence comparisons suggest that the four putative kinases represent the Drosophila homologs of vertebrate cdk4/6, cdk5, PCTAIRE, and PITSLRE kinases. Although the similarity between human and Drosophila homologs is extensive in the case of cdk5, PCTAIRE, and PITSLRE kinases (78%, 58%, and 65% identity in the kinase domain), only limited conservation is observed for Drosophila cdk4 (47% identity) (Sauer, 1996).


Cyclin-dependent kinase 4: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 21 October 2000

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