Cyclin E constrains Cdk5 activity to regulate synaptic plasticity and memory formation

Cyclin E is a component of the core cell cycle machinery, and it drives cell proliferation by regulating entry and progression of cells through the DNA synthesis phase. Cyclin E expression is normally restricted to proliferating cells. However, high levels of cyclin E are expressed in the adult brain. The function of cyclin E in quiescent, postmitotic nervous system remains unknown. Here we use a combination of in?vivo quantitative proteomics and analyses of cyclin E knockout mice to demonstrate that in terminally differentiated neurons cyclin E forms complexes with Cdk5 and controls synapse function by restraining Cdk5 activity. Ablation of cyclin E led to?a decreased number of synapses, reduced number and volume of dendritic spines, and resulted in impaired synaptic plasticity and memory formation in cyclin E-deficient animals. These results reveal a cell cycle-independent role for a core cell cycle protein, cyclin E, in synapse function and memory.     

Cdk2 knockout mice are viable

BACKGROUND: Cyclin-dependent kinases (Cdks) and their cyclin regulatory subunits control cell growth and division. Cdk2/cyclin E complexes are thought to be required because they phosphorylate the retinoblastoma protein and drive cells through the G1/S transition into the S phase of the cell cycle. In addition, Cdk2 associates with cyclin A, which itself is essential for cell proliferation during early embryonic development. RESULTS: In order to study the functions of Cdk2 in vivo, we generated Cdk2 knockout mice. Surprisingly, these mice are viable, and therefore Cdk2 is not an essential gene in the mouse. However, Cdk2 is required for germ cell development; both male and female Cdk2(-/-) mice are sterile. Immunoprecipitates of cyclin E1 complexes from Cdk2(-/-) spleen extracts displayed no activity toward histone H1. Cyclin A2 complexes were active in primary mouse embryonic fibroblasts (MEFs), embryo extracts and in spleen extracts from young animals. In contrast, there was little cyclin A2 kinase activity in immortalized MEFs and spleen extracts from adult animals. Cdk2(-/-) MEFs proliferate but enter delayed into S phase. Ectopic expression of Cdk2 in Cdk2(-/-) MEFs rescued the delayed entry into S phase. CONCLUSIONS: Although Cdk2 is not an essential gene in the mouse, it is required for germ cell development and meiosis. Loss of Cdk2 affects the timing of S phase, suggesting that Cdk2 is involved in regulating progression through the mitotic cell cycle.     

Retinoblastoma protein (pRb), but not p107 or p130, is required for maintenance of enterocyte quiescence and differentiation in small intestine

The function of retinoblastoma protein (pRb) in the regulation of small intestine epithelial cell homeostasis has been challenged by several groups using various promoter-based Cre transgenic mouse lines. Interestingly, different pRb deletion systems yield dramatically disparate small intestinal phenotypes. These findings confound the function of pRb in this dynamic tissue. In this study, Villin-Cre transgenic mice were crossed with Rb (flox/flox) mice to conditionally delete pRb protein in small intestine enterocytes. We discovered a novel hyperplasia phenotype as well as ectopic cell cycle reentry within villus enterocytes in the small intestine. This phenotype was not seen in other pRb family member (p107 or p130) null mice. Using a newly developed crypt/villus isolation method, we uncovered that expression of pRb was undetectable, whereas proliferating cell nuclear antigen, p107, cyclin E, cyclin D3, Cdk2, and Cdc2 were dramatically increased in pRb-deficient villus cells. Cyclin A, cyclin D1, cyclin D2, and Cdk4/6 expression was not affected by absent pRb expression. pRb-deficient villus cells appeared capable of progressing to mitosis but with higher rates of apoptosis. However, the cycling villus enterocytes were not completely differentiated as gauged by significant reduction of intestinal fatty acid-binding protein expression. In summary, pRb, but not p107 or p130, is required for maintaining the postmitotic villus cell in quiescence, governing the expression of cell cycle regulatory proteins, and completing of absorptive enterocyte differentiation in the small intestine.     

Cyclin D3 and c-MYC control glucocorticoid-induced cell cycle arrest but not apoptosis in lymphoblastic leukemia cells

Glucocorticoids (GC) induce cell cycle arrest and apoptosis in lymphoblastic leukemia cells. To investigate cell cycle effects of GC in the absence of obscuring apoptotic events, we used human CCRF-CEM leukemia cells protected from cell death by transgenic bcl-2. GC treatment arrested these cells in the G1 phase of the cell cycle due to repression of cyclin D3 and c-myc. Cyclin E and Cdk2 protein levels remained high, but the kinase complex was inactive due to increased levels of bound p27(Kip1). Conditional expression of cyclin D3 and/or c-myc was sufficient to prevent GC-induced G1 arrest and p27(Kip1) accumulation but, importantly, did not interfere with the induction of apoptosis. The combined data suggest that repression of both, c-myc and cyclin D3, is necessary to arrest human leukemia cells in the G1 phase of the cell division cycle, but that neither one is required for GC-induced apoptosis.     

A Distinct Expression Pattern of Cyclin K in Mammalian Testes Suggests a Functional Role in Spermatogenesis

Germ cell and embryonic stem cells are inextricably linked in many aspects. Remarkably both can generate all somatic cell types in organisms. Yet the molecular regulation accounting for these similarities is not fully understood. Cyclin K was previously thought to associate with CDK9 to regulate gene expression. However, we and others have recently shown that its cognate interacting partners are CDK12 and CDK13 in mammalian cells. We further demonstrated that cyclin K is essential for embryonic stem cell maintenance. In this study, we examined the expression of cyclin K in various murine and human tissues. We found that cyclin K is highly expressed in mammalian testes in a developmentally regulated manner. During neonatal spermatogenesis, cyclin K is highly expressed in gonocytes and spermatogonial stem cells. In adult testes, cyclin K can be detected in spermatogonial stem cells but is absent in differentiating spermatogonia, spermatids and spermatozoa. Interestingly, the strongest expression of cyclin K is detected in primary spermatocytes. In addition, we found that cyclin K is highly expressed in human testicular cancers. Knockdown of cyclin K in a testicular cancer cell line markedly reduces cell proliferation. Collectively, we suggest that cyclin K may be a novel molecular link between germ cell development, cancer development and embryonic stem cell maintenance.     

Cdk2 plays a critical role in hepatocyte cell cycle progression and survival in the setting of cyclin D1 expression in vivo

Cdk2 was once believed to play an essential role in cell cycle progression, but cdk2-/- mice have minimal phenotypic abnormalities. In this study, we examined the role of cdk2 in hepatocyte proliferation, centrosome duplication, and survival. Cdk2-/- hepatocytes underwent mitosis and had normal centrosome content after mitogen stimulation. Unlike wild-type cells, cdk2-/- liver cells failed to undergo centrosome overduplication in response to ectopic cyclin D1 expression. After mitogen stimulation in culture or partial hepatectomy in vivo, cdk2-/- hepatocytes demonstrated diminished proliferation. Cyclin D1 is a key mediator of cell cycle progression in hepatocytes, and transient expression of this protein is sufficient to promote robust proliferation of these cells in vivo. In cdk2-/- mice and animals treated with the cdk2 inhibitor seliciclib, cyclin D1 failed to induce hepatocyte cell cycle progression. Surprisingly, cdk2 ablation or inhibition led to massive hepatocyte and animal death following cyclin D1 transfection. In a transgenic model of chronic hepatic cyclin D1 expression, seliciclib induced hepatocyte injury and animal death, suggesting that cdk2 is required for survival of cyclin D1-expressing cells even in the absence of substantial proliferation. In conclusion, our studies demonstrate that cdk2 plays a role in liver regeneration. Furthermore, it is essential for centrosome overduplication, proliferation, and survival of hepatocytes that aberrantly express cyclin D1 in vivo. These studies suggest that cdk2 may warrant further investigation as a target for therapy of liver tumors with constitutive cyclin D1 expression.     

Cyclin D1 transgene impedes lymphocyte maturation and collaborates in lymphomagenesis with the myc gene.

Cyclin D1 is the regulatory subunit of certain protein kinases thought to advance the G1 phase of the cell cycle. Deregulated cyclin D1 expression has been implicated in several human neoplasms, most consistently in centrocytic B lymphoma, where the cyclin D1 gene usually has been translocated to an immunoglobulin locus. To determine directly whether constitutive cyclin D1 expression is lymphomagenic, transgenic mice were generated having the cyclin D1 gene linked to an immunoglobulin enhancer. Despite abundant transgene expression, their lymphocytes were normal in cell cycle activity, size and mitogen responsiveness, but young transgenic animals contained fewer mature B- and T-cells. Although spontaneous tumours were infrequent, lymphomagenesis was much more rapid in mice that co-expressed the cyclin D1 transgene and a myc transgene than in mice expressing either transgene alone. Moreover, the spontaneous lymphomas of myc transgenic animals often ectopically expressed the endogenous cyclin D1 gene. These findings indicate that this G1 cyclin can modulate differentiation and collaborate with myc-like genes in oncogenesis.     

Effect of elevated levels of ornithine decarboxylase on cell cycle progression in skin.

By crossing TG.AC v-Ha-ras and K6/ODC transgenic mice, we found previously that an activated ras and follicular ornithine decarboxylase (ODC) overexpression cooperate to generate spontaneous tumors in the skin. Cellular proliferation was dramatically increased in the K6/ODC transgenic skin, as evidenced by elevated proliferating cell nuclear antigen and Ki67 expression compared with nontransgenic littermates. Keratinocytes isolated from transgenic skin also displayed increased clonal growth. Paradoxically, expression of the growth inhibition-associated proteins p53, p21Waf1, p27Klp1, and Bax was increased with ODC overexpression in the skin. ODC overexpression did not affect cyclin D/cyclin-dependent kinase 4 (Cdk4)-dependent phosphorylation of retinoblastoma protein but stimulated cyclin E/Cdk2 and cyclin A/Cdk2-associated kinase activity, with minimal effect on the levels of these proteins. Thus, ODC/polyamine-induced activation of cyclin E/Cdk2 and cyclin A/Cdk2-associated kinase activity may cooperate with the ras induction of cyclin D/Cdk4/6-associated retinoblastoma protein phosphorylation to not only stimulate proliferation but ultimately contribute to tumor development.     

Cell-cycle regulation and mammalian gametogenesis: a lesson from the unexpected

The progression of mammalian gametogenesis requires a precise balance between cell-cycle activities and elimination of defective gametogenic cells to ensure the perpetuation of species. Both spermatogonia and oogonia are stem cell populations committed to meiosis with the aim of generating haploid gametes for fertilization. At puberty, mitotically dividing spermatogonial cell cohorts maintain the ability of cell renewal and occupy niches in the seminiferous tubule. In contrast, mitotically dividing oogonial cell cohorts produced in the fetal ovary, are exclusively committed to meiosis and produce primordial follicles housing a primary oocyte surrounded by somatic follicular cells. A consistent physiological event during mammalian gametogenesis is the disposal of spermatogenic cells by apoptosis and ovarian follicles by atresia. Cyclin-dependent kinases (Cdks) and their cyclin partners coordinate the activities of the cell cycle. An additional cell-cycle regulatory component is the centrosome. The centrosome harbors regulatory proteins controlling the normal progression of the cell cycle. Changes in individual centrosome proteins can lead to cell-cycle arrest and a decrease in the genomic protective function of p53 that promotes apoptosis. Disruption of cyclin A1, Cdk2, and Cdk4 expression in transgenic mice results in infertility and gonadal atrophy. Cdk-cyclin complexes interact with regulatory proteins, which may fine-tune the activities of the complex. One of the many regulatory proteins is p12, a 115 amino acid growth suppressor polypeptide designated p12(CDK2AP1), partner of Cdk2 and with binding affinity to DNA polymerase alpha/primase. Overexpression of p12 is associated with testicular and ovarian atrophy without affecting fertility. Ectopic expression of p12 was driven by the keratin 14 promoter. Keratin 14 is the pairing partner of keratin 5 and both keratins are expressed in testis. The efficiency of keratin promoters in driving ectopic gonadal gene expression, the association of gonadal atrophy with the ectopic expression of a Cdk2 regulatory protein and the centrosome, as a reservoir of cell-cycle regulatory proteins, open new experimental opportunities to address still lingering questions concerning cell differentiation and division during mammalian gametogenesis.     

Sister chromatids fail to separate during an induced endoreplication cycle in Drosophila embryos

When mitosis is bypassed, as in some cancer cells or in natural endocycles, sister chromosomes remain paired and produce four-stranded diplochromosomes or polytene chromosomes. Cyclin/Cdk1 inactivation blocks entry into mitosis and can reset G2 cells to G1, allowing another round of replication. Reciprocally, persistent expression of Cyclin A/Cdk1 or Cyclin E/Cdk2 blocks Drosophila endocycles. Inactivation of Cyclin A/Cdk1 by mutation or overexpression of the Cyclin/Cdk1 inhibitor, Roughex (Rux), converts the 16(th) embryonic mitotic cycle to an endocycle; however, we show that Rux expression fails to convert earlier cell cycles unless Cyclin E is also downregulated. Following induction of a Rux transgene in Cyclin E mutant embryos during G2 of cell cycle 14 (G2(14)), Cyclins A, B, and B3 disappeared and cells reentered S phase. This rereplication produced diplochromosomes that segregated abnormally at a subsequent mitosis. Thus, like the yeast CKIs Rum1 and Sic1, Drosophila Rux can reset G2 cells to G1. The observed cyclin destruction suggests that cell cycle resetting by Rux was associated with activation of the anaphase-promoting complex (APC), while the presence of diplochromosomes implies that this activation of APC outside of mitosis was not sufficient to trigger sister disjunction.     

Cyclin E2, a Novel G1 Cyclin That Binds Cdk2 and Is Aberrantly Expressed in Human Cancers

A novel cyclin gene was discovered by searching an expressed sequence tag database with a cyclin box profile. The human cyclin E2 gene encodes a 404-amino-acid protein that is most closely related to cyclin E. Cyclin E2 associates with Cdk2 in a functional kinase complex that is inhibited by both p27^Kip1 and p21^Cip1. The catalytic activity associated with cyclin E2 complexes is cell cycle regulated and peaks at the G₁/S transition. Overexpression of cyclin E2 in mammalian cells accelerates G₁, demonstrating that cyclin E2 may be rate limiting for G₁ progression. Unlike cyclin E1, which is expressed in most proliferating normal and tumor cells, cyclin E2 levels were low to undetectable in nontransformed cells and increased significantly in tumor-derived cells. The discovery of a novel second cyclin E family member suggests that multiple unique cyclin E-CDK complexes regulate cell cycle progression.     

Cdk2 as a master of S phase entry: fact or fake?

It has long been believed that Cdk2 and its activator cyclin E play essential roles in the progression of the mitotic cell cycle. However, recent studies using knockout mouse models revealed that neither Cdk2 nor cyclin E are essential in vivo. The purpose of this Perspective is to compare both Cdk2 and cyclin E knockout mice models and to discuss potential mechanisms driving the cell cycle in the absence of Cdk2 or cyclin E. Particular emphasis is placed on possible non-catalytic roles of cyclin E, the expression and activity of the second cyclin binding partner of Cdk2, cyclin A, as well as on the expression and degradation of the Cdk2 inhibitor p27Kip1 in the absence of Cdk2.     

Proteolytic cleavage of cyclin E leads to inactivation of associated kinase activity and amplification of apoptosis in hematopoietic cells

Cyclin E/Cdk2 is a critical regulator of cell cycle progression from G(1) to S in mammalian cells and has an established role in oncogenesis. Here we examined the role of deregulated cyclin E expression in apoptosis. The levels of p50-cyclin E initially increased, and this was followed by a decrease starting at 8 h after treatment with genotoxic stress agents, such as ionizing radiation. This pattern was mirrored by the cyclin E-Cdk2-associated kinase activity and a time-dependent expression of a novel p18-cyclin E. p18-cyclin E was induced during apoptosis triggered by multiple genotoxic stress agents in all hematopoietic tumor cell lines we have examined. The p18-cyclin E expression was prevented by Bcl-2 overexpression and by the general caspase and specific caspase 3 pharmacologic inhibitors zVAD-fluoromethyl ketone (zVAD-fmk) and N-acetyl-Asp-Glu-Val-Asp-aldehyde (DEVD-CHO), indicating that it was linked to apoptosis. A p18-cyclin E(276-395) (where cyclin E(276-395) is the cyclin E fragment containing residues 276 to 395) was reconstituted in vitro, with mutagenesis experiments, indicating that the caspase-dependent cleavage was at amino acid residues 272 to 275. Immunoprecipitation analyses of the ectopically expressed cyclin E(1-275), cyclin E(276-395) deletion mutants, and native p50-cyclin E demonstrated that caspase-mediated cyclin E cleavage eliminated interaction with Cdk2 and therefore inactivated the associated kinase activity. Overexpression of cyclin E(276-395), but not of several other cyclin E mutants, specifically induced phosphatidylserine exposure and caspase activation in a dose-dependent manner, which were inhibited in Bcl-2-overexpressing cells or in the presence of zVAD-fmk. Apoptosis and generation of p18-cyclin E were significantly inhibited by overexpressing the cleavage-resistant cyclin E mutant, indicating a functional role for caspase-dependent proteolysis of cyclin E for apoptosis of hematopoietic tumor cells.     

Cyclin D3 compensates for loss of cyclin D2 in mouse B-lymphocytes activated via the antigen receptor and CD40

Cyclin D2 is the only D-type cyclin expressed in mature mouse B-lymphocytes, and its expression is associated with retinoblastoma protein (pRB) and pRB-related protein phosphorylation and induction of E2F activity, as B-cells enter the cell cycle following stimulation via surface IgM and/or CD40. Cyclin D-dependent kinase activity is required for cell proliferation, yet cyclin D2(-/-) mice have normal levels of mature B-lymphocytes. Here we show that B-lymphocytes from cyclin D2(-/-) mice can proliferate in response to anti-IgM and anti-CD40, but the time taken to enter S-phase is longer than for the corresponding cyclin D2(+/+) cells. This is due to the compensatory induction of cyclin D3, but not cyclin D1, which causes pRb phosphorylation on CDK4-specific sites. This is the first demonstration that loss of a D-type cyclin causes specific expression and functional compensation by another member of the family in vivo and provides a rationale for the presence of mature B-lymphocytes in cyclin D2(-/-) mice.     

SCAPER, a novel cyclin A-interacting protein that regulates cell cycle progression

Cyclin A/Cdk2 plays an important role during S and G2/M phases of the eukaryotic cell cycle, but the mechanisms by which it regulates cell cycle events are not fully understood. We have biochemically purified and identified SCAPER, a novel protein that specifically interacts with cyclin A/Cdk2 in vivo. Its expression is cell cycle independent, and it associates with cyclin A/Cdk2 at multiple phases of the cell cycle. SCAPER localizes primarily to the endoplasmic reticulum. Ectopic expression of SCAPER sequesters cyclin A from the nucleus and results specifically in an accumulation of cells in M phase of the cell cycle. RNAi-mediated depletion of SCAPER decreases the cytoplasmic pool of cyclin A and delays the G1/S phase transition upon cell cycle re-entry from quiescence. We propose that SCAPER represents a novel cyclin A/Cdk2 regulatory protein that transiently maintains this kinase in the cytoplasm. SCAPER could play a role in distinguishing S phase- from M phase-specific functions of cyclin A/Cdk2.     

Overexpression of Bcl-W in the testis disrupts spermatogenesis: revelation of a role of BCL-W in male germ cell cycle control

To explore physiological roles of BCL-W, a prosurvival member of the BCL-2 protein family, we generated transgenic (TG) mice overexpressing Bcl-w driven by a chicken beta-actin promoter. Male Bcl-w TG mice developed normally but were infertile. The adult TG testes displayed disrupted spermatogenesis with various severities ranging from thin seminiferous epithelium containing less germ cells to Sertoli cell-only appearance. No overpopulation of any type of germ cells was observed during testicular development. In contrast, the developing TG testes displayed decreased number of spermatogonia, degeneration, and detachment of spermatocytes and Sertoli cell vacuolization. The proliferative activity of germ cells was significantly reduced during testicular development and spermatogenesis, as determined by in vivo and in vitro 5'-bromo-2'deoxyuridine incorporation assays. Sertoli cells were structurally and functionally normal. The degenerating germ cells were TUNEL-negative and no typical apoptotic DNA ladder was detected. Our data suggest that regulated spatial and temporal expression of BCL-W is required for normal testicular development and spermatogenesis, and overexpression of BCL-W inhibits germ cell cycle entry and/or cell cycle progression leading to disrupted spermatogenesis.