Interactive Fly, Drosophila

Interaction of Dacapo homologs with the cyclin/cdk complex: Promotion of the G1/S transition

The regulation of cell-cycle entry has been investigated in C. elegans, taking advantage of its largely invariant and completely described pattern of somatic cell divisions. In a genetic screen, mutations in cyd-1 cyclin D and cdk-4 Cdk4/6 were identified. Recent results have indicated that during Drosophila development, cyclin D-dependent kinases regulate cell growth rather than cell division. However, the data presented here indicate that C. elegans cyd-1 primarily controls G1 progression. To investigate whether cyd-1 and cdk-4 solely act to overcome G1 inhibition by retinoblastoma family members, double mutants were constructed that completely eliminate the function of the retinoblastoma family and cyclin D-Cdk4/6 kinases. Inactivation of lin-35 Rb, the single Rb-related gene in C. elegans, substantially reduces the DNA replication and cell-division defects in cyd-1 and cdk-4 mutant animals. These results demonstrate that lin-35 Rb is an important negative regulator of G1/S progression and probably a downstream target for cyd-1 and cdk-4. However, since the suppression by lin-35 Rb is not complete, cyd-1 and cdk-4 probably have additional targets. An additional level of control over G1 progression is provided by Cip/Kip kinase inhibitors. lin-35 Rb and cki-1, a member of the CIP/KIP family of cyclin-dependent kinase inhibitors, contribute non-overlapping levels of G1/S inhibition in C. elegans. Surprisingly, loss of cki-1, but not lin-35, results in precocious entry into S phase. It is suggested that a rate limiting role for cki-1 Cip/Kip rather than lin-35 Rb explains the lack of cell-cycle phenotype of lin-35 mutant animals (Boxem, 2001).

This study examines in vivo the role and functional interrelationships of components regulating exit from the G1 resting phase into the DNA synthetic (S) phase of the cell cycle. The approach made use of several key experimental attributes of the developing mouse lens, namely its strong dependence on pRb in maintenance of the postmitotic state, the down-regulation of cyclins D and E and up-regulation of the p57(KIP2) inhibitor in the postmitotic lens fiber cell compartment, and the ability to target transgene expression to this compartment. These attributes provide an ideal in vivo context from which to examine the consequences of forced cyclin expression and/or of loss of p57(KIP2) inhibitor function in a cellular compartment. This location permits an accurate quantitation of cellular proliferation and apoptosis rates in situ. Despite substantial overlap in cyclin transgene expression levels, D-type and E cyclins exhibit clear functional differences in promoting entry into S phase. In general, forced expression of the D-type cyclins is more efficient than cyclin E in driving lens fiber cells into S phase. In the case of cyclins D1 and D2, ectopic proliferation requires their enhanced nuclear localization through CDK4 coexpression. High nuclear levels of cyclin E and CDK2, while not sufficient to promote efficient exit from G1, act synergistically with ectopic cyclin D/CDK4. The functional differences between D-type and E cyclins is most evident in the p57(KIP2)-deficient lens wherein cyclin D overexpression induces a rate of proliferation equivalent to that of the pRb null lens, while overexpression of cyclin E does not increase the rate of proliferation over that induced by the loss of p57(KIP2) function. These in vivo analyses provide strong biological support for the prevailing view that the antecedent actions of cyclin D/CDK4 act cooperatively with cyclin E/CDK2 and antagonistically with p57(KIP2) to regulate the G1/S transition in a cell type highly dependent on pRb (Gomez Lahoz, 1999).

Normal fibroblasts are dependent on adhesion to a substrate for cell cycle progression. Adhesion-deprived Rat1 cells arrest in the G1 phase of the cell cycle, with low cyclin E-dependent kinase activity, low levels of cyclin D1 protein, and high levels of the cyclin-dependent kinase inhibitor p27kip1. To understand the signal transduction pathway underlying adhesion-dependent growth, it is important to know whether prevention of any one of these down-regulation events under conditions of adhesion deprivation is sufficient to prevent the G1 arrest. To that end, sublines of Rat1 fibroblasts were used, capable of expressing cyclin E, cyclin D1, or both in an inducible manner. Ectopic expression of cyclin D1 is sufficient to allow cells to enter S phase in an adhesion-independent manner. In contrast, cells expressing exogenous cyclin E at a level high enough to overcome the p27kip1-imposed inhibition of cyclin E-dependent kinase activity still arrest in G1 when deprived of adhesion. Moreover, expression of both cyclins D1 and E in the same cells does not confer any additional growth advantage upon adhesion deprivation compared to the expression of cyclin D1 alone. Exogenously expressed cyclin D1 is down-regulated under conditions of adhesion deprivation, despite the fact that it was expressed from a heterologous promoter. The ability of cyclin D1-induced cells to enter S phase in an adhesion-independent manner disappears as soon as cyclin D1 proteins disappear. These results suggest that adhesion-dependent cell cycle progression is mediated through cyclin D1, at least in Rat1 fibroblasts (Resnitzky, 1997).

The synthesis of cyclin D1 and its assembly with cyclin-dependent kinase 4 (CDK4) to form an active complex is a rate-limiting step in progression through the G1 phase of the cell cycle. Using an activated allele of mitogen-activated protein kinase kinase 1 (MEK1), it has been shown that this kinase plays a significant role in positively regulating the expression of cyclin D1. This was found both in quiescent serum-starved cells and in cells expressing dominant-negative Ras. Despite the observation that cyclin D1 is a target of MEK1, in cycling cells it has been found that activated MEK1, but not cyclin D1, is capable of overcoming a G1 arrest induced by Ras inactivation. Either wild-type or catalytically inactive CDK4 cooperates with cyclin D1 in reversing the G1 arrest induced by inhibition of Ras activity. In quiescent NIH 3T3 cells expressing either ectopic cyclin D1 or activated MEK1, cyclin D1 is able to efficiently associate with CDK4; however, the complex is inactive. A significant percentage of the cyclin D1-CDK4 complexes are associated with p27 in serum-starved activated MEK1 or cyclin D1 cell lines. Reduction of p27 levels by expression of antisense p27 allows for S-phase entry from quiescence in NIH 3T3 cells expressing ectopic cyclin D1, but not in parental cells (Ladha, 1998).

Elevated levels of the p21(WAF1) (p21) cyclin-dependent kinase inhibitor induce growth arrest. A panel of monoclonal antibodies against human p21 has been characterized in an effort to understand the dynamic regulatory interactions between this and other cellular proteins during the cell cycle. The kinase activity of cyclin A/Cdk2 associated with p21 is significantly lower than that of cyclin A/Cdk2 free of p21, suggesting that p21 abolishes its activity in vivo, and the use of multiple antibodies has enabled a study of the molecular architecture of p21 complexes in vivo. In addition, human fibroblasts released from a quiescent state display abundant amounts of p21 devoid of associated proteins ("free" p21), the levels of which decrease as cells approach S phase. Cyclin A levels increase as the amount of monomeric p21 decreases, resulting in an excess of cyclin A/Cdk2 complexes that are not bound to, or inactivated by, p21. These data strengthen the notion that the G1-to-S phase transition in human fibroblasts occurs when the concentration of cyclin A/Cdk2 surpasses that of p21 (Cai, 1998).

CDK inhibitors are thought to prevent cell proliferation by negatively regulating cyclin-CDK complexes. The opposite is also true: cyclin-CDK complexes in mammalian cells can promote cell cycle progression by directly down-regulating CDK inhibitors. Expression of cyclin E-CDK2 in murine fibroblasts causes phosphorylation of the CDK inhibitor p27Kip1 on T187, and cyclin E-CDK2 can directly phosphorylate p27 T187 in vitro. Cyclin E-CDK2-dependent phosphorylation of p27 results in elimination of p27 from the cell, allowing cells to transit from G1 to S phase. Moreover, mutation of T187 in p27 to alanine creates a p27 protein that causes a G1 block resistant to cyclin E whose level of expression is not modulated by cyclin E. A kinetic analysis of the interaction between p27 and cyclin E-CDK2 explains how p27 can be regulated by the same enzyme it targets for inhibition. p27 interacts with cyclin E-CDK2 in at least two distinct ways: one resulting in p27 phosphorylation and release, the other in tight binding and cyclin E-CDK2 inhibition. The binding of ATP to the CDK governs which of these two states predominate. At low ATP (< 50 microM) p27 is primarily a CDK inhibitor, but at ATP concentrations approaching physiological levels (> 1 mM) p27 is more likely to be a substrate. Thus, p27 is identified as a biologically relevant cyclin E-CDK2 substrate, the physiological consequences of p27 phosphorylation has been demonstrated, and a kinetic model has been developed to explain how p27 can be both an inhibitor and a substrate of cyclin E-CDK2 (Sheaff, 1997).

A constitutively active form of mitogen-activated protein kinase kinase (MEK1) was synthesized under control of a zinc-inducible promoter in NIH 3T3 fibroblasts. Zinc treatment of serum-starved cells activates extracellular signal-regulated protein kinases (ERKs) and induces expression of cyclin D1. Newly synthesized cyclin D1 assembles with cyclin-dependent kinase-4 (CDK4) to form holoenzyme complexes that inefficiently phosphorylate the retinoblastoma protein. Activation of the MEK1/ERK pathway triggers neither degradation of the CDK inhibitor kinase inhibitory protein-1 (p27Kip1) nor leads to activation of cyclin E- and A-dependent CDK2, and such cells do not enter the DNA synthetic (S) phase of the cell division cycle. In contrast, zinc induction of active MEK1 in cells also engineered to ectopically overexpress cyclin D1 and CDK4 subunits, generates levels of cyclin D-dependent retinoblastoma protein kinase activity approximating those achieved in cells stimulated by serum. In this setting, p27Kip1 is mobilized into complexes containing cyclin D1; cyclin E- and A-dependent CDK2 complexes are activated, and serum-starved cells enter S phase. Thus, although the activity of p27Kip1 normally is canceled through a serum-dependent degradative process, overexpressed cyclin D1-CDK complexes sequester p27Kip1 and reduce the effective inhibitory threshold through a stoichiometric mechanism. A fraction of these cells complete S phase and divide, but they are unable to continuously proliferate, indicating that other serum-responsive factors ultimately become rate limiting for cell cycle progression. Therefore, the MEK/ERK pathway not only acts transcriptionally to induce the cyclin D1 gene but functions posttranslationally to regulate cyclin D1 assembly with CDK4 and to thereby help cancel p27Kip1-mediated inhibition (Cheng, 1998).

The calmodulin-dependent protein kinase-II (CaMK-II) inhibitor KN-93 reversibly arrests mouse and human cells in the G1 phase of the cell cycle. The stimulation of Ca(2+)-independent (autonomous) CaMK-II enzymatic activity, a barometer of in situ activated CaMK-II, is prevented by the same KN-93 concentrations that cause G1 phase arrest. KN-93 causes the retinoblastoma protein pRB to become dephosphorylated and the activity of both cdk2 and cdk4, two potential pRb kinases, to decrease. Neither the activity of p42MAP kinase, an early response G1 signaling molecule, nor the phosphorylation status or DNA-binding capability of the transcription factors serum response factor and cAMP responsive element-binding protein is altered during this G1 arrest. The protein levels of cyclin-dependent kinase 2 (cdk2) and cdk4 are unaffected during this G1 arrest and the total cellular levels of the cdk inhibitors p21cip1 and p27kip1 are not increased. Instead, the cdk4 activity decreases resulting from KN-93 are the result of a 75% decrease in cyclin D1 levels. In contrast, cyclin A and E levels are relatively constant. Cdk2 activity decreases are primarily the result of enhanced p27kip1 association with cdk2/cyclin E. All of these phenomena are unaffected by KN-93's inactive analog, KN-92, and are reversible upon KN-93 washout. The kinetics of recovery from cell cycle arrest are similar to those reported for other G1 phase blockers. These results suggest a mechanism by which G1 Ca2+ signals can be linked via calmodulin-dependent phosphorylations to the cell cycle-controlling machinery through cyclins and cdk inhibitors (Morris, 1998).

Cyclin E-Cdk2 kinase activation is an essential step in Myc-induced proliferation. It is presumed that this requires sequestration of G1 cell cycle inhibitors p27Kip1 and p21Cip1 (Ckis) via a Myc-induced protein. This sequestration is shown to be mediated by the protein synthesis rate of induction of cyclin D1 and/or cyclin D2. Consistent with this, primary cells from cyclin D1-/- and cyclin D2-/- mouse embryos, unlike wild-type controls, do not respond to Myc with increased proliferation, although they undergo accelerated cell death in the absence of serum. Myc sensitivity of cyclin D1-/- cells can be restored by retroviruses expressing either cyclins D1, D2 or a cyclin D1 mutant that forms kinase-defective, Cki-binding cyclin-cdk complexes. Thus, the sequestration function of D cyclins appears essential for Myc-induced cell cycle progression but dispensable for apoptosis (Perez-Roger, 1999).

The rate of the induction of cyclin D1 and/or cyclin D2 protein synthesis leads to the preferential association of p27Kip1 and p21Cip1 with cyclin D-Cdk complexes. At the same time Myc also induces cyclin E protein synthesis; the rates of induction help to promote a net gain of newly formed Cki-free cyclin E-Cdk2 complexes. These complexes become active concomitant with phosphorylation of the kinase subunit by CAK. Consistent with this model of dynamic equilibrium, cyclin E-Cdk2 kinase activity can be controlled by changes in the rates of cyclin D synthesis. Moreover, as shown with a cyclin D mutant that forms kinase-defective Cki-binding cyclin D-Cdk complexes, this link between cyclins D-Cdk and cyclin E-Cdk2 is independent of cyclin D-Cdk activity, but correlates with the ability of cyclin D-Cdk complexes to bind or sequester Ckis. This is strongly supported by the fact that the deficiency of cyclin D1-/- mouse embryo cells to respond to Myc with increased proliferation is restored by expression of the same cyclin D mutant. Consistent with these findings, transient over-expression of either catalytically inactive cyclin D-Cdk, or cyclin E-Cdk2 complexes can rescue the cell cycle inhibitory effect of a dominant-negative Mad-Myc chimera. It is concluded that due to the nature of physical interactions between cyclin D-Cdks and the cell cycle inhibitors p27Kip1 and p21Cip1, cyclin D-Cdk complexes can fulfil a dual function as cell cycle kinases and as buffers for sequestration or release of cell cycle inhibitors (Perez-Roger, 1999 and references therein).

Table of contents

dacapo: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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