Activation of cyclin-dependent kinases by Myc mediates induction of cyclin A, but not apoptosis.

The activation of conditional alleles of Myc induces both cell proliferation and apoptosis in serum-deprived RAT1 fibroblasts. Entry into S phase and apoptosis are both preceded by increased levels of cyclin E- and cyclin D1-dependent kinase activities. To assess which, if any, cellular responses to Myc depend on active cyclin-dependent kinases (cdks), we have microinjected expression plasmids encoding the cdk inhibitors p16, p21 or p27, and have used a specific inhibitor of cdk2, roscovitine. Expression of cyclin A, which starts late in G1 phase, served as a marker for cell cycle progression. Our data show that active G1 cyclin/cdk complexes are both necessary and sufficient for induction of cyclin A by Myc. In contrast, neither microinjection of cdk inhibitors nor chemical inhibition of cdk2 affected the ability of Myc to induce apoptosis in serum-starved cells. Further, in isoleucine-deprived cells, Myc induces apoptosis without altering cdk activity. We conclude that Myc acts upstream of cdks in stimulating cell proliferation and also that activation of cdks and induction of apoptosis are largely independent events that occur in response to induction of Myc.     

Hepatocyte growth factor releases mink epithelial cells from transforming growth factor beta1-induced growth arrest by restoring Cdk6 expression and cyclin E-associated Cdk2 activity

Transforming growth factor beta (TGF-beta) potently suppresses Mv1Lu mink epithelial cell growth, whereas hepatocyte growth factor (HGF) counteracts TGF-beta-mediated growth inhibition and induces Mv1Lu cell proliferation (J. Taipale and J. Keski-Oja, J. Biol. Chem. 271:4342-4348, 1996). By addressing the cell cycle regulatory mechanisms involved in HGF-mediated release of Mv1Lu cells from TGF-beta inhibition, we show that increased DNA replication is accompanied by phosphorylation of the retinoblastoma protein and alternative regulation of cyclin-Cdk-inhibitor complexes. While TGF-beta treatment decreased the expression of Cdk6, this effect was counteracted by HGF, followed by partial restoration of cyclin D2-associated kinase activity. Notably, HGF failed to prevent TGF-beta induction of p15 and its association with Cdk6. However, HGF reversed the TGF-beta-mediated decrease in Cdk6-associated p27 and cyclin D2-associated Cdk6, suggesting that HGF modifies the TGF-beta response at the level of G1 cyclin complex formation. Counteraction of TGF-beta regulation of Cdk6 by HGF may in turn affect the association of p27 with Cdk2-cyclin E complexes. Though HGF did not differentially regulate the total levels of p27 in TGF-beta-treated cells, p27 immunodepletion experiments suggested that upon treatment with both growth factors, less p27 is associated with Cdk2-cyclin E complexes, in parallel with restoration of the active form of Cdk2 and the associated kinase activity. The results demonstrate that HGF intercepts TGF-beta cell cycle regulation at multiple points, affecting both G1 and G1-S cyclin kinase activities.     

Multifaceted regulation of cell cycle progression by estrogen: regulation of Cdk inhibitors and Cdc25A independent of cyclin D1-Cdk4 function

Estrogens induce proliferation of estrogen receptor (ER)-positive MCF-7 breast cancer cells by stimulating G(1)/S transition associated with increased cyclin D1 expression, activation of cyclin-dependent kinases (Cdks), and phosphorylation of the retinoblastoma protein (pRb). We have utilized blockade of cyclin D1-Cdk4 complex formation through adenovirus-mediated expression of p16(INK4a) to demonstrate that estrogen regulates Cdk inhibitor expression and expression of the Cdk-activating phosphatase Cdc25A independent of cyclin D1-Cdk4 function and cell cycle progression. Expression of p16(INK4a) inhibited G(1)/S transition induced in MCF-7 cells by 17-beta-estradiol (E(2)) with associated inhibition of both Cdk4- and Cdk2-associated kinase activities. Inhibition of Cdk2 activity was associated with delayed removal of Cdk-inhibitory activity in early G(1) and decreased cyclin A expression. Cdk-inhibitory activity and expression of both p21(Cip1) and p27(Kip1) was decreased, however, in both control and p16(INK4a)-expressing cells 20 h after estrogen treatment. Expression of Cdc25A mRNA and protein was induced by E(2) in control and p16(INK4a)-expressing MCF-7 cells; however, functional activity of Cdc25A was inhibited in cells expressing p16(INK4a). Inhibition of Cdc25A activity in p16(INK4a)-expressing cells was associated with depressed Cdk2 activity and was reversed in vivo and in vitro by active Cdk2. Transfection of MCF-7 cells with a dominant-negative Cdk2 construct inhibited the E(2)-dependent activation of ectopic Cdc25A. Supporting a role for Cdc25A in estrogen action, antisense CDC25A oligonucleotides inhibited estrogen-induced Cdk2 activation and DNA synthesis. In addition, inactive cyclin E-Cdk2 complexes from p16(INK4a)-expressing, estrogen-treated cells were activated in vitro by treatment with recombinant Cdc25A and in vivo in cells overexpressing Cdc25A. The results demonstrate that functional association of cyclin D1-Cdk4 complexes is required for Cdk2 activation in MCF-7 cells and that Cdk2 activity is, in turn, required for the in vivo activation of Cdc25A. These studies establish Cdc25A as a growth-promoting target of estrogen action and further indicate that estrogens independently regulate multiple components of the cell cycle machinery, including expression of p21(Cip1) and p27(Kip1).     

Complex mechanisms underlying impaired activation of Cdk4 and Cdk2 in replicative senescence: roles of p16, p21, and cyclin D1

Numerous changes in gene expression are known to occur during replicative senescence, including changes in genes involved in the cell cycle control. In the present study, we have found a severe impairment in the activation of Cdk2 and Cdk4 in response to mitogens in senescent human fibroblasts and determined the molecular basis for this. Although Cdk4 protein was constitutively expressed in senescent cells at the same level as in early-passage young cells, it was found to be complexed with a distinct set of Cdk inhibitors. Cdk4 derived from early passage quiescent cells was effectively activated by incubation with cyclin D1 and Cdk-activating kinase (CAK) in vitro, whereas Cdk4 from senescent cells was not. Cdk2 protein was dramatically decreased in senescent cells and complexed primarily with cyclin D1 and p21. This cyclin D1-bound Cdk2 was not activated by CAK either in vivo or in vitro, implicating cyclin D1 as an inhibitor of Cdk2 activation. Thus, one of the underlying molecular events involved in replicative senescence is the impaired activation of Cdk4 and Cdk2 due to increased binding of p16 to Cdk4 and increased association of Cdk2 with cyclin D1 and p21.     

The proto-oncogene c-myc acts through the cyclin-dependent kinase (Cdk) inhibitor p27(Kip1) to facilitate the activation of Cdk4/6 and early G(1) phase progression

Progression through the early G(1) phase of the cell cycle requires mitogenic stimulation, which ultimately leads to the activation of cyclin-dependent kinases 4 and 6 (Cdk4/6). Cdk4/6 activity is promoted by D-type cyclins and opposed by Cdk inhibitor proteins. Loss of c-myc proto-oncogene function results in a defect in the activation of Cdk4/6. c-myc(-/-) cells express elevated levels of the Cdk inhibitor p27(Kip1) and reduced levels of Cdk7, the catalytic subunit of Cdk-activating kinase. We show here that in normal (c-myc(+/+)) cells, the majority of cyclin D-Cdk4/6 complexes are assembled with p27 and remain inactive during cell cycle progression; their function is presumably to sequester p27 from Cdk2 complexes. A small fraction of Cdk4/6 protein was found in lower molecular mass catalytically active complexes. Conditional overexpression of p27 in c-myc(+/+) cells caused inhibition of Cdk4/6 activity and elicited defects in G(0)-to-S phase progression very similar to those seen in c-myc(-/-) cells. Overexpression of cyclin D1 in c-myc(-/-) cells rescued the defect in Cdk4/6 activity, indicating that the limiting factor is the number of cyclin D-Cdk4/6 complexes. Cdk-activating kinase did not rescue Cdk4/6 activity. We propose that the defect in Cdk4/6 activity in c-myc(-/-) cells is caused by the elevated levels of p27, which convert the low abundance activable cyclin D-Cdk4/6 complexes into unactivable complexes containing higher stoichiometries of p27. These observations establish p27 as a physiologically relevant regulator of cyclin D-Cdk4/6 activity as well as mechanistically a target of c-Myc action and provide a model by which c-Myc influences the early-to-mid G(1) phase transition.     

Redistribution of the CDK inhibitor p27 between different cyclin.CDK complexes in the mouse fibroblast cell cycle and in cells arrested with lovastatin or ultraviolet irradiation.

The cyclin-dependent kinase (CDK) inhibitor p27 binds and inhibits the kinase activity of several CDKs. Here we report an analysis of the behavior and partners of p27 in Swiss 3T3 mouse fibroblasts during normal mitotic cell cycle progression, as well as in cells arrested at different stages in the cycle by growth factor deprivation, lovastatin treatment, or ultraviolet (UV) irradiation. We found that the level of p27 is elevated in cells arrested in G0 by growth factor deprivation or contact inhibition. In G0, p27 was predominantly monomeric, although some portion was associated with residual cyclin A.Cdk2. During G1, all of p27 was associated with cyclin D1.Cdk4 and was then redistributed to cyclin A.Cdk2 as cells entered S phase. The loss of the monomeric p27 pool as cyclins accumulate in G1 is consistent with the in vivo and in vitro data showing that p27 binds better to cyclin.CDK complexes than to monomeric CDKs. In growing cells, the majority of p27 was associated with cyclin D1 and the level of p27 was significantly lower than the level of cyclin D1. In cells arrested in G1 with lovastatin, cyclin D1 was degraded and p27 was redistributed to cyclin A.Cdk2. In contrast to p21 (which is a p27-related CDK inhibitor and is induced by UV irradiation), the level of p27 was reduced after UV irradiation, but because cyclin D1 was degraded more rapidly than p27, there was a transient increase in binding of p27 to cyclin A.Cdk2. These data suggest that cyclin D1.Cdk4 acts as a reservoir for p27, and p27 is redistributed from cyclin D1.Cdk4 to cyclin A.Cdk2 complexes during S phase, or when cells are arrested by growth factor deprivation, lovastatin treatment, or UV irradiation. It is likely that a similar principle of redistribution of p27 is used by the cell in other instances of cell cycle arrest.     

Protein kinase A regulates expression of p27(kip1) and cyclin D3 to suppress proliferation of leukemic T cell lines.

Peripheral homeostasis and tolerance requires the suppression or removal of excessive or harmful T lymphocytes. This can occur either by apoptosis through active antigen-induced death or cytokine withdrawal. Alternatively, T cell activation can be suppressed by agents that activate the cAMP-dependent protein kinase (PKA) signaling pathway, such as prostaglandin E2. Stimulation of PKA inhibits lymphocyte proliferation and immune effector functions. Here we have investigated the mechanism by which activation of PKA induces inhibition of proliferation in human leukemic T cell lines. Using a variety of agents that stimulate PKA, we can arrest Jurkat and H9 leukemic T cells in the G(1) phase of the cell cycle, whereas cell viability is hardly affected. This G(1) arrest is associated with an inhibition of cyclin D/Cdk and cyclin E/Cdk kinase activity. Interestingly, expression of cyclin D3 is rapidly reduced by PKA activation, whereas expression of the Cdk inhibitor p27(kip1) is induced. Ectopic expression of cyclin D3 can override the growth suppression induced by PKA activation to some extent, indicating that growth inhibition of leukemic T cells by PKA activation is partially dependent on down-regulation of cyclin D3 expression. Taken together our data suggest that immunosuppression by protein kinase A involves regulation of both cyclin D3 and p27(kip1) expression.     

Ras links growth factor signaling to the cell cycle machinery via regulation of cyclin D1 and the Cdk inhibitor p27KIP1.

Activation of growth factor receptors by ligand binding initiates a cascade of events leading to cell growth and division. Progression through the cell cycle is controlled by cyclin-dependent protein kinases (Cdks), but the mechanisms that link growth factor signaling to the cell cycle machinery have not been established. We report here that Ras proteins play a key role in integrating mitogenic signals with cell cycle progression through G1. Ras is required for cell cycle progression and activation of both Cdk2 and Cdk4 until approximately 2 h before the G1/S transition, corresponding to the restriction point. Analysis of Cdk-cyclin complexes indicates that Ras signaling is required both for induction of cyclin D1 and for downregulation of the Cdk inhibitor p27KIP1. Constitutive expression of cyclin D1 circumvents the requirement for Ras signaling in cell proliferation, indicating that regulation of cyclin D1 is a critical target of the Ras signaling cascade.     

Dna damage-induced G(1) arrest in hematopoietic cells is overridden following phosphatidylinositol 3-kinase-dependent activation of cyclin-dependent kinase 2

Exposure of hematopoietic cells to DNA-damaging agents induces p53-independent cell cycle arrest at a G(1) checkpoint. Previously, we have shown that this growth arrest can be overridden by cytokine growth factors, such as erythropoietin or interleukin-3, through activation of a phosphatidylinositol 3-kinase (PI 3-kinase)/Akt-dependent signaling pathway. Here, we show that gamma-irradiated murine myeloid 32D cells arrest in G(1) with active cyclin D-cyclin-dependent kinase 4 (Cdk4) but with inactive cyclin E-Cdk2 kinases. The arrest was associated with elevated levels of the Cdk inhibitors p21(Cip1) and p27(Kip1), yet neither was associated with Cdk2. Instead, irradiation-induced inhibition of cyclin E-Cdk2 correlated with absence of the activating threonine-160 phosphorylation on Cdk2. Cytokine treatment of irradiated cells induced Cdk2 phosphorylation and activation, and cells entered into S phase despite sustained high-level expression of p21 and p27. Notably, the PI 3-kinase inhibitor, LY294002, completely blocked cytokine-induced Cdk2 activation and cell growth in irradiated 32D cells but not in nonirradiated cells. Together, these findings demonstrate a novel mechanism underlying the DNA damage-induced G(1) arrest of hematopoietic cells, that is, inhibition of Cdk2 phosphorylation and activation. These observations link PI 3-kinase signaling pathways with the regulation of Cdk2 activity.     

Incomplete folding upon binding mediates Cdk4/cyclin D complex activation by tyrosine phosphorylation of inhibitor p27 protein

p27(Kip1) (p27), an intrinsically disordered protein, regulates the various Cdk/cyclin complexes that control cell cycle progression. The kinase inhibitory domain of p27 contains a cyclin-binding subdomain (D1), a Cdk-binding subdomain (D2), and a linker helix subdomain that connects D1 and D2. Here, we report that, despite extensive sequence conservation between Cdk4/cyclin D1 (hereafter Cdk4/cyclin D) and Cdk2/cyclin A, the thermodynamic details describing how the individual p27 subdomains contribute to equally high affinity binding to these two Cdk/cyclin complexes are strikingly different. Differences in enthalpy/entropy compensation revealed that the D2 subdomain of p27 folds incompletely when binding Cdk4/cyclin D versus Cdk2/cyclin A. Incomplete binding-induced folding exposes tyrosine 88 of p27 for phosphorylation by the nonreceptor tyrosine kinase Abl. Importantly, tyrosine phosphorylation (of p27) relieves Cdk inhibition by p27, enabling cell cycle entry. Furthermore, the interaction between a conserved hydrophobic patch on cyclin D and subdomain D1 is much weaker than that with cyclin A; consequently, a construct containing subdomains D1 and LH (p27-D1LH) does not inhibit substrate binding to Cdk4/cyclin D as it does to Cdk2/cyclin A. Our results provide a mechanism by which Cdk4 (within the p27/Cdk4/cyclin D complex) is poised to be activated by extrinsic mitogenic signals that impinge upon p27 at the earliest stage of cell division. More broadly, our results further illustrate the regulatory versatility of intrinsically disordered proteins.     

Cdk4 activation is dependent on the subunit rearrangement in the complexes

Although several factors have been implicated in the regulation of Cdk4 activity, little is known regarding the contributions of cyclin-dependent kinase inhibitors (CKIs) in Cdk4 activation in the mid G1 phase. Using a mouse macrophage cell line (Bac1.2F5), we found that most of Cdk4 bound to p15 when cells were in a quiescent state. Following CSF-1 stimulation, Cdk4 bound to cyclin D1 and then to p21, concomitant with the dissociation of p15 from the complexes. The activation of Cdk4 correlated well with p21 binding to the complexes, and the majority of active Cdk4 complexes contained p21. During regeneration of mouse liver after partial hepatectomy, Cdk4 activity coincided precisely with ternary complex formation of cyclin D1/Cdk4/p21. Using the baculovirus expression system, we succeeded in reconstituting a capacity for Cdk4 activation in insect cells, forming an active cyclin D1/Cdk4/p21 ternary complex. Taken together, it is suggested that p21 and cyclin D1 act cooperatively as activators of Cdk4 through the release of CKIs of the INK4 family.     

Cyclins D1 and D2 mediate myc-induced proliferation via sequestration of p27(Kip1) and p21(Cip1).

Cyclin E-Cdk2 kinase activation is an essential step in Myc-induced proliferation. It is presumed that this requires sequestration of G(1) cell cycle inhibitors p27(Kip1) and p21(Cip1) (Ckis) via a Myc-induced protein. We provide biochemical and genetic evidence to show that this sequestration is mediated via induction of cyclin D1 and/or cyclin D2 protein synthesis rates. Consistent with this conclusion, 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 forming kinase-defective, Cki-binding cyclin-cdk complexes. The sequestration function of D cyclins thus appears essential for Myc-induced cell cycle progression but dispensable for apoptosis.     

Mechanism of cell cycle entry mediated by the intrinsically disordered protein p27(Kip1)

p27(Kip1) (p27), a prototypical intrinsically disordered protein (IDP), regulates eukaryotic cell division through interactions with cyclin-dependent kinase (Cdk)/cyclin complexes. The activity, stability, and subcellular localization of p27 are regulated by phosphorylation. We illustrate how p27 integrates regulatory signals from several non-receptor tyrosine kinases (NRTKs) to activate Cdk4 and initiate cell cycle entry. Unmodified p27 potently inhibits Cdk/cyclin complexes, including Cdk4/cyclin D (IC(50), 1 nM). Some NRTKs (e.g., Abl) phosphorylate p27 on Tyr 88, which facilitates a second modification on Tyr 74 by another NRTK (e.g., Src). Importantly, this second modification causes partial reactivation of Cdk4 within ternary complexes containing doubly Tyr phosphorylated p27. Partial activation of Cdk4 initiates entry into the cell division cycle. Therefore, p27's disordered features enable NRTKs to sequentially promote a phosphorylation cascade that controls cell fate. Beyond cell cycle control, these results illustrate general concepts regarding why IDPs are well-suited for roles in signaling and regulation in biological systems.