Spy1 interacts with p27Kip1 to allow G1/S progression

Progression through the G1/S transition commits cells to synthesize DNA. Cyclin dependent kinase 2 (CDK2) is the major kinase that allows progression through G1/S phase and subsequent replication events. p27 is a CDK inhibitor (CKI) that binds to CDK2 to prevent premature activation of this kinase. Speedy (Spy1), a novel cell cycle regulatory protein, has been found to prematurely activate CDK2 when microinjected into Xenopus oocytes and when expressed in mammalian cells. To determine the mechanism underlying Spy1-induced proliferation in mammalian cell cycle regulation, we used human Spy1 as bait in a yeast two-hybrid screen to identify interacting proteins. One of the proteins isolated was p27; this novel interaction was confirmed both in vitro, using bacterially expressed and in vitro translated proteins, and in vivo, through the examination of endogenous and transfected proteins in mammalian cells. We demonstrate that Spy1 expression can overcome a p27-induced cell cycle arrest to allow for DNA synthesis and CDK2 histone H1 kinase activity. In addition, we utilized p27-null cells to demonstrate that the proliferative effect of Spy1 depends on the presence of endogenous p27. Our data suggest that Spy1 associates with p27 to promote cell cycle progression through the G1/S transition.     

Speedy: a novel cell cycle regulator of the G2/M transition

Stage VI Xenopus oocytes are suspended at the G2/M transition of meiosis I, and represent an excellent system for the identification and examination of cell cycle regulatory proteins. Essential cell cycle regulators such as MAPK, cyclins and mos have the ability to induce oocyte maturation, causing the resumption of the cell cycle from its arrested state. We have identified the product of a novel Xenopus gene, Speedy or Spy1, which is able to induce rapid maturation of Xenopus oocytes, resulting in the induction of germinal vesicle breakdown (GVBD) and activation of M-phasepromoting factor (MPF). Spy1 activates the MAPK pathway in oocytes, and its ability to induce maturation is dependent upon this pathway. Spy1-induced maturation occurs much more rapidly than maturation induced by other cell cycle regulators including progesterone, mos or Ras, and does not require any of these proteins or hormones, indicating that Spy1-induced maturation proceeds through a novel regulatory pathway. In addition, we have shown that Spy1 physically interacts with cdk2, and prematurely activates cdk2 kinase activity. Spy1 therefore represents a novel cell cycle regulatory protein, inducing maturation through the activation of MAPK and MPF, and also leading to the premature activation of cdk2.     

Spy1 expression prevents normal cellular responses to DNA damage: inhibition of apoptosis and checkpoint activation

Spy1 is the originally identified member of the Speedy/Ringo family of vertebrate cell cycle regulators, which can control cell proliferation and survival through the atypical activation of cyclin-dependent kinases. Here we report a role for Spy1 in apoptosis and checkpoint activation in response to UV irradiation. Using an inducible system allowing for regulated expression of Spy1, we show that Spy1 expression prevents activation of caspase-3 and suppresses apoptosis in response to UV irradiation. Spy1 expression also allows for UV irradiation-resistant DNA synthesis and permits cells to progress into mitosis, as demonstrated by phosphorylation on histone H3, indicating that Spy1 expression can inhibit the S-phase/replication and G2/M checkpoints. We demonstrate that Spy1 expression inhibits phosphorylation of Chk1, RPA, and histone H2A.X, which may directly contribute to the decrease in apoptosis and checkpoint bypass. Furthermore, mutation of the conserved Speedy/Ringo box, known to mediate interaction with CDK2, abrogates the ability of Spy1 to inhibit apoptosis and the phosphorylation of Chk1 and RPA. The data presented indicate that Spy1 expression allows cells to evade checkpoints and apoptosis and suggest that Spy1 regulation of CDK2 is important for the response to DNA damage.     

Identification and Comparative Analysis of Multiple Mammalian Speedy/Ringo Proteins

In addition to their activation via binding to cyclins, cyclin-dependent kinases (CDKs)can be activated via binding to a novel cell cycle regulator termed Speedy or Ringo, whichshows no apparent similarity to cyclins. The first Speedy/Ringo protein was found to beessential for Xenopus oocyte maturation and a human homolog (Spy1, herein calledSpeedy/Ringo A1) regulates S-phase entry and cell survival after DNA damage in culturedsomatic cells. We have identified a Speedy/Ringo-like gene in the most primitive branchingclade of chordates (Ciona intestinalis), as well as four mammalian homologs. Of the mammalianproteins, two, Speedy/Ringo A and C, bind to Cdc2 and Cdk2, whereas Speedy/Ringo B bindspreferentially to Cdc2. Despite their distinct CDK-binding preferences, both Speedy/Ringo Aand B can promote the maturation of Xenopus oocytes and all three Speedy/Ringo proteins canbind to and activate CDKs in vivo. These mammalian Speedy/Ringo proteins exhibit distincttissue expression patterns, though all three are enriched in testis, consistent with the initialobservation that Xenopus Speedy/Ringo functions during meiosis. Speedy/Ringo A is widelyexpressed in tissues and cell lines. Speedy/Ringo B expression appears to be testis-specific.Speedy/Ringo C is expressed in diverse tissues, particularly those that undergo polyploidization.All Speedy/Ringo proteins share a highly conserved ~140-aa domain we term the Speedy/Ringobox that is essential for CDK binding. Point mutations in this domain abolish CDK binding.Besides the central Speedy/Ringo box, Speedy/Ringo A contains a C-terminal portion, whichpromotes CDK activation, and an N-terminal portion, which is dispersible for both CDK bindingand activation but that influences protein expression. The existence of this growing family ofCDK activators suggests that Speedy/Ringo proteins may play as complex a role in cell cyclecontrol as the diverse family of cyclins.     

Speedy/Ringo C regulates S and G2 phase progression in human cells

Cyclin-dependent kinases (CDKs) control cell cycle transitions and progression. In addition to their activation via binding to cyclins, CDKs can be activated via binding to an unrelated class of cell cycle regulators termed Speedy/Ringo (S/R) proteins. Although mammals contain at least five distinct Speedy/Ringo homologues, the specific functions of members of this growing family of CDK activators remain largely unknown. We investigated the cell cycle roles of human Speedy/Ringo C in HEK293 cells. Down-regulation of Speedy/Ringo C by RNA interference delayed S and G2 progression whereas ectopic expression had the opposite effect, reducing S and G2/M populations. Double thymidine arrest and release experiments showed that overexpression of Speedy/Ringo C promoted late S phase progression. Using a novel three-color FACS protocol to determine the length of G2 phase, we found that the suppression of Speedy/Ringo C by RNAi prolonged G2 phase by ~30 min whereas ectopic expression of Speedy/Ringo C shortened G2 phase by ~25 min. In addition, overexpression of Speedy/Ringo C disrupted the G2 DNA damage checkpoint, increased cell death and caused a cell cycle delay at the G1-to-S transition. These observations indicate that CDK-Speedy/Ringo C complexes positively regulate cell cycle progression during the late S and G2 phases of the cell cycle.     

Regulation of the cell cycle by the cdk2 protein kinase in cultured human fibroblasts

In mammalian cells inhibition of the cdc2 function results in arrest in the G2-phase of the cell cycle. Several cdc2-related gene products have been identified recently and it has been hypothesized that they control earlier cell cycle events. Here we have studied the relationship between activation of one of these cdc2 homologs, the cdk2 protein kinase, and the progression through the cell cycle in cultured human fibroblasts. We found that cdk2 was activated and specifically localized to the nucleus during S phase and G2. Microinjection of affinity-purified anti-cdk2 antibodies but not of affinity-purified anti-cdc2 antibodies, during G1, inhibited entry into S phase. The specificity of these effects was demonstrated by the fact that a plasmid-driven cdk2 overexpression counteracted the inhibition. These results demonstrate that the cdk2 protein kinase is involved in the activation of DNA synthesis.     

Telomerase regulation during entry into the cell cycle in normal human T cells.

Telomerase activity is involved in telomere length maintenance. Leukocytes, unlike many human somatic tissues, have detectable telomerase activity. These cells provide a normal human cell type in which to study telomerase. We studied the regulation of telomerase activity and the telomerase RNA component as leukocytes were stimulated to enter the cell cycle. In primary human leukocytes stimulated with phytohemagglutinin, telomerase activity increased > 10-fold as naturally quiescent cells entered the cell cycle. Antibodies to the T cell receptor (TCR)/CD3 complex and the costimulatory CD28 receptor induced telomerase activity in a T cell-enriched population of cells. Rapamycin, an immunosuppressant that blocks TCR/CD3 signal transduction pathways and cdk2 activation, blocked telomerase induction. Hydroxyurea, an inhibitor of S phase, did not block cdk2 kinase activity or telomerase activation. In summary, telomerase is regulated in G1 phase as normal human T cells enter the cell cycle.     

Spy1 Enhances Phosphorylation and Degradation of the Cell Cycle Inhibitor p27

The cyclin dependent kinase inhibitor (CKI) p27Kip1 binds to cyclin E/CDK2 complexes and prevents premature S-phase entry. During late G₁ and throughout S phase, p27 phosphorylation at T187 leads to its subsequent degradation, which relieves CDK2 inhibition to promote cell cycle progression. However, critical events that trigger CDK2 complexes to phosphorylate p27 remain unclear. Utilizing recombinant proteins, we demonstrate that human Speedy (Spy1) activates CDK2 to phosphorylate p27 at T187 in vitro. Addition of Spy1 or Spy1/CDK2 to a preformed, inhibited cyclin E/CDK2/p27 complex also promoted this phosphorylation. Furthermore, Spy1 protected cyclin E/CDK2 from p27 inhibition toward histone H1, in vitro. Inducible Spy1 expression in U2OS cells reduced levels of endogenous p27 and exogenous p27WT, but not a p27T187A mutant. Additionally, Spy1 expression in synchronized HeLa cells enhanced T187 phosphorylation and degradation of endogenous p27 in late G₁ and throughout S phase. Our studies provide evidence that Spy1 expression enhances CDK2-dependent p27 degradation during late G₁ and throughout S phase.     

Spy1, a unique cell cycle regulator,alters viability in ALS motor neurons and cell lines in response to mutant SOD1-induced DNA damage

Increasing evidence indicates that DNA damage and p53 activation play major roles in the pathological process of motor neuron death in amyotrophic lateral sclerosis (ALS). Human SpeedyA1 (Spy1), a member of the Speedy/Ringo family, enhances cell proliferation and promotes tumorigenesis. Further studies have demonstrated that Spy1 promotes cell survival and inhibits DNA damage-induced apoptosis. We showed that the Spy1 expression levels were substantially decreased in ALS motor neurons compared with wild-type controls both in vivo and in vitro by qRT-PCR, western blotting, and Immunoassay tests. In addition, we established that over-expression of human SOD1 mutant G93A led to a decreased expression of Spy1. Furthermore, DNA damage response was activated in SOD1^G93A-transfected cells (mSOD1 cells). Moreover, decreased Spy1 expression reduced cell viability and further activated the DNA damage response in mSOD1 cells. In contrast, increased Spy1 expression improved cell viability and inhibited the DNA damage response in mSOD1 cells. These results suggest that Spy1 plays a protective role in ALS motor neurons. Importantly, these findings provide a novel direction for therapeutic options for patients with ALS as well as for trial designs, such as investigating the role of oncogenic proteins in ALS.     

Peripheral Nerve Lesion Induces an Up-regulation of Spy1 in Rat Spinal Cord

Spy1, as a member of the Speedy/RINGO family and a novel activator of cyclin-dependent kinases, was shown to promote cell cycle progression and cell survival in response to DNA damage. While its expression and roles in nervous system lesion and repair were still unknown. Here, we performed an acute sciatic nerve injury model in adult rats and studied the dynamic changes of Spy1 expression in lumbar spinal cord. Temporally, Spy1 expression was increased shortly after sciatic nerve crush and peaked at day 2. Spatially, Spy1 was widely expressed in the lumbar spinal cord including neurons and glial cells. While after injury, Spy1 expression was increased predominantly in astrocytes and microglia, which were largely proliferated. Moreover, there was a concomitant up-regulation of CDK2 activity and down-regulation of p27. Collectively, we hypothesized peripheral nerve injury induced an up-regulation of Spy1 in lumbar spinal cord, which was associated with glial proliferation. Ye Huang and Yonghua Liu contributed equally to this work.     

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.     

Estrogen-dependent cyclin E-cdk2 activation through p21 redistribution.

In order to elucidate the mechanisms by which estrogens and antiestrogens modulate the growth of breast cancer cells, we have characterized the changes induced by estradiol that occur during the G1 phase of the cell cycle of MCF-7 human mammary carcinoma cells. Addition of estradiol relieves the cell cycle block created by tamoxifen treatment, leading to marked activation of cyclin E-cdk2 complexes and phosphorylation of the retinoblastoma protein within 6 h. Cyclin D1 levels increase significantly while the levels of cyclin E, cdk2, and the p21 and p27 cdk inhibitors are relatively constant. However, the p21 cdk inhibitor shifts from its association with cyclin E-cdk2 to cyclin D1-cdk4, providing an explanation for the observed activation of the cyclin E-cdk2 complexes. These results support the notion that cyclin D1 has an important role in steroid-dependent cell proliferation and that estrogen, by regulating the activities of G1 cyclin-dependent kinases, can control the proliferation of breast cancer cells.     

Cdk7- and Cdc25A-independent dephosphorylation of Cdk2 during phorbol ester-mediated cell cycle arrest in U937 cells

The molecular mechanism underlying protein kinase C (PKC)-mediated cell cycle arrest is poorly understood. We undertook to characterize phorbol ester-activated PKC-mediated cell cycle arrest. Treatment with phorbol ester inhibited cell growth of human histiocytic lymphoma U937 cells with 83% of the cells arrested in G1 phase. Reduced activity of cdk2 correlated with cdk2 dephosphorylation and accumulation of cdk2 inhibitor p21Waf in phorbol ester-treated cells. Dephosphorylation of cdk2 was not associated with cdk7 and cdc25A activity in phorbol ester-treated cells. Protein phosphatase inhibitor assays suggest that the dephosphorylation of cdk2 results in the activation of a specific protein tyrosine phosphatase. Thus, dephosphorylation of cdk2 as well as accumulation of cdk2 inhibitor is likely to contribute to the G1 phase arrest in phorbol ester-treated in U937 cells.     

Dominant negative E2F inhibits progression of the cell cycle after the midblastula transition in Xenopus

The cleavage cycle, which is initiated by fertilization, consists of only S and M phases, and the gap phases (G1 and G2) appear after the midblastula transition (MBT) in the African clawed frog, Xenopus laevis. During early development in Xenopus, we examined the E2F activity, which controls transition from the G1 to S phase in the somatic cell cycle. Gel retardation and transactivation assays revealed that, although the E2F protein was constantly present throughout early development, the E2F transactivation activity was induced in a stage-specific manner, that is, low before MBT and rapidly increased after MBT. Introduction of the recombinant dominant negative E2F (dnE2F), but not the control, protein into the 2-cell stage embryos specifically suppressed E2F activation after MBT. Cells in dnE2F-injected embryos appeared normal before MBT, but ceased to proliferate and eventually died at the gastrula. These cells contained decreased cdk activity with enhanced inhibitory phosphorylation of Cdc2 at Tyr15. Thus, E2F activity is required for cell cycle progression and cell viability after MBT, but not essential for MBT transition and developmental progression during the cleavage stage.     

IFN-gamma inhibits human airway smooth muscle cell proliferation by modulating the E2F-1/Rb pathway

Elucidating the factors that inhibit the increase in airway smooth muscle (ASM) mass may be of therapeutic benefit in asthma. Here, we investigated whether interferon-gamma (IFN-gamma), a potent inducer of growth arrest in various cell types, regulates mitogen-induced ASM cell proliferation. IFN-gamma (1-100 U/ml) was found to markedly decrease both DNA synthesis and ASM cell number induced by the mitogens epidermal growth factor (EGF) and thrombin. Interestingly, IFN-gamma had no effect on mitogen-induced activation of three major mitogenic signaling pathways, phosphatidylinositol 3-kinase, p70(S6k), or mitogen-activated protein kinases. Mitogen-induced expression of cell cycle regulator cyclin D1 was increased by IFN-gamma, whereas no effect was observed on degradation of p27(Kip1). Expression array analysis of 23 cell cycle-related genes showed that IFN-gamma inhibited EGF-induced increases in E2F-1 expression, whereas induction of c-myc, cyclin D2, Egr-1, and mdm2 were unaffected. Induction of E2F-1 protein and Rb hyperphosphorylation after mitogen stimulation was also suppressed by IFN-gamma. In addition, IFN-gamma decreased activation of cdk2 and expression of cyclin E, upstream signaling molecules responsible for Rb hyperphosphorylation in the late G1 phase. IFN-gamma also increased levels of IFI 16 protein, whose mouse homolog p202 has been associated with growth inhibition. Together, our data indicate that IFN-gamma is an effective inhibitor of ASM cell proliferation by blocking transition from G1-to-S phase by acting at two different levels: modulation of cdk2/cyclin E activation and inhibition of E2F-1 gene expression.     

Speedy/RINGO Regulation of CDKs in Cell Cycle,Checkpoint Activation and Apoptosis

Speedy/RINGO family members bind and activate cyclin dependent kinases (CDKs), although these proteins have no homology to known cyclin proteins. Members of this family are required for and enhance meiotic maturation, in addition to having novel roles in regulating the mitotic mammalian cell cycle and the DNA damage response. Here we discuss how the specialized functions of these proteins differ from classical cyclin-mediated activation of CDKs. Through atypical activation of CDKs, bypass of conventional inhibitory mechanisms, and unique substrate selection, Speedy/RINGO proteins contribute to cell cycle, checkpoint, and apoptotic regulation. Furthermore, we address the recently established correlation between Spy1 and cancer in terms of the specialized functions of the Speedy/RINGO family.     

The kinetic origins of the restriction point in the mammalian cell cycle

A detailed model mechanism for the G1/S transition in the mammalian cell cycle is presented and analysed by computer simulation to investigate whether the kinetic origins of the restriction point (R-point) can be identified. The R-point occurs in mid-to-late G1 phase and marks the transition between mitogen-dependent to mitogen-independent progression of the cell cycle. For purposes of computer simulations, the R-point is defined as the first point in time after mitosis where cutting off mitogen stimulation does not prevent the cell reaching the threshold activity of cyclin-E/cdk2 required for entry into S phase. The key components of the network that generate a dynamic switching behaviour associated with the R-point include a positive feedback loop between cyclin-E/cdk2 and Cdc25A, along with the mutually negative interaction between the cdk inhibitor p27Kip1 and cyclin-E/cdk2. Simulations of the passage through the R-point were carried out and the factors affecting the position of the R-point in G1 are determined. The detailed model also shows various points in the network where the activation of cyclin-E/cdk2 can be initiated with or without the involvement of the retinoblastoma protein.     

The expression and characterization of cyclin-dependent kinase 6 during the activation of murine G0 T-cells.

Recent evidence strongly suggest that the D type cyclins with cdk4 and cdk6 form holoenzymes that regulate cell cycle events earlier in G1 than cyclin E/cdk2 complexes which functions near the G1/S transition. In human T lymphocytes cdk6 has been shown to be the initial retinoblastoma protein kinase detectable at mid G1. Following activation of splenic derived murine G0T-cells, cdk6, cyclin D2 and D3 specific mRNAs were detected early in G1 and reached maximal levels prior to or near G1/S. The phosphorylation of retinoblastoma protein from T-cells was detected very early in G1 and was associated mainly with cdk6/cyclin D2 complexes which accounted for a minor portion of the total cellular cdk6 contained in the cytoplasmic fraction of T-cells and mostly in the catalytically inactive form.