Presence Of Roscovitine Research Articles

Exploring the role of Cdk5 on striatal synaptic plasticity in a 3-NP-induced model of early stages of Huntington’s disease

Impaired mitochondrial function has been associated with the onset of neurodegenerative diseases. Specifically, certain mitochondrial toxins, such as 3-nitropropionic acid (3-NP), initiate cellular changes within the striatum that closely resemble the pathology observed in Huntington’s disease (HD). Among the pivotal signaling molecules contributing to neurodegeneration, cyclin-dependent kinase 5 (Cdk5) stands out. In particular, Cdk5 has been implicated not only in cellular pathology but also in the modulation of synaptic plasticity. Given its widespread presence in the striatum, this study seeks to elucidate the potential role of Cdk5 in the induction of corticostriatal synaptic plasticity in murine striatal cells subjected to subchronic doses of 3-NP in vivo, aiming to mimic the early stages of HD. Immunostaining analyses revealed an increase in Cdk5 in tissues from animals treated with 3-NP, without a significant change in protein levels. Regarding striatal plasticity, long-term depression (LTD) was induced in both control and 3-NP cells when recorded in voltage clamp mode. The Cdk5 inhibitor roscovitine-reduced LTD in most cells. A minority subset of cells exhibited long-term potentiation (LTP) generation in the presence of roscovitine. The inhibitor of D1 receptors SCH23390 prevented LTP in three of nine cells, implying that MSN cells lacking D1/PKA activation were capable of LTP induction when Cdk5 was also blocked. Nevertheless, the co-administration of H89, a PKA inhibitor, along with roscovitine, prevented the generation of any type of plasticity in all recorded cells. These findings show the impact of 3-NP treatment on striatal plasticity and suggest that Cdk5 during early neurodegeneration may attenuate signaling pathways that lead neurons to increase their activity.

Cooperativity Between Orthosteric Inhibitors and Allosteric Inhibitor 8-Anilino-1-Naphthalene Sulfonic Acid (ANS) in Cyclin-Dependent Kinase 2

While kinases have been attractive targets to combat many diseases, including cancer, selective kinase inhibition has been challenging, because of the high degree of structural homology in the active site, where many kinase inhibitors bind. We have previously discovered that 8-anilino-1-naphthalene sulfonic acid (ANS) binds an allosteric pocket in cyclin-dependent kinase 2 (Cdk2). Here, we detail the positive cooperativity between ANS and orthosteric Cdk2 inhibitors dinaciclib and roscovitine, which increase the affinity of ANS toward Cdk2 5-fold to 10-fold, and the relatively noncooperative effects of ATP. We observe these effects using a fluorescent binding assay and heteronuclear single quantum correlation nuclear magnetic resonance (HSQC NMR), where we noticed a shift from fast exchange to slow exchange upon ANS titration in the presence of roscovitine but not with an ATP mimic. The discovery of cooperative relationships between orthosteric and allosteric kinase inhibitors could further the development of selective kinase inhibitors in general.

GLUTATHIONE AND GLUTAREDOXIN IN ROSCOVITINE-MEDIATED INHIBITION OF BREAST CANCER CELL PROLIFERATION

Background: Breast tumors are number one cause of cancer morbidity and mortality among women around the world, and Russia is not an exception. Many proteins that control proliferation of immortalized cells are redox-regulated, which is essential for modulating cellular proliferative activity, especially during tumor growth. Studying the role of glutaredoxin and glutathione in cell cycle phase distribution will allow not only to identify the molecular targets regulating cell proliferation, but also to develop methods of diagnosis and targeted therapy of socially sensitive diseases, including breast cancer, in the future.Aims: To evaluate the role of glutathione and glutaredoxin in the molecular mechanisms regulating MCF-7 breast cancer cell proliferation under the effects of roscovitine, a cyclin-dependent protein kinase inhibitor.Materials and methods: The MCF-7 cell line (human breast adenocarcinoma) was used in the study. The cell culture was incubated in the presence and absence of roscovitine in the final concentration of 20 µmol for 18 h. The production of reactive oxygen species, the distribution of cells between cell cycle phases and the amount of Annexin V positive cells were determined using flow cytometry. The concentrations of total, reduced and oxidized glutathione, protein SH groups and protein-bound glutathione were measured by spectrophotometry. The levels of glutaredoxin, cyclin E and cyclin-dependent protein kinases were estimated by Western blotting with monoclonal antibodies.Results: The effects of roscovitine in the MCF-7 cells resulted in cell cycle arrest in G2/М phases with the decreased levels of cyclin E and cyclin-dependent protein kinase 2. It was accompanied by activation of programmed cell death. In tumor cells incubated in the presence of roscovitine, oxidative stress was triggered, which was accompanied by the elevated generation of reactive oxygen species, the decrease in the concentration of reduced glutathione, and the rise in the level of glutaredoxin. It contributed to the increase in protein glutathionylation against the backdrop of the decreased SH group concentration.Conclusions: Breast cancer cell proliferation under the effects of roscovitine is reduced following not only the decrease in the cyclin level and cyclin-dependent protein kinase activity, but also the shift in the intracellular oxidant/antioxidant ratio. Roscovitine-induced oxidative stress in the MCF-7 cells contributed to protein glutathionylation with the changes in the protein structure and functions. It results in impaired cell cycle progression, indicating a possibility to regulate cellular proliferation through modulating functional properties of redox-dependent proteins using the glutathione/glutaredoxin system.

Cyclic AMP and Polyamines Overcome Inhibition by Myelin-Associated Glycoprotein through eIF5A-Mediated Increases in p35 Expression and Activation of Cdk5.

Inhibitory molecules associated with CNS myelin, such as myelin-associated glycoprotein (MAG), represent major obstacles to axonal regeneration following CNS injury. Our laboratory has shown that elevating levels of intracellular cAMP, via application of the nonhydrolyzable analog dibutyryl cAMP (dbcAMP), can block the inhibitory effects of MAG and myelin. We have also shown that elevation of cAMP results in upregulation of arginase I and increased polyamine synthesis. Treatment with putrescine or spermidine blocks myelin-mediated inhibition of neurite outgrowth, but the mechanism underlying this effect has not yet been elucidated. Here we show that cyclin-dependent kinase 5 (Cdk5) is required for dbcAMP and putrescine to overcome MAG-mediated inhibition. The ability of dbcAMP and putrescine to overcome inhibition by MAG is abolished in the presence of roscovitine, a Cdk inhibitor that has greater selectivity for Cdk5, and expression of dominant negative Cdk5 abolishes the ability of dbcAMP or putrescine to enhance neurite outgrowth in the presence of MAG. Importantly, dbcAMP and putrescine increase expression of p35, the neuron-specific activator of Cdk5, and rat DRG neurons transduced with HSV overexpressing p35 can overcome inhibition by MAG. The upregulation of p35 by putrescine is also reflected in increased localization of p35 to neurites and growth cones. Last, we show that putrescine upregulates p35 expression by serving as a substrate for hypusine modification of eIF5A, and that this hypusination is necessary for putrescine's ability to overcome inhibition by MAG. Our findings reveal a previously unknown mechanism by which polyamines may encourage regeneration after CNS injury.

Gene expression in sheep cumulus-oocyte complexes meiotically inhibited with roscovitine

The majority of mammalian oocytes destined for in vitro maturation (IVM) have not undergone all molecular and structural changes necessary for competence acquisition to support the fertilization and early embryogenesis. In this context, different methods able to provide a transient arrest of meiosis resumption have been tested in order to improve the in vitro developmental potential of oocytes. Based on that, our study aimed to evaluate the effect of temporary meiosis inhibition using roscovitine on gene expression in sheep oocytes and cumulus cells. For this, cumulus-oocyte complexes (COCs) were cultured for 6h in modified TCM199 medium with (Rosco) and without (Control) 75μM roscovitine. Subsequently, they were in vitro matured for a further 18h in inhibitor-free TCM199 medium supplemented with gonadotropins. At 0, 6 and 24h of culture, nuclear status of oocytes and expression of selected genes were evaluated by Hoescht staining and qRT-PCR, respectively. The analysis of oocyte chromatin organization revealed that roscovitine efficiently inhibited the meiosis of sheep oocytes for 6h and its action was completely reversed after 18h of in vitro maturation in inhibitor-free medium. Besides, no detrimental effect on cumulus expansion was observed. The expression profile of most investigated genes in cumulus cells (PTX3, GREM1, GLUT1, PTGS2, ALK5, ALK6) and oocytes (ZAR1, NLRP5, SOD1, BMP15, GDF9) was similar between Control and Rosco treatments and the ratio BCL2/BAX was maintained in both cell types even in the presence of roscovitine. These results indicate that reversible meiotic arrest promoted by roscovitine, at the concentration and exposure time studied, neither impaired nor improved the expression of investigated genes in sheep oocytes and cumulus cells. Moreover, the efficiency of temporary meiotic arrest and the absence of deleterious effect on COCs suggest that roscovitine provides a useful method for transportation or manipulation of sheep oocytes at onset of maturation. However, further investigations are necessary to evaluate the benefits of roscovitine treatment on in vitro development of sheep embryos and its effects on cellular ultrastructure.

Meiotic arrest of sheep oocytes using roscovitine under different medium compositions

Attempts to maintain the meiotic arrest under suitable culture conditions have been performed in order to improve the quality of oocytes in vitro matured. So, our study aimed to evaluate the potential of roscovitine, an inhibitor of cyclin-dependent kinases, to reversibly arrest the meiosis of sheep oocytes in medium supplemented with gonadotropins and/or serum. Cumulus-oocyte complexes (COCs) were treated, for 20h, with 75μM roscovitine (Rosco) in basic maturation medium (Basic) containing 10% fetal bovine serum (FBS) and 0.1IU/mL FSH plus 0.1IU/mL LH (Enriched). After this, COCs were in vitro matured (IVM) for a further 18h in the presence of gonadotropins and serum. At 20 and 38h of culture, cumulus expansion and nuclear maturation were assessed under stereomicroscope and by Hoechst 33342 staining, respectively. Our findings demonstrate that total cumulus expansion occurred only in medium containing gonadotropins and serum. In the presence of roscovitine, however, the cumulus expansion was inhibited in a not fully reversible manner, independently of medium composition. The GV rate in the Rosco FBS group (64.6%) was significantly higher than that of Rosco Enriched (51.8%); however, both did not differ from that observed in the Rosco Basic (56.2%). In all roscovitine treatments, the meiotic arrest was reversible after the additional culture for 18h in inhibitor-free medium. Besides, significant proportion of oocytes from Basic, FBS and Enriched groups reached the MII stage after 20 and 38h of culture. Therefore, we can infer that (i) gonadotropins and serum did not affect the reversible meiotic arrest promoted by roscovitine in sheep oocytes; (ii) the maximum efficiency of meiosis inhibition occurred in serum-supplemented medium; (iii) roscovitine action on cumulus cells was not fully reversible independently of medium composition.

The Cdk5 Inhibitor Roscovitine Increases LTP Induction in Corticostriatal Synapses

In corticostriatal synapses, LTD (long-term depression) and LTP (long-term potentiation) are modulated by the activation of DA (dopamine) receptors, with LTD being the most common type of long-term plasticity induced using the standard stimulation protocols. In particular, activation of the D1 signaling pathway increases cAMP/PKA (protein kinase A) phosphorylation activity and promotes an increase in the amplitude of glutamatergic corticostriatal synapses. However, if the Cdk5 (cyclin-dependent kinase 5) phosphorylates the DARPP-32 (dopamine and cAMP-regulated phosphoprotein of 32 kDa) at Thr75, DARPP-32 becomes a strong inhibitor of PKA activity. Roscovitine is a potent Cdk5 inhibitor; it has been previously shown that acute application of Roscovitine increases striatal transmission via Cdk5/DARPP-32. Since DARPP-32 controls long-term plasticity in the striatum, we wondered whether switching off CdK5 activity with Roscovitine contributes to the induction of LTP in corticostriatal synapses. For this purpose, excitatory population spikes and whole cell EPSC (excitatory postsynaptic currents) were recorded in striatal slices from C57/BL6 mice. Experiments were carried out in the presence of Roscovitine (20 μM) in the recording bath. Roscovitine increased the amplitude of excitatory population spikes and the percentage of population spikes that exhibited LTP after HFS (high-frequency stimulation; 100Hz). Results obtained showed that the mechanisms responsible for LTP induction after Cdk5 inhibition involved the PKA pathway, DA and NMDA (N-methyl-D-aspartate) receptor activation, L-type calcium channels activation and the presynaptic modulation of neurotransmitter release.

Cdk5 regulates Rap1 activity

Rap1 signaling is important for migration, differentiation, axonal growth, and during neuronal polarity. Rap1 can be activated by external stimuli, which in turn regulates specific guanine nucleotide exchange factors such as C3G, among others. Cdk5 functions are also important to neuronal migration and differentiation. Since we found that pharmacological inhibition of Cdk5 by using roscovitine reduced Rap1 protein levels in COS-7 cells and also C3G contains three putative phosphorylation sites for Cdk5, we examined whether the Cdk5-dependent phosphorylation of C3G could affect Rap1 expression and activity. We co-transfected C3G and tet-OFF system for p35 over-expression, an activator of Cdk5 activity into COS-7 cells, and then we evaluated phosphorylation in serine residues in C3G by immunoprecipitation and Western blot. We found that p35 over-expression increased C3G-serine-phosphorylation while inhibition of p35 expression by tetracycline or inhibition of Cdk5 activity with roscovitine decreased it. Interestingly, we found that MG-132, a proteasome inhibitor, rescue Rap1 protein levels in the presence of roscovitine. Besides, C3G-serine-phosphorylation and Rap1 protein levels were reduced in brain from Cdk5−/− as compared with the Cdk5+/+ brain. Finally, we found that p35 over-expression increased Rap1 activity while inhibition of p35 expression by tetracycline or roscovitine decreased Rap1 activity. These results suggest that Cdk5-mediated serine-phosphorylation of C3G may control Rap1 stability and activity, and this may potentially impact various neuronal functions such as migration, differentiation, and polarity.

Physiological responses and endogenous cytokinin profiles of tissue-cultured ‘Williams’ bananas in relation to roscovitine and an inhibitor of cytokinin oxidase/dehydrogenase (INCYDE) treatments

The effect of supplementing either meta-topolin (mT) or N(6)-benzyladenine (BA) requiring cultures with roscovitine (6-benzylamino-2-[1(R)-(hydroxymethyl)propyl]amino-9-isopropylpurine), a cyclin-dependent kinase (CDK) and N-glucosylation inhibitor, and INCYDE (2-chloro-6-(3-methoxyphenyl)aminopurine), an inhibitor of cytokinin (CK) degradation, on the endogenous CK profiles and physiology of banana in vitro was investigated. Growth parameters including multiplication rate and biomass were recorded after 42 days. Endogenous CK levels were quantified using UPLC-MS/MS while the photosynthetic pigment and phenolic contents were evaluated spectrophotometrically. The highest regeneration rate (93 %) was observed in BA + roscovitine while mT + INCYDE plantlets produced most shoots. Treatment with BA + roscovitine had the highest shoot length and biomass. Although not significant, there was a higher proanthocyanidin level in BA + roscovitine treatments compared to the control (BA). The levels of total phenolics and flavonoids were significantly higher in mT + roscovitine treatment than in the mT-treated regenerants. The presence of roscovitine and/or INCYDE had no significant effect on the photosynthetic pigments of the banana plantlets. Forty-seven aromatic and isoprenoid CKs categorized into nine CK-types were detected at varying concentrations. The presence of mT + roscovitine and/or INCYDE increased the levels of O-glucosides while 9-glucosides were higher in the presence of BA. Generally, the underground parts had higher CK levels than the aerial parts; however, the presence of INCYDE increased the level of CK quantified in the aerial parts. From a practical perspective, the use of roscovitine and INCYDE in micropropagation could be crucial in the alleviation of commonly observed in vitro-induced physiological abnormalities.

Roscovitine-induced apoptosis of H1299 cells depends on functional status of p53

Roscovitine, an inhibitor of cyclin-dependent kinases, is promising anticancer agent. Its antiproliferative and cytotoxic effects can be mediated by the p53 signaling pathway. To define the role of p53 in roscovitine-induced cell response, we prepared H1299/p53 cell lines inducibly expressing specific variants of p53 (p53wt and hotspot R175H, temperature-dependent P98A, A159V, S215G, Y220C, Y234C mutants). In the presence of roscovitine, each cell line variant behaved in specific way reflecting activity of the p53 protein. Roscovitine decreased production of the cell cycle inhibitor p21 and induced apoptosis. This effect was the most efficient in cells expressing p53wt protein with full activity. The cell expressing partially and conditionally active p53 mutants responded to roscovitine less efficiently. The cells expressing p53 mutants A159V and Y234C were very sensitive to roscovitine but their response was clearly temperature-dependent. The cells expressing P98A, S215G and Y220C p53 mutants exhibited only weak sensitivity to roscovitine and underwent apoptosis in low frequency. In principle, each td p53 mutant responded to roscovitine in distinct way. We showed clearly that the impact of roscovitine on H1299 cells depends on functional status of p53 they produce. This suggests that patients with tumors exhibiting specific p53 variants can benefit from the roscovitine therapy.

The Auto-ubiquitylation of E3 Ubiquitin-protein Ligase Chfr at G2 Phase Is Required for Accumulation of Polo-like Kinase 1 and Mitotic Entry in Mammalian Cells

The E3 ubiquitin-protein ligase Chfr is a mitotic stress checkpoint protein that delays mitotic entry in response to microtubule damage; however, the molecular mechanism by which Chfr accomplishes this remains elusive. Here, we show that Chfr levels are elevated in response to microtubule-damaging stress. Moreover, G(2)/M transition is associated with cell cycle-dependent turnover of Chfr accompanied by high autoubiquitylation activity, suggesting that regulation of Chfr levels and auto-ubiquitylation activity are functionally significant. To test this, we generated Chfr mutants Chfr-K2A and Chfr-K5A in which putative lysine target sites of auto-ubiquitylation were replaced with alanine. Chfr-K2A did not undergo cell cycle-dependent degradation, and its levels remained high during G(2)/M phase. The elevated levels of Chfr-K2A caused a significant reduction in phosphohistone H3 levels and cyclinB1/Cdk1 kinase activities, leading to mitotic entry delay. Notably, polo-like kinase 1 levels at G(2) phase, but not at S phase, were ∼2-3-fold lower in cells expressing Chfr-K2A than in wild-type Chfr-expressing cells. Consistent with this, ubiquitylation of Plk1 at G(2) phase was accelerated in Chfr-K2A-expressing cells. In contrast, Aurora A levels remained constant, indicating that Plk1 is a major target of Chfr in controlling the timing of mitotic entry. Indeed, overexpression of Plk1 in Chfr-K2A-expressing cells restored cyclin B1/Cdk1 kinase activity and promoted mitotic entry. Collectively, these data indicate that Chfr auto-ubiquitylation is required to allow Plk1 to accumulate to levels necessary for activation of cyclin B1/Cdk1 kinase and mitotic entry. Our results provide the first evidence that Chfr auto-ubiquitylation and degradation are important for the G(2)/M transition.

Inhibition of cyclin-dependent kinase 5 but not of glycogen synthase kinase 3-β prevents neurite retraction and tau hyperphosphorylation caused by secretable products of human T-cell leukemia virus type I-infected lymphocytes

Human T-cell leukemia virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a neurodegenerative disease characterized by selective loss of axons and myelin in the corticospinal tracts. This central axonopathy may originate from the impairment of anterograde axoplasmic transport. Previous work showed tau hyperphosphorylation at T(181) in cerebrospinal fluid of HAM/TSP patients. Similar hyperphosphorylation occurs in SH-SY5Y cells incubated with supernatant from MT-2 cells (HTLV-I-infected lymphocytes secreting viral proteins, including Tax) that produce neurite shortening. Tau phosphorylation at T(181) is attributable to glycogen synthase kinase 3-β (GSK3-β) and cyclin-dependent kinase 5 (CDK5) activation. Here we investigate whether neurite retraction in the SH-SY5Y model associates with concurrent changes in other tau hyperphosphorylable residues. Threonine 181 turned out to be the only tau hyperphosphorylated residue. We also evaluate the role of GSK3-β and CDK5 in this process by using specific kinase inhibitors (LiCl, TDZD-8, and roscovitine). Changes in both GSK3-β active and inactive forms were followed by measuring the regulatory phosphorylable sites (S(9) and Y(216) , inactivating and activating phosphorylation, respectively) together with changes in β-catenin protein levels. Our results showed that LiCl and TDZD-8 were unable to prevent MT-2 supernatant-mediated neurite retraction and also that neither Y(216) nor S(9) phosphorylations were changed in GSK3-β. Thus, GSK3-β seems not to play a role in T(181) hyperphosphorylation. On the other hand, the CDK5 involvement in tau phosphorylation was confirmed by both the increase in its enzymatic activity and the absence of MT-2 neurite retraction in the presence of roscovitine or CDK5 siRNA transfection.

Antiapoptotic effects of roscovitine on camptothecin-induced DNA damage in neuroblastoma cells

In the present study dopaminergic neuroblastoma B65 cells were exposed to Camptothecin (CPT) (0.5-10 μM), either alone or in the presence of roscovitine (ROSC). The results show that CPT induces apoptosis through the activation of ataxia telangiectasia mutated (ATM)-induced cell-cycle alteration in neuroblastoma B65 cells. The apoptotic process is mediated through the activation of cystein proteases, namely calpain/caspases. However, whereas a pan-caspase inhibitor, zVADfmk, inhibited CPT-mediated apoptosis, a calpain inhibitor, calpeptin, did not prevent cell death. Interestingly, CPT also induces CDK5 activation and ROSC (25 μM) blocked CDK5, ATM activation and apoptosis (as measured by caspase-3 activation). By contrast, selective inhibition of ATM, by KU55933, and non-selective inhibition, by caffeine, did not prevent CPT-mediated apoptosis. Thus, we conclude that CDK5 is activated in response to DNA damage and that CDK5 inhibition prevents ATM and p53ser15 activation. However, pharmacological inhibition of ATM using KU55933 and caffeine suggests that ATM inhibition by ROSC is not the only mechanism that might explain the anti-apoptotic effects of this drug in this apoptosis model. Our findings have a potential clinical implication, suggesting that combinatory drugs in the treatment of cancer activation should be administered with caution.

Raman micro-spectroscopic analysis of cultured HCT116 colon cancer cells in the presence of roscovitine

Raman micro-spectroscopic analysis of cultured HCT116 colon cancer cells in the presence of roscovitine, [seliciclib, 2-(1-ethyl-2-hydroxy-ethylamino)-6-benzylamino-9-isopropylpurine], a promising drug candidate in cancer therapy, has been performed for the first time. The aim of this study was to investigate modulations in colon cancer cells induced by roscovitine. Raman spectra of the cultured HCT116 colon cancer cells treated with roscovitine at different concentrations (0, 5, 10, 25 and 50 μM) were recorded in the range 400–1850 cm −1. It was shown that the second derivative profile of the experimental spectrum gives valuable information about the wavenumbers and band widths of the vibrational modes of cell components, and it eliminates the appearance of false peaks arising from incorrect baseline corrections. In samples containing roscovitine, significant spectral changes were observed in the intensities of characteristic protein and DNA bands, which indicate roscovitine-induced apoptosis. Roscovitine-induced apoptosis was also assessed by flow cytometry analysis, and analysis of propidium iodide staining. We observed some modifications in amide I and III bands, which arise from alterations in the secondary structure of cell proteins caused by the presence of roscovitine.

Time Course ofIn VitroMaturation of Compact Cumulus Horse Oocytes after Roscovitine‐Induced Meiotic Inhibition: Effects on the Coordination Between Nuclear and Cytoplasmic Maturation

This study was carried out to evaluate the usefulness of a pre-maturation step in improving the coordination between cytoplasmic and nuclear maturation of horse compact cumulus oocytes by the addition of roscovitine (ROSC). Oocytes were collected by scraping and pre-cultured for 18 h in a maturation medium TCM199 supplemented with pyruvate, LH, FSH, insulin growth factor (IGF), epidermal growth factor (EGF), insulin, transferrin and selenium (IVM-ROSC) or in a simple medium (M199-ROSC). After pre-maturation, oocytes from both the groups were in part denuded and fixed-stained and in part in vitro matured to assess the kinetic of in vitro maturation (IVM). The nuclear progression and the cytoskeletal organization of microfilaments and cortical granules (CG) of treated and untreated oocytes were assessed by fluorescent probes. Oocytes immediately fixed after recovery and oocytes pre-cultured in M199-ROSC for 18 h did not show metaphase II (MII) plates, whereas in IVM-ROSC group, 6/69 oocytes (8.7%) showed MII plates. After inhibition, during maturation kinetics at 11, 18 and 29 h, maturation rate of M199-ROSC group progressively increased and at 29 h of IVM, reached the maturation rate of control group (13/66, 19.7% vs 31/125, 24.8%). No statistically significant differences in cytoplasmic maturation were found. The number of MII plates after 29 h of IVM, was significantly higher (p < 0.05) in IVM-ROSC group (34/90) compared with M199-ROSC (13/66) and control groups (31/125) as well as the number of oocytes with microfilaments and CG distributed in cortical region (25/34 vs 3/13 and 7/31 respectively). Our results showed that pre-culturing in the presence of Roscovitine in a fully supplemented maturation medium containing gonadotropins and growth factors partially suppressed the meiotic maturation, but established a more suitable environment for improving cytoplasmic maturation of horse compact cumulus oocytes as defined by microfilaments and CG configuration.

A 24-Residue Peptide (p5), Derived from p35, the Cdk5 Neuronal Activator, Specifically Inhibits Cdk5-p25 Hyperactivity and Tau Hyperphosphorylation

The activity of Cdk5-p35 is tightly regulated in the developing and mature nervous system. Stress-induced cleavage of the activator p35 to p25 and a p10 N-terminal domain induces deregulated Cdk5 hyperactivity and perikaryal aggregations of hyperphosphorylated Tau and neurofilaments, pathogenic hallmarks in neurodegenerative diseases, such as Alzheimer disease and amyotrophic lateral sclerosis, respectively. Previously, we identified a 125-residue truncated fragment of p35 called CIP that effectively and specifically inhibited Cdk5-p25 activity and Tau hyperphosphorylation induced by Aβ peptides in vitro, in HEK293 cells, and in neuronal cells. Although these results offer a possible therapeutic approach to those neurodegenerative diseases assumed to derive from Cdk5-p25 hyperactivity and/or Aβ induced pathology, CIP is too large for successful therapeutic regimens. To identify a smaller, more effective peptide, in this study we prepared a 24-residue peptide, p5, spanning CIP residues Lys(245)-Ala(277). p5 more effectively inhibited Cdk5-p25 activity than did CIP in vitro. In neuron cells, p5 inhibited deregulated Cdk5-p25 activity but had no effect on the activity of endogenous Cdk5-p35 or on any related endogenous cyclin-dependent kinases in HEK293 cells. Specificity of p5 inhibition in cortical neurons may depend on the p10 domain in p35, which is absent in p25. Furthermore, we have demonstrated that p5 reduced Aβ(1-42)-induced Tau hyperphosphorylation and apoptosis in cortical neurons. These results suggest that p5 peptide may be a unique and useful candidate for therapeutic studies of certain neurodegenerative diseases.

Cdc2-mediated Phosphorylation of CLIP-170 Is Essential for Its Inhibition of Centrosome Reduplication

CLIP-170, the founding member of microtubule "plus ends tracking" proteins, is involved in many critical microtubule-related functions, including recruitment of dynactin to the microtubule plus ends and formation of kinetochore-microtubule attachments during metaphase. Although it has been reported that CLIP-170 is a phosphoprotein, neither have individual phosphorylation sites been identified nor have the associated kinases been extensively studied. Herein, we identify Cdc2 as a kinase that phosphorylates CLIP-170. We show that Cdc2 interacts with CLIP-170 mediating its phosphorylation on Thr(287) in vivo. Significantly, expression of CLIP-170 with a threonine 287 to alanine substitution (T287A) results in its mislocalization, accumulation of Plk1 and cyclin B, and block of the G2/M transition. Finally, we found that depletion of CLIP-170 leads to centrosome reduplication and that Cdc2 phosphorylation of CLIP-170 is required for the process. These results demonstrate that Cdc2-mediated phosphorylation of CLIP-170 is essential for the normal function of this protein during cell cycle progression.

Inhibition of the Cyclin-Dependent Kinases at the Beginning of Human Cytomegalovirus Infection Specifically Alters the Levels and Localization of the RNA Polymerase II Carboxyl-Terminal Domain Kinases cdk9 and cdk7 at the Viral Transcriptosome

We previously reported that defined components of the host transcription machinery are recruited to human cytomegalovirus immediate-early (IE) transcription sites, including cdk9 and cdk7 (S. Tamrakar, A. J. Kapasi, and D. H. Spector, J. Virol. 79:15477-15493, 2005). In this report, we further document the complexity of this site, referred to as the transcriptosome, through identification of additional resident proteins, including viral UL69 and cellular cyclin T1, Brd4, histone deacetylase 1 (HDAC1), and HDAC2. To examine the role of cyclin-dependent kinases (cdks) in the establishment of this site, we used roscovitine, a specific inhibitor of cdk1, cdk2, cdk7, and cdk9, that alters processing of viral IE transcripts and inhibits expression of viral early genes. In the presence of roscovitine, IE2, cyclin T1, Brd4, HDAC1, and HDAC2 accumulate at the transcriptosome. However, accumulation of cdk9 and cdk7 was specifically inhibited. Roscovitine treatment also resulted in decreased levels of cdk9 and cdk7 RNA. There was a corresponding reduction in cdk9 protein but only a modest decrease in cdk7 protein. However, overexpression of cdk9 does not compensate for the effects of roscovitine on cdk9 localization or viral gene expression. Delaying the addition of roscovitine until 8 h postinfection prevented all of the observed effects of the cdk inhibitor. These data suggest that IE2 and multiple cellular factors needed for viral RNA synthesis accumulate within the first 8 h at the viral transcriptosome and that functional cdk activity is required for the specific recruitment of cdk7 and cdk9 during this time interval.

Calcium-mediated Transient Phosphorylation of Tau and Amyloid Precursor Protein Followed by Intraneuronal Amyloid-β Accumulation

Intraneuronal accumulation of hyperphosphorylated protein tau in paired helical filaments together with amyloid-beta peptide (Abeta) deposits confirm the clinical diagnosis of Alzheimer disease. A common cellular mechanism leading to the production of these potent toxins remains elusive. Here we show that, in cultured neurons, membrane depolarization induced a calcium-mediated transient phosphorylation of both microtubule-associated protein tau and amyloid precursor protein (APP), followed by a dephosphorylation of these proteins. Phosphorylation was mediated by glycogen synthase kinase 3 and cyclin-dependent kinase 5 protein kinases, while calcineurin was responsible for dephosphorylation. Following the transient phosphorylation of APP, intraneuronal Abeta accumulated and induced neurotoxicity. Phosphorylation of APP on Thr-668 was indispensable for intraneuronal accumulation of Abeta. Our data demonstrate that an increase in cytosolic calcium concentration induces modifications of neuronal metabolism of APP and tau, similar to those found in Alzheimer disease.

Different Mechanisms of CDK5 and CDK2 Activation as Revealed by CDK5/p25 and CDK2/Cyclin A Dynamics

A detailed analysis is presented of the dynamics of human CDK5 in complexes with the protein activator p25 and the purine-like inhibitor roscovitine. These and other findings related to the activation of CDK5 are critically reviewed from a molecular perspective. In addition, the results obtained on the behavior of CDK5 are compared with data on CDK2 to assess the differences and similarities between the two kinases in terms of (i) roscovitine binding, (ii) regulatory subunit association, (iii) conformational changes in the T-loop following CDK/regulatory subunit complex formation, and (iv) specificity in CDK/regulatory subunit recognition. An energy decomposition analysis, used for these purposes, revealed why the binding of p25 alone is sufficient to stabilize the extended active T-loop conformation of CDK5, whereas the equivalent conformational change in CDK2 requires both the binding of cyclin A and phosphorylation of the Thr(160) residue. The interaction energy of the CDK5 T-loop with p25 is about 26 kcal.mol(-1) greater than that of the CDK2 T-loop with cyclin A. The binding pattern between CDK5 and p25 was compared with that of CDK2/cyclin A to find specific regions involved in CDK/regulatory subunit recognition. The analyses performed revealed that the alphaNT-helix of cyclin A interacts with the alpha6-alpha7 loop and the alpha7 helix of CDK2, but these regions do not interact in the CDK5/p25 complex. Further differences between the CDK5/p25 and CDK2/cyclin A systems studied are discussed with respect to their specific functionality.