CDK5 activator p35 downregulates E-cadherin precursor independently of CDK5

Dysfunction of E-cadherins often results in metastasis of cancerous cells. Here we show that p35, a critical regulator of cyclin-dependent kinase 5 (CDK5), specifically depletes the precursor form of E-cadherin, but not the mature form, by using a precursor-specific antibody. Most intriguingly, this downregulation of precursor E-cadherin by p35 is unequivocally independent of CDK5. Moreover, we found that p35 forms complexes with E-cadherin proteins. We also found that p35 co-expression can target E-cadherin to lysosomes and that p35-triggered disappearance of E-cadherin precursor can be blocked specifically by lysosomal protease inhibitors, indicating that p35 induces endocytosis and subsequent degradation of precursor E-cadherin.     

黄色羊驼皮肤细胞周期素依赖蛋白激酶5(CDK5)完整CDS区结构域分析

用RT-PCR法获得了黄色羊驼皮肤的CDK5完整CDS区,其长度为891bp,与其他哺乳动物相比,高度保守。通过对其结构域分析,发现羊驼CDK5编码区在第10-203位氨基酸间含有多个激活位点如ATP结合位点和P25底物结合位点(?)第1-9位氨基酸和第204-292位氨基酸为两个非活性部位,其中在第204-292位氨基酸中,亮氨酸排列与亮氨酸拉链结构相似,而且位于羊驼CDK5的C-端,可能是与基因转录调控有关。     

Cyclin K inhibits HIV-1 gene expression and replication by interfering with cyclin-dependent kinase 9 (CDK9)-cyclin T1 interaction in Nef-dependent manner

Human immunodeficiency virus-1 (HIV-1) exploits a number of host cellular factors for successful survival and propagation. The viral protein Nef plays an important role in HIV-1 pathogenesis by interacting with various cellular proteins. In the present work, we identified Cyclin K (CycK) as a novel Nef-interacting protein, and for the first time, we showed that CycK inhibits HIV-1 gene expression and replication in a Nef-dependent manner. The positive elongation factor b complex comprising cyclin-dependent kinase 9 (CDK9) and Cyclin T1 is a critical cellular complex required for viral gene expression and replication. Enhanced expression of CycK in the presence of Nef induced CycK-CDK9 binding, which prevented CDK9-Cyclin T1 complex formation and nuclear translocation of CDK9, resulting in inhibition of HIV-1 long terminal repeat-driven gene expression. Furthermore, this effect of CycK was not observed with Nef-deleted virus, indicating the importance of Nef in this phenomenon. Finally, silencing of CycK in HIV-1-infected cells resulted in increased translocation of CDK9 into the nucleus, leading to increased viral gene expression and replication. These data also suggest that endogenous CycK might act as an inhibitory factor for HIV-1 gene expression and replication in T-cells. Thus, our results clearly demonstrate that CycK utilizes HIV-1 Nef protein to displace CycT1 from the positive elongation factor b complex, resulting in inhibition of HIV-1 gene expression and replication.     

p35 and Rac1 underlie the neuroprotection and cognitive improvement induced by CDK5 silencing

CDK5 plays an important role in neurotransmission and synaptic plasticity in the normal function of the adult brain, and dysregulation can lead to Tau hyperphosphorylation and cognitive impairment. In a previous study, we demonstrated that RNAi knock down of CDK5 reduced the formation of neurofibrillary tangles (NFT) and prevented neuronal loss in triple transgenic Alzheimer's mice. Here, we report that CDK5 RNAi protected against glutamate‐mediated excitotoxicity using primary hippocampal neurons transduced with adeno‐associated virus 2.5 viral vector eGFP‐tagged scrambled or CDK5 shRNA‐miR during 12 days. Protection was dependent on a concomitant increase in p35 and was reversed using p35 RNAi, which affected the down‐stream Rho GTPase activity. Furthermore, p35 over‐expression and constitutively active Rac1 mimicked CDK5 silencing‐induced neuroprotection. In addition, 3xTg‐Alzheimer's disease mice (24 months old) were injected in the hippocampus with scrambled or CDK5 shRNA‐miR, and spatial learning and memory were performed 3 weeks post‐injection using ‘Morris’ water maze test. Our data showed that CDK5 knock down induced an increase in p35 protein levels and Rac activity in triple transgenic Alzheimer's mice, which correlated with the recovery of cognitive function; these findings confirm that increased p35 and active Rac are involved in neuroprotection. In summary, our data suggest that p35 acts as a mediator of Rho GTPase activity and contributes to the neuroprotection induced by CDK5 RNAi.

     

Mutation of a conserved CDK site converts a metazoan Elongation Factor 1Bbeta subunit into a replacement for yeast eEF1Balpha

Elongation factor subunit eEF1B (formerly EF-1 in plants and EF-1 in animals) was identified and cloned in a screen for proteins from pea that interact with a cyclin-dependent kinase (CDK). CDKs are enzymes that regulate progression through meiotic and mitotic cell cycles in eukaryotes. eEF1B and the related protein eEF1B (formerly EF-1' in plants and EF-1 in animals and fungi) can catalyze GTP/GDP exchange on the G-protein eEF1A (formerly EF-1 in plants, animals and fungi) during the elongation phase of protein synthesis in eukaryotes. Recombinant Cdc2 and its native homologues from pea extracts associated both in vitro and in vivo with eEF1B. A Cdc2-cyclin B complex phosphorylated recombinant plant eEF1Bs, but not eEF1B. These interactions between CDK and eEF1B prompted investigations into the in vivo consequences of this relationship. Expression of cDNAs encoding rice or pea eEF1B subunits failed to complement a Saccharomyces cerevisiae mutant deleted for the eEF1B gene, as was previously observed for the human eEF1B. However, replacement of Thr91, the sole consensus CDK phosphorylation site in pea eEF1B, with alanine allowed the pea protein to substitute for eEF1B function in vivo. In addition, this rescued strain was severely cold sensitive, and more sensitive to translational inhibitors than wild-type yeast. Taken together, these results suggest a physiological connection between the cyclin-dependent class of kinases and a translational elongation factor in mitotic cells, and provide the first in vivo evidence that an altered form of eEF1B can serve as the guanine nucleotide exchange factor for eEF1A.Communicated by C. P. Hollenberg     

A dinoflagellate CDK5-like cyclin-dependent kinase

Background information. Mitosis during the dinoflagellate cell cycle is unusual in that the nuclear envelope remains intact and segregation of the permanently condensed chromosomes uses a cytoplasmic mitotic spindle. To examine regulation of the dinoflagellate cell cycle in the context of these unusual nuclear features, it is necessary to isolate and characterize cell cycle regulators such as CDK (cyclin‐dependent kinase). Results. We report the characterization of a CDK from the dinoflagellate Lingulodinium polyedrum. This CDK reacts with an anti‐PSTAIRE antibody and was identified by protein microsequencing after partial purification. The protein microsequence shows homology toward the Pho85/CDK5 clade of CDKs. Neither the amount nor the phosphorylation state changed over the course of the cell cycle, in agreement with results reported for CDK5 family members in other systems. Conclusions. We conclude we have probably isolated a dinoflagellate CDK5‐like protein. The data reported here support the identification of this protein as a CDK5 homologue, and suggest that dinoflagellates may contain several CDK families.     

Design, synthesis, and testing of an 6-O-linked series of benzimidazole based inhibitors of CDK5/p25

Alzheimer’s disease (AD) is a progressive neurodegenerative disease resulting in cognitive and behavioral impairment. The two classic pathological hallmarks of AD include extraneuronal deposition of amyloid ?? (A??) and intraneuronal formation of neurofibrillary tangles (NFTs). NFTs contain hyperphosphorylated tau. Tau is the major microtubule-associated protein in neurons and stabilizes microtubules (MTs). Cyclin dependent kinase 5 (CDK5), when activated by the regulatory binding protein p25, phosphorylates tau at a number of proline-directed serine/threonine residues, resulting in formation of phosphorylated tau as paired helical filaments (PHFs) then in subsequent deposition of PHFs as NFTs. Beginning with the structure of Roscovitine, a moderately selective CDK5 inhibitor, we sought to conduct structural modifications to increase inhibitory potency of CDK5 and increase selectivity over a similar enzyme, cyclin dependent kinase 2 (CDK2). The design, synthesis, and testing of a series of 1-isopropyl-4-aminobenzyl-6-ether-linked benzimidazoles is presented.     

p27Kip1 Mediates Addiction of Ovarian Cancer Cells to MYCC (c-MYC) and Their Dependence on MYC Paralogs

The MYCC (c-MYC) gene is amplified in 30–60% of human ovarian cancers. We assessed the functional significance of MYCC amplification by siRNA inhibition of MYCC or MYC paralogs in a panel of ovarian cancer cell lines expressing varying levels of MYCC. Inactivation of MYCC inhibited cell proliferation and induced replicative senescence only in lines with amplified MYCC, indicating that these cells are addicted to continued MYCC overexpression. In contrast, siRNA knockdown of all three MYC isoforms inhibited proliferation of MYCC non-amplified ovarian cancer cells without inducing replicative senescence, and did not inhibit the proliferation of telomerase-immortalized ovarian surface epithelial cells. The arrest induced by MYCC knockdown was accompanied by an increase in the level of the Cdk inhibitor p27^Kip1 and a decrease in cyclin A expression and Cdk2 activity, and could be reversed by RNAi knockdown of p27^Kip1 or Rb, or by overexpression of cyclin A/Cdk2. The arrest induced by knockdown of all three MYC isoforms could similarly be reversed by p27^Kip1 knockdown. Our findings indicate that the addiction of MYCC-amplified ovarian cancer cells to MYCC differs from the dependence of MYCC non-amplified cancer cells on MYC paralogs, but both are mediated, at least in part, by p27^Kip1. They also suggest that growth of ovarian cancers may be blocked by inhibition of MYCC or MYC paralogs.     

Expression and Activity of Cyclin-Dependent Kinase 5/p35 in Adult Rat Peripheral Nervous System

Abstract: In spite of the clarification in the temporal and spatial expression pattern of a cyclin-dependent kinase (Cdk) 5 and its neuron-specific activator, p35, in the CNS, it remains to be elucidated in the PNS. In addition, it is not known whether Cdk5 activity exists in the PNS. Therefore, we have examined their expression and activity in the PNS by immunoblot analysis, immunohistochemistry, and in vitro kinase assay. Immunoblot analysis indicated the expression of Cdk5 and p35 proteins in dorsal root ganglion (DRG) and sciatic nerve alike in the CNS. By immunohistochemistry, both proteins were shown to be present in the cell body and axon (sciatic nerve) of both DRG neurons and anterior horn cells. A co-immunoprecipitation study indicated the in vivo association between Cdk5 and p35 in both DRG and sciatic nerve. However, Cdk5 kinase activity was found only in DRG, but not in sciatic nerve. These results suggest that Cdk5 kinase activity exists and functions physiologically in the PNS and may be regulated by unknown mechanisms other than the availability of p35 as reported in developing brains.     

Phosphorylation of Synaptic GTPase-activating Protein (synGAP) by Ca2+/Calmodulin-dependent Protein Kinase II (CaMKII) and Cyclin-dependent Kinase 5 (CDK5) Alters the Ratio of Its GAP Activity toward Ras and Rap GTPases

synGAP is a neuron-specific Ras and Rap GTPase-activating protein (GAP) found in high concentrations in the postsynaptic density (PSD) fraction from the mammalian forebrain. We have previously shown that, in situ in the PSD fraction or in recombinant form in Sf9 cell membranes, synGAP is phosphorylated by Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), another prominent component of the PSD. Here, we show that recombinant synGAP (r-synGAP), lacking 102 residues at the N terminus, can be purified in soluble form and is phosphorylated by cyclin-dependent kinase 5 (CDK5) as well as by CaMKII. Phosphorylation of r-synGAP by CaMKII increases its HRas GAP activity by 25% and its Rap1 GAP activity by 76%. Conversely, phosphorylation by CDK5 increases r-synGAP''s HRas GAP activity by 98% and its Rap1 GAP activity by 20%. Thus, phosphorylation by both kinases increases synGAP activity; CaMKII shifts the relative GAP activity toward inactivation of Rap1, and CDK5 shifts the relative activity toward inactivation of HRas. GAP activity toward Rap2 is not altered by phosphorylation by either kinase. CDK5 phosphorylates synGAP primarily at two sites, Ser-773 and Ser-802. Phosphorylation at Ser-773 inhibits r-synGAP activity, and phosphorylation at Ser-802 increases it. However, the net effect of concurrent phosphorylation of both sites, Ser-773 and Ser-802, is an increase in GAP activity. synGAP is phosphorylated at Ser-773 and Ser-802 in the PSD fraction, and its phosphorylation by CDK5 and CaMKII is differentially regulated by activation of NMDA-type glutamate receptors in cultured neurons.     

Expression of cell-cycle regulator CDK2-associating protein 1 (p12CDK2AP1) in transgenic mice induces testicular and ovarian atrophy in vivo

The novel cell-cycle regulator p12(CDK2AP1) (p12) gene encodes a cyclin-dependent kinase 2 (CDK2) partner that participates in cell-cycle regulation, apoptosis, and proliferation. CDK2 has been implicated in maintenance of gonadal homeostasis, as knockout mice display reproductive abnormalities. To investigate the role of p12 in homeostasis of gonadal tissues in vivo, we generated a transgenic mouse model driven by the human keratin 14 promoter, reported to target transgene expression to gonadal tissues and also stratified epithelia. Overexpression of the transgene was associated with a gonadal atrophy phenotype in mice of both sexes, yet fertility was not impaired. Histological evaluation of testes showed seminiferous tubule degeneration and decreased tubule diameter. Female transgenic mice had small ovaries, with a higher number of atretic follicles/mm(2) as compared to control nontransgenic mice. Also observed was increased germ cell apoptosis in both sexes (TUNEL). These results suggest that overexpression of p12 leads to testicular and ovarian abnormalities, a phenotype closely related to that of cdk2-/- mice. In combination, these observations suggest that the p12/CDK2 signaling pathways are carefully orchestrated to maintain proper gonadal tissue homeostasis. We suggest that the mechanisms of this regulation may be through p12-mediated altered expression of gonadal-specific genes and apoptotic pathways.     

Molecular Cloning and Characterization of Galactosylceramide Expression Factor-1 (GEF-1)

A rat brain cDNA clone has been isolated using a eukaryotic cell transient expression system with anti-galactosylceramide (GalCer) monoclonal antibody (MAb), that induces GalCer expression in COS-7 cells. The protein was designated as GalCer expression factor-1 (GEF-1). The deduced amino acid sequences revealed a strikingly high homology to a mouse hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), but no homology to UDP-galactose: ceramide galactosyltransferase. COS-7 cells transfected with the cDNA clone showed dramatic morphological changes and cell growth suppression. Overexpression of GEF-1 in MDCK (MDCK/GEF-1) cells showed GalCer-derived sulfatide expression as well as morphological changes, but not cell growth suppression. The enzyme activity and the mRNA level of CGT increased significantly in MDCK/GEF-1 cells compared with control cells. Taking these results together, it is suggested that GEF-1 may play an important role in regulating GalCer and sulfatide expression in the epithelial cells as well as in the brain.     

Molecular Cloning,Sequence, and Expression of Mouse Flavin-Containing Monooxygenases 1 and 5 (FMO1 and FMO5)

Full-length cDNA clones encoding FMO1 and FMO5 have been isolated from a library constructed with mRNA from the liver of a female CD-1 mouse. The derived sequence of FMO1 contains 2310 bases: 1596 in the coding region, 301 in the 5′-flanking region, and 413 in the 3′-flanking region. The sequence for FMO5 consists of 3168 bases; 1599 in the coding region, 812 in the 5′-flanking region, and 757 in the 3′-flanking region. The sequence of FMO1 encodes a protein of 532 amino acids with a predicted molecular weight of 59.9 kDa and shows 83.3% identity to human FMO1 and 83–94% identity to other FMO1 homologs. FMO5 encodes a protein of 533 amino acids with a predicted molecular weight of 60.0 kDa and 84.1% identity to human FMO5 and 83–84% identity to other FMO5 orthologs. Two GxGxxG putative pyrophosphate binding domains exist beginning at positions 9 and 191 for FMO1, and 10 and 192 for FMO5. Mouse FMO1 and FMO5 were expressed in E. coli and show similar mobility to the native proteins as determined by SDS-PAGE. The expressed FMO1 protein showed activity toward methimazole, and FMO5 was active toward n -octylamine. In addition, FMO1 was shown to metabolize radiolabeled phorate, whereas FMO5 showed no activity toward phorate. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 12: 205–212, 1998     

Overexpression of DOC-1R Inhibits Cell Cycle G1/S Transition by Repressing CDK2 Expression and Activation

DOC-1R (deleted in oral cancer-1 related) is a novel putative tumor suppressor. This study investigated DOC-1R antitumor activity and the underlying molecular mechanisms. Cell phenotypes were assessed using flow cytometry, BrdU incorporation and CDK2 kinase assays in DOC-1R overexpressing HeLa cells. In addition, RT-PCR and Western blot assays were used to detect underlying molecular changes in these cells. The interaction between DOC-1R and CDK2 proteins was assayed by GST pull-down and immunoprecipitation-Western blot assays. The data showed that DOC-1R overexpression inhibited G1/S phase transition, DNA replication and suppressed CDK2 activity. Molecularly, DOC-1R inhibited CDK2 expression at the mRNA and protein levels, and there were decreased levels of G1-phase cyclins (cyclin D1 and E) and elevated levels of p21, p27, and p53 proteins. Meanwhile, DOC-1R associated with CDK2 and inhibited CDK2 activation by obstructing its association with cyclin E and A. In conclusion, the antitumor effects of DOC-1R may be mediated by negatively regulating G1 phase progression and G1/S transition through inhibiting CDK2 expression and activation.     

Inhibitor of cyclin-dependent kinase (CDK) interacting with cyclin A1 (INCA1) regulates proliferation and is repressed by oncogenic signaling

The cell cycle is driven by the kinase activity of cyclin·cyclin-dependent kinase (CDK) complexes, which is negatively regulated by CDK inhibitor proteins. Recently, we identified INCA1 as an interaction partner and a substrate of cyclin A1 in complex with CDK2. On a functional level, we identified a novel cyclin-binding site in the INCA1 protein. INCA1 inhibited CDK2 activity and cell proliferation. The inhibitory effects depended on the cyclin-interacting domain. Mitogenic and oncogenic signals suppressed INCA1 expression, whereas it was induced by cell cycle arrest. We established a deletional mouse model that showed increased CDK2 activity in spleen with altered spleen architecture in Inca1(-/-) mice. Inca1(-/-) embryonic fibroblasts showed an increase in the fraction of S-phase cells. Furthermore, blasts from acute lymphoid leukemia and acute myeloid leukemia patients expressed significantly reduced INCA1 levels highlighting its relevance for growth control in vivo. Taken together, this study identifies a novel CDK inhibitor with reduced expression in acute myeloid and lymphoid leukemia. The molecular events that control the cell cycle occur in a sequential process to ensure a tight regulation, which is important for the survival of a cell and includes the detection and repair of genetic damage and the prevention of uncontrolled cell division.