Dietary polyphenols increase paraoxonase 1 gene expression by an aryl hydrocarbon receptor-dependent mechanism

Human paraoxonase 1 (PON-1) is a serum high-density lipoprotein-associated enzyme mainly secreted by the liver. It has endogenous and exogenous substrates and displays protective properties with respect to cardiovascular disease and organophosphate intoxication. In the HuH7 human hepatoma cell line, PON-1 activity and mRNA levels were increased by dietary polyphenolic compounds such as quercetin but also by toxic ligands of the aryl hydrocarbon receptor (AhR) such as 3-methylcholanthrene (3-MC). However, the 2,3,7,8-tetrachlorobenzo(p)dioxin (TCDD) was a poor inducer. Transient and stable transfection assays indicated that these compounds increased the PON-1 gene promoter activity in an AhR-dependent manner, since their effect was inhibited by 7-keto-cholesterol and AhR-directed short interfering RNA. Deletions and mutations studies showed that a xenobiotic responsive element (XRE)-like sequence within the PON-1 promoter mediated the effect of 3-MC and quercetin. In contrast with consensus XREs from the cytochrome P450 1A1 gene, the PON-1 XRE-like element mediated preferentially the effect of quercetin compared to the results seen with TCDD. Furthermore, AhR binding to this element was preferentially activated by quercetin. These observations provide a molecular mechanism for the regulation of the cardioprotective enzyme PON-1 by polyphenols. They suggest also that AhR ligands may differentially regulate gene expression depending on the DNA target sequence.     

Cdk1 is essential for mammalian cyclosome/APC regulation

The cyclosome/APC (anaphase-promoting complex), the major component of cell-cycle-specific ubiquitin-mediated proteolysis of mitotic cyclins and of other cell cycle proteins, is essential for sister chromatid separation and for exit from mitosis. Cyclosome activity and substrate specificity are modulated by phosphorylation and by transient interactions with Fizzy/cdc20 (Fzy) and Fizzy-related/Hct1/Cdh1 (Fzr). This regulation has been studied so far in Drosophila embryos, in yeast, and in cell-free extracts in vitro. Studying cyclosome regulation in mammalian cells in vivo we found that both Fzr overexpression and Cdk1 inhibition can override the prometaphase checkpoint. We further show that Fzr activation of the cyclosome is negatively regulated by Cdk1. Finally, we show that the mammalian cdc14 phosphatase, like its budding yeast homologue, plays a role in cyclosome pathway regulation. These results suggest that Cdk1 is essential for coupling various activities of the cyclosome and in particular for preventing Fzr from short-circuiting the spindle pole checkpoint. Cdk1-cyclin B is thus an inhibitor, activator, and substrate of the cyclosome.     

PP2ACdc55 regulates G1 cyclin stability

Maintaining accurate progression through the cell cycle requires the proper temporal expression and regulation of cyclins. The mammalian D-type cyclins promote G₁-S transition. D1 cyclin protein stability is regulated through its ubiquitylation and resulting proteolysis catalyzed by the SCF E3 ubiquitin ligase complex containing the F-box protein, Fbx4. SCF E3-ligase-dependent ubiquitylation of D1 is trigged by an increase in the phosphorylation status of the cyclin. As inhibition of ubiquitin-dependent D1 degradation is seen in many human cancers, we set out to uncover how D-type cyclin phosphorylation is regulated. Here we show that in S. cerevisiae, a heterotrimeric protein phosphatase 2A (PP2A^Cdc55) containing the mammalian PPP2R2/PR55 B subunit ortholog Cdc55 regulates the stability of the G₁ cyclin Cln2 by directly regulating its phosphorylation state. Cells lacking Cdc55 contain drastically reduced Cln2 levels caused by degradation due to cdk-dependent hyperphosphorylation, as a Cln2 mutant unable to be phosphorylated by the yeast cdk Cdc28 is highly stable in cdc55-null cells. Moreover, cdc55-null cells become inviable when the SCF^Grr1 activity known to regulate Cln2 levels is eliminated or when Cln2 is overexpressed, indicating a critical relationship between SCF and PP2A functions in regulating cell cycle progression through modulation of G₁-S cyclin degradation/stability. In sum, our results indicate that PP2A is absolutely required to maintain G₁-S cyclin levels through modulating their phosphorylation status, an event necessary to properly transit through the cell cycle.     

p53-independent regulation of cyclin B1 in normal human fibroblasts during UV-induced G2-arrest

Recently we demonstrated, using normal human fibroblasts (NHFs), that UVc radiation induces a G2/M arrest which was even more pronounced when p53 expression was inhibited. So, the aim of this study was to evaluate in NHFs the relationship between UV-induced G2/M arrest and cyclin B1 regulation and to investigate if p53 could contribute to the cyclin B1 regulation in these conditions. Following exposure of asynchronous NHFs to UV light, we showed that the induced G2/M arrest was accompanied by a dose-dependent down-regulation of cyclin B1 mRNA as evaluated by RT-PCR. Concomitantly, using flow cytometric analysis, we observed a strong accumulation of cyclin B1 protein which was correlated to the apparition of the G2/M arrest. In order to study the contribution of p53 to the cyclin B1 accumulation in response to UV exposure, we inhibited p53 induction using p53 antisense oligonucleotides. We found that the inhibition of p53 protein induction after UV exposure had no effect on the level of cyclin B1 mRNA. Moreover, although inhibition of p53 protein induction increased the number of the cells in the G2-M phase, the mean content of cyclin B1 protein was not augmented in these cells. These results indicate clearly that the induction of p53 protein following UV exposure does not regulate the level of cyclin B1 mRNA or protein in normal cells.     

Human cyclin E, a nuclear protein essential for the G1-to-S phase transition.

Cyclin E was first identified by screening human cDNA libraries for genes that would complement G1 cyclin mutations in Saccharomyces cerevisiae and has subsequently been found to have specific biochemical and physiological properties that are consistent with it performing a G1 function in mammalian cells. Most significantly, the cyclin E-Cdk2 complex is maximally active at the G1/S transition, and overexpression of cyclin E decreases the time it takes the cell to complete G1 and enter S phase. We have now found that mammalian cells express two forms of cyclin E protein which differ from each other by the presence or absence of a 15-amino-acid amino-terminal domain. These proteins are encoded by alternatively spliced mRNAs and are localized to the nucleus during late G1 and early S phase. Fibroblasts engineered to constitutively overexpress either form of cyclin E showed elevated cyclin E-dependent kinase activity and a shortened G1 phase of the cell cycle. The overexpressed cyclin E protein was detected in the nucleus during all cell cycle phases, including G0. Although the cyclin E protein could be overexpressed in quiescent cells, the cyclin E-Cdk2 complex was inactive. It was not activated until 6 to 8 h after readdition of serum, 4 h earlier than the endogenous cyclin E-Cdk2. This premature activation of cyclin E-Cdk2 was consistent with the extent of G1 shortening caused by cyclin E overexpression. Microinjection of affinity-purified anti-cyclin E antibodies during G1 inhibited entry into S phase, whereas microinjection performed near the G1/S transition was ineffective. These results demonstrate that cyclin E is necessary for entry into S phase. Moreover, we found that cyclin E, in contrast to cyclin D1, was required for the G1/S transition even in cells lacking retinoblastoma protein function. Therefore, cyclins E and D1 control two different transitions within the human cell cycle.     

Cyclin-dependent kinase 16/PCTAIRE kinase 1 is activated by cyclin Y and is essential for spermatogenesis

Cyclin-dependent kinase 16 (CDK16, PCTK1) is a poorly characterized protein kinase, highly expressed in the testis and the brain. Here, we report that CDK16 is activated by membrane-associated cyclin Y (CCNY). Treatment of transfected human cells with the protein kinase A (PKA) activator forskolin blocked, while kinase inhibition promoted, CCNY-dependent targeting of CDK16-green fluorescent protein (GFP) to the cell membrane. CCNY binding to CDK16 required a region upstream of the kinase domain and was found to be inhibited by phosphorylation of serine 153, a potential PKA phosphorylation site. Thus, in contrast to other CDKs, CDK16 is regulated by phosphorylation-controlled cyclin binding. CDK16 isolated from murine testis was unphosphorylated, interacted with CCNY, and exhibited kinase activity. To investigate the function of CDK16 in vivo, we established a conditional knockout allele. Mice lacking CDK16 developed normally, but male mice were infertile. Spermatozoa isolated from their epididymis displayed thinning and elongation of the annulus region, adopted a bent shape, and showed impaired motility. Moreover, CDK16-deficient spermatozoa had malformed heads and excess residual cytoplasm, suggesting a role of CDK16 in spermiation. Thus, CDK16 is a membrane-targeted CDK essential for spermatogenesis.     

G protein-coupled receptor kinase 2-mediated phosphorylation of ezrin is required for G protein-coupled receptor-dependent reorganization of the actin cytoskeleton

G protein-coupled receptor kinase 2 (GRK2) phosphorylates and desensitizes activated G protein-coupled receptors (GPCRs). Here, we identify ezrin as a novel non-GPCR substrate of GRK2. GRK2 phosphorylates glutathione S-transferase (GST)-ezrin, but not an ezrin fusion protein lacking threonine 567 (T567), in vitro. These results suggest that T567, the regulatory phosphorylation site responsible for maintaining ezrin in its active conformation, represents the principle site of GRK2-mediated phosphorylation. Two lines of evidence indicate that GRK2-mediated ezrin-radixinmoesin (ERM) phosphorylation serves to link GPCR activation to cytoskeletal reorganization. First, in Hep2 cells muscarinic M1 receptor (M1MR) activation causes membrane ruffling. This ruffling response is ERM dependent and is accompanied by ERM phosphorylation. Inhibition of GRK2, but not rho kinase or protein kinase C, prevents ERM phosphorylation and membrane ruffling. Second, agonist-induced internalization of the beta2-adrenergic receptor (beta2AR) and M1MR is accompanied by ERM phosphorylation and localization of phosphorylated ERM to receptor-containing endocytic vesicles. The colocalization of internalized beta2AR and phosphorylated ERM is not dependent on Na+/H+ exchanger regulatory factor binding to the beta2AR. Inhibition of ezrin function impedes beta2AR internalization, further linking GPCR activation, GRK activity, and ezrin function. Overall, our results suggest that GRK2 serves not only to attenuate but also to transduce GPCR-mediated signals.     

A Cdc28 mutant uncouples G1 cyclin phosphorylation and ubiquitination from G1 cyclin proteolysis

Proteolysis of the yeast G(1) cyclins is triggered by their Cdc28-dependent phosphorylation. Phosphorylated Cln1 and Cln2 are ubiquitinated by the SCF-Grr1 complex and then degraded by the 26 S proteasome. In this study, we identified a cak1 allele in a genetic screen for mutants that stabilize the yeast G(1) cyclins. Further characterization showed that Cln2HA was hypophosphorylated, unable to bind Cdc28, and stabilized in cak1 mutants at the restrictive temperature. Hypophosphorylation of Cln2HA could thus explain its stabilization. To test this possibility, we expressed a Cak1-independent mutant of Cdc28 (Cdc28-43244) in cak1 mutants and found that Cln2HA phosphorylation was restored, but surprisingly, the phospho-Cln2HA was stabilized. When bound to Cdc28-43244, Cln2HA was recognized and polyubiquitinated by SCF-Grr1. The Cdc28-43244 mutant thus reveals an unexpected complexity in the degradation of polyubiquitinated Cln2HA by the proteasome.     

Migratory localization of cyclin D2-Cdk4 complex suggests a spatial regulation of the G1-S transition

The association of the cyclin D-Cdk (DC) complex with retinoblastoma protein (pRb) is required for the G1-S transition of the cell cycle. Cyclin synthesis, nuclear localization and degradation are control mechanisms for the transition, but regulation of the DC complex nuclear import also contributes to the transition. Analysis of the timing of the G1-S transition in mammalian cell lines revealed acceleration with overexpression of cyclin D2 and Cdk4. Immunolocalization assays revealed that cyclin D2 and Cdk4 formed a complex in the cytoplasm and approached the nucleus. They accumulated on the cytosolic surfaces of the nuclear pores and then were arrested at the nuclear membrane before the nucleus reached a critical size. Finally, the complex was released into the nucleus and colocalized with pRb there, which led to pRb phosphorylation and DNA synthesis. The translocalization depended on the G1-S transition. In contrast, a truncated cyclin D2 that was not able to fully associate with Cdk4 lost the ability for release into the nucleus. This pattern of translocalization suggests a spatial separation of the cyclin D-Cdk complex from pRb and DNA in the nucleus to regulate the G1-S transition.     

The D-type cyclin CYCD3;1 is limiting for the G1-to-S-phase transition in Arabidopsis

The G1-to-S-phase transition is a key regulatory point in the cell cycle, but the rate-limiting component in plants is unknown. Overexpression of CYCLIN D3;1 (CYCD3;1) in transgenic plants increases mitotic cycles and reduces endocycles, but its effects on cell cycle progression cannot be unambiguously determined. To analyze the cell cycle roles of plant D-type cyclins, we overexpressed CYCD3;1 in Arabidopsis thaliana cell suspension cultures. Changes in cell number and doubling time were insignificant, but cultures exhibited an increased proportion of G2- over G1-phase cells, as well as increased G2 arrest in response to stationary phase and sucrose starvation. Synchronized cultures confirm that CYCD3;1-expressing (but not CYCD2;1-expressing) cells show increased G2-phase length and delayed activation of mitotic genes such as B-type cyclins, suggesting that CYCD3;1 has a specific G1/S role. Analysis of putative cyclin-dependent kinase phosphorylation sites within CYCD3;1 shows that mutating Ser-343 to Ala enhances CYCD3;1 potency without affecting its rate of turnover and results in a fivefold increase in the level of cell death in response to sucrose removal. We conclude that CYCD3;1 dominantly drives the G1/S transition, and in sucrose-depleted cells the decline in CYCD3;1 levels leads to G1 arrest, which is overcome by ectopic CYCD3;1 expression. Ser-343 is likely a key residue in modulating CYCD3;1 activity in response to sucrose depletion.     

Rca1 inhibits APC-Cdh1(Fzr) and is required to prevent cyclin degradation in G2.

We demonstrate that Rca1 is an essential inhibitor of the anaphase-promoting complex/cyclosome (APC) in Drosophila. APC activity is restricted to mitotic stages and G1 by its activators Cdc20-Fizzy (Cdc20(Fzy)) and Cdh1-Fizzy-related (Cdh1(Fzr)), respectively. In rca1 mutants, cyclins are degraded prematurely in G2 by APC-Cdh1(Fzr)-dependent proteolysis, and cells fail to execute mitosis. Overexpression of Cdh1(Fzr) mimics the rca1 phenotype, and coexpression of Rca1 blocks this Cdh1(Fzr) function. We show that Rca1 and Cdh1(Fzr) are in a complex that also includes the APC component Cdc27. Previous studies have shown that phosphorylation of Cdh1 prevents its interaction with the APC. Our data reveal a different mode of APC regulation by Rca1 at the G2 stage, when low Cdk activity is unable to inhibit Cdh1(Fzr) interaction.