Cyclin-dependent kinase 2 nucleocytoplasmic translocation is regulated by extracellular regulated kinase

Activation of cyclin-dependent kinase 2 (CDK2)-cyclin E in the late G(1) phase of the cell cycle is important for transit into S phase. In Chinese hamster embryonic fibroblasts (IIC9) phosphatidylinositol 3-kinase and ERK regulate alpha-thrombin-induced G(1) transit by their effects on cyclin D1 protein accumulation (Phillips-Mason, P. J., Raben, D. M., and Baldassare, J. J. (2000) J. Biol. Chem. 275, 18046-18053). Here, we show that ERK also affects CDK2-cyclin E activation by regulating the subcellular localization of CDK2. Ectopic expression of cyclin E rescues the inhibition of alpha-thrombin-induced activation of CDK2-cyclin E and transit into S phase brought about by treatment of IIC9 cells with LY29004, a selective inhibitor of mitogen stimulation of phosphatidylinositol 3-kinase activity. However, cyclin E expression is ineffectual in rescuing these effects when ERK activation is blocked by treatment with PD98059, a selective inhibitor of MEK activation of ERK. Investigation into the mechanistic reasons for this difference found the following. 1) Although treatment with LY29004 inhibits alpha-thrombin-stimulated nuclear localization, ectopic expression of cyclin E rescues CDK2 translocation. 2) In contrast to treatment with LY29004, ectopic expression of cyclin E fails to restore alpha-thrombin-stimulated nuclear CDK2 translocation in IIC9 cells treated with PD98059. 3) CDK2-cyclin E complexes are not affected by treatment with either inhibitor. These data indicate that, in addition to its effects on cyclin D1 expression, ERK activity is an important controller of the translocation of CDK2 into the nucleus where it is activated.     

Role of histone deacetylase in the expression of CTP:phosphocholine cytidylyltransferase alpha

Histone acetylation plays an important role in chromatin remodeling and gene expression. The molecular mechanisms involved in cell-specific expression of CTP:phosphocholine cytidylyltransferase alpha (CTalpha) are not fully understood. In this study, we investigated whether or not histone deacetylation is involved in repression of CTalpha expression in quiescent C3H10T1/2 mouse embryo fibroblasts. We have examined the contributions of the Sp1 and E2F binding sites in the repression of CTalpha gene expression. Immunoprecipitation experiments showed that histone deacetylase 1 (HDAC1) and HDAC activity are associated with Sp1 in serum-starved cells or during serum stimulation. However, HDAC1 association with E2F was only detected in serum-starved cells. By chromatin immunoprecipitation assays, we detected both direct and indirect association of HDAC1 with the CTalpha promoter. Treatment with the HDAC inhibitor trichostatin A induced CTalpha expression. Our data suggest that HDAC1 plays a critical role in CTalpha repression and that Sp1 and E2F may serve as key targets for HDAC1-mediated CTalpha repression in fibroblasts.     

Angiotensin II-induced transcriptional activation of the cyclin D1 gene is mediated by Egr-1 in CHO-AT(1A) cells

Cyclin D1 protein expression is regulated by mitogenic stimuli and is a critical component in the regulation of G(1) to S phase progression of the cell cycle. Angiotensin II (Ang II) binds to specific G protein-coupled receptors and is mitogenic in Chinese hamster ovary cells stably expressing the rat vascular Ang II type 1A receptor (CHO-AT(1A)). We recently reported that in these cells, Ang II induced cyclin D1 promoter activation and protein expression in a phosphatidylinositol 3-kinase (PI3K)-, SHP-2-, and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK)-dependent manner (Guillemot, L., Levy, A., Zhao, Z. J., Béréziat, G., and Rothhut, B. (2000) J. Biol. Chem. 275, 26349-26358). In this report, transfection studies using a series of deleted cyclin D1 promoters revealed that two regions between base pairs (bp) -136 and -96 and between bp -29 and +139 of the human cyclin D1 promoter contained regulatory elements required for Ang II-mediated induction. Mutational analysis in the -136 to -96 bp region provided evidence that a Sp1/early growth response protein (Egr) motif was responsible for cyclin D1 promoter activation by Ang II. Gel shift and supershift studies showed that Ang II-induced Egr-1 binding involved de novo protein synthesis and correlated well with Egr-1 promoter activation. Both U0126 (an inhibitor of the MAPK/ERK kinase MEK) and wortmannin (an inhibitor of PI3K) abrogated Egr-1 endogenous expression and Egr-1 promoter activity induced by Ang II. Moreover, using a co-transfection approach, we found that Ang II induction of Egr-1 promoter activity was blocked by dominant-negative p21(ras), Raf-1, and tyrosine phosphatase SHP-2 mutants. Identical effects were obtained when inhibitors and dominant negative mutants were tested on the -29 to +139 bp region of the cyclin D1 promoter. Taken together, these findings demonstrate that Ang II-induced cyclin D1 up-regulation is mediated by the activation and specific interaction of Egr-1 with the -136 to -96 bp region of the cyclin D1 promoter and by activation of the -29 to +139 bp region, both in a p21(ras)/Raf-1/MEK/ERK-dependent manner, and also involves PI3K and SHP-2.     

Expression of cyclin E renders cyclin D-CDK4 dispensable for inactivation of the retinoblastoma tumor suppressor protein, activation of E2F, and G1-S phase progression

The activation of CDK2-cyclin E in late G1 phase has been shown to play a critical role in retinoblastoma protein (pRb) inactivation and G1-S phase progression of the cell cycle. The phosphatidylinositol 3-OH-kinase inhibitor LY294002 has been shown to block cyclin D1 accumulation, CDK4 activity and, thus, G1 progression in alpha-thrombin-stimulated IIC9 cells (Chinese hamster embryonic fibroblasts). Our previous results show that expression of cyclin E rescues S phase progression in alpha-thrombin-stimulated IIC9 cells treated with LY294002, arguing that cyclin E renders CDK4 activity dispensable for G1 progression. In this work we investigate the ability of alpha-thrombin-induced CDK2-cyclin E activity to inactivate pRb in the absence of prior CDK4-cyclin D1 activity. We report that in the absence of CDK4-cyclin D1 activity, CDK2-cyclin E phosphorylates pRb in vivo on at least one residue and abolishes pRb binding to E2F response elements. We also find that expression of cyclin E rescues E2F activation and cyclin A expression in cyclin D kinase-inhibited, alpha-thrombin-stimulated cells. Furthermore, the rescue of E2F activity, cyclin A expression, and DNA synthesis by expression of E can be blocked by the expression of either CDK2(D145N) or RbDeltaCDK, a constitutively active mutant of pRb. However, restoring four known cyclin E-CDK2 phosphorylation sites to RbDeltaCDK renders it susceptible to inactivation in late G1, as assayed by E2F activation, cyclin A expression, and S phase progression. These data indicate that CDK2-cyclin E, without prior CDK4-cyclin D activity, can phosphorylate and inactivate pRb, activate E2F, and induce DNA synthesis.     

Hepatic CTP:phosphocholine cytidylyltransferase-alpha is a critical predictor of plasma high density lipoprotein and very low density lipoprotein

CTP:phosphocholine cytidylyltransferase (CT) is the key regulatory enzyme in the CDP-choline pathway for the biosynthesis of phosphatidylcholine (PC). We previously generated a mouse in which the hepatic CTalpha gene was specifically inactivated by the cre/loxP procedure. In CTalpha knock-out mice, plasma high density lipoprotein (HDL) and very low density lipoprotein (VLDL) levels were markedly lower than in wild type mice (Jacobs, R. L., Devlin, C., Tabas, I., and Vance, D. E. (2004) J. Biol. Chem. 279, 47402-47410.) To investigate the mechanism(s) responsible for the decrease in plasma lipoprotein levels, we isolated primary hepatocytes from knock-out and wild type mice. ABCA1 expression was reduced in knock-out hepatocytes and apoAI-dependent cholesterol, and PC efflux was impaired. When knock-out hepatocytes were infected with an adenovirus expressing CTalpha, apoAI-dependent PC efflux returned partially, whereas cholesterol efflux and ABCA1 levels were not restored to normal levels. Adenoviral expression of CTalpha did not increase VLDL secretion in knock-out hepatocytes, even though cellular PC levels returned to normal. However, in vivo adenoviral delivery of CTalpha normalized plasma HDL and VLDL levels in knock-out mice. The observations demonstrate that hepatic PC biosynthesis is a key player in maintaining plasma VLDL and HDL, and further underscores the importance of the liver in HDL formation.     

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.