Promoter activities of pGL4.10-<i>ek1</i>(-1966/+1) construct in HepG2, HCT116 and MCF-7 cells.

<p>Each bar represents the mean ± SEM of triplicate samples from three independent experiments. (** <i>p</i> < 0.01; ***<i>p</i> < 0.001 vs. promoterless pGL4.10[<i>luc2</i>]).</p

Effect of TSA on the activities of wild type and Sp(-40/-31) mutated <i>ek1</i> minimal promoters.

<p>A. Promoter activity of pGL4.10-<i>ek1</i>(-69/+1) reporter construct in HCT116 cells treated with the indicated concentrations of TSA for 24 hours (*<i>p</i> < 0.001; **<i>p</i> < 0.01; ***<i>p</i> < 0.05 vs. DMSO control; #<i>p</i> < 0.05, significant within TSA treatment group). B. Promoter activity of pGL4.10-<i>ek1</i>(-69/+1) reporter construct in HCT116 cells treated with 1 μM of TSA for the indicated time points (*<i>p</i> < 0.05 vs. DMSO control). C. Activities of wild type <i>ek1</i> minimal promoter and Sp(-40/-31)-mutated <i>ek1</i> minimal promoter after treatment with 1 μM of TSA for 24 hours. Each bar represents the mean ± SEM of triplicate samples from three independent experiments. D. Effect of TSA on <i>ek1</i> gene expression in HCT116 cells. HCT116 cells were treated with 1 μM of TSA for 24 hours (**<i>p</i> < 0.01 vs. DMSO control). Each bar represents the mean ± SEM of triplicate samples from three independent experiments.</p

Effects TSA treatment on the binding of Sp1, Sp3 and RNA polymerase II to the <i>ek1</i> minimal promoter region.

<p>ChIP analysis was performed to confirm the interaction of (A) Sp proteins and (B) RNA polymerase II with the promoter under 1 μM TSA treatment for 24 hours. PCR amplification products were resolved on 2% (w/v) agarose gel and visualized by EtBr staining. Band intensities were quantitated with Image J 1.42 and the relative intensities (compared to negative control) of PCR products from Sp1 and Sp3 immunoprecipitates were plotted. Each bar represents standard error of means (SEM) from two independent experiments. M: GeneRuler^™ DNA Ladder Mix; T: total input sample (unprocessed chromatin); P: positive control (amplified using GAPDH primers) and N: pre-immune normal rabbit IgG (negative control).</p

Effect of TSA on the activity of wild type <i>ek1</i> minimal promoter in HepG2 and HCT116 cells.

<p>Cells were treated with 1 μM of TSA for 24 hours (**<i>p</i> < 0.01 vs. DMSO control). Each bar represents the mean ± SEM of triplicate samples from three independent experiments.</p

ChIP analysis of <i>ek1</i> minimal promoter region for the binding of Sp1 and Sp3 in HCT116 and HepG2 cells.

<p>PCR amplification products were resolved on 2% (w/v) agarose gel and visualized by EtBr staining. Band intensities were quantitated with Image J 1.42 and the relative intensities (compared to negative control) of PCR products from Sp1 and Sp3 immunoprecipitates were plotted. Each bar represents standard error of means (SEM) from two independent experiments. M: GeneRuler^™ DNA Ladder Mix; T: total input sample (unprocessed chromatin); P: positive control (amplified using GAPDH Primers) and N: pre-immune normal rabbit IgG (negative control).</p

Primers used for generating promoter-luciferase constructs and PCR site-directed mutagenesis.

<p>Primers used for generating promoter-luciferase constructs and PCR site-directed mutagenesis.</p

Phosphorylation of Human Choline Kinase Beta by Protein Kinase A: Its Impact on Activity and Inhibition

<div><p>Choline kinase beta (CKβ) is one of the CK isozymes involved in the biosynthesis of phosphatidylcholine. CKβ is important for normal mitochondrial function and muscle development as the lack of the <i>ckβ</i> gene in human and mice results in the development of muscular dystrophy. In contrast, CKα is implicated in tumorigenesis and has been extensively studied as an anticancer target. Phosphorylation of human CKα was found to regulate the enzyme’s activity and its subcellular location. This study provides evidence for CKβ phosphorylation by protein kinase A (PKA). <i>In vitro</i> phosphorylation of CKβ by PKA was first detected by phosphoprotein staining, as well as by in-gel kinase assays. The phosphorylating kinase was identified as PKA by Western blotting. CKβ phosphorylation by MCF-7 cell lysate was inhibited by a PKA-specific inhibitor peptide, and the intracellular phosphorylation of CKβ was shown to be regulated by the level of cyclic adenosine monophosphate (cAMP), a PKA activator. Phosphorylation sites were located on CKβ residues serine-39 and serine-40 as determined by mass spectrometry and site-directed mutagenesis. Phosphorylation increased the catalytic efficiencies for the substrates choline and ATP about 2-fold, without affecting ethanolamine phosphorylation, and the S39D/S40D CKβ phosphorylation mimic behaved kinetically very similar. Remarkably, phosphorylation drastically increased the sensitivity of CKβ to hemicholinium-3 (HC-3) inhibition by about 30-fold. These findings suggest that CKβ, in concert with CKα, and depending on its phosphorylation status, might play a critical role as a druggable target in carcinogenesis.</p></div

Oligonucleotides used in this study.

<p>Oligonucleotides used in this study.</p

Effect of PKA phosphorylation and phosphorylation mimic mutation of CKβ on the sensitivity to HC-3 inhibition.

<p>The activities of unphosphorylated, <i>in vitro</i> phosphorylated, and S39D/S40D-mutant CKβ were measured by PK-LDH-dependent coupled-enzyme assays as described in the experimental procedures, with 4 mM choline as substrate and the indicated HC-3 inhibitor concentrations. Each bar represents the standard error of the mean (SEM) from three independent experiments.</p

Identification of PKA as the protein kinase responsible for the phosphorylation of CKβ.

<p>A) In-gel kinase assay of CKβ with MCF-7, HepG2, and HCT-116 as the sources of protein kinase PKA. Two milligrams of CKβ were used as phosphorylation substrate in the gel, and 60 μg of cell lysate were applied in each lane. After electrophoresis, the gel was washed, incubated in denaturation and renaturation buffers as described in the experimental procedures, followed by the kinase reaction by incubating the gel with 50 μM of radiolabelled ATP for 4 hours. The reaction was terminated by incubating the gel in stop buffer. CKβ phosphorylation was detected by autoradiography. B) Effect of specific PKA peptide inhibitor (PKI) on the activity of the 40 kDa protein kinase. The in-gel kinase assay was run as described above with 15 μg/mL of PKI included in the kinase buffer. MCF-7 cell lysates were run in duplicate lanes. C) Western blot detection of the 40 kDa protein kinase by a specific anti-PKA antibody. A replicated gel of the in-gel kinase assay before the kinase reaction step was blotted onto a nitrocellulose membrane followed by immunodetection with 1:5,000 dilution of anti-PKA catalytic subunit polyclonal antibody.</p