Nutritional modulation of CK2 in Saccharomyces cerevisiae: regulating the activity of a constitutive enzyme

CK2 is a highly conserved protein kinase involved in different cellular processes, which shows a higher activity in actively proliferating mammalian cells and in various types of cancer and cancer cell lines. We recently demonstrated that CK2 activity is strongly influenced by growth rate in yeast cells as well. Here, we extend our previous findings and show that, in cells grown in either glucose or ethanol-supplemented media, CK2 presents no alteration in K(m) for both the ATP and the peptide substrate RRRADDSDDDDD, while a significant increase in V (max) is observed. In chemostat-grown cells, no difference of CK2 activity was observed in cells grown at the same dilution rate in media supplemented with either ethanol or glucose, excluding the contribution of carbon metabolism on CK2 activity. By using the eIF2 beta-derived peptide, which can be phosphorylated by the holoenzyme but not by the free catalytic subunits, we show that the holoenzyme activity requires the concurrent presence of both beta and beta' encoding genes. Finally, conditions of nitrogen deprivation leading to a G0-like arrest result in a decrease of total CK2 activity, but have no effect on the activity of the holoenzyme. These findings newly indicate a regulatory role of beta and beta' subunits of CK2 in the nutrient response

Enhanced amino acid utilization sustains growth of cells lacking Snf1/AMPK

AbstractThe metabolism of proliferating cells shows common features even in evolutionary distant organisms such as mammals and yeasts, for example the requirement for anabolic processes under tight control of signaling pathways. Analysis of the rewiring of metabolism, which occurs following the dysregulation of signaling pathways, provides new knowledge about the mechanisms underlying cell proliferation.The key energy regulator in yeast Snf1 and its mammalian ortholog AMPK have earlier been shown to have similar functions at glucose limited conditions and here we show that they also have analogies when grown with glucose excess. We show that loss of Snf1 in cells growing in 2% glucose induces an extensive transcriptional reprogramming, enhances glycolytic activity, fatty acid accumulation and reliance on amino acid utilization for growth. Strikingly, we demonstrate that Snf1/AMPK-deficient cells remodel their metabolism fueling mitochondria and show glucose and amino acids addiction, a typical hallmark of cancer cells

List of EC₅₀ values of AL-9 for different HCV genotypes.

<p>Huh7.5 cells replicating subgenomic replicons of genotype 1b or 2a (Con1-SR and JFH-A4, respectively) or Huh7.5 cells infected with the chimeric virus J6/JFH were treated with AL-9 for three days and intracellular viral RNA was measured by real time PCR. The data are representative of at least three independent experiments, and the standard deviations are shown.</p>*<p>CC₅₀ measured in uninfected Huh7.5 cells.</p

Reversibility of HCV-induced changes in PI4P subcellular distribution.

<p>JFH-A4 cells were incubated for 14 days with the HCV RdRP inhibitor HCV-796 (2 µM) or the HCV NS3/4A protease inhibitor MK-5172 (0.2 µM). Cure from HCV was controlled by detection of NS5A with a specific NS5A antibody (red, right column). As control, untreated Huh7.5 cells or JFH-A4 cells were used. Cells were fixed and PI4P (green) was detected in the internal membranes (IM, left column) or in the plasma membrane (PM, central column). For internal membrane staining giantin (red) was used as a specific marker for Golgi membranes. Nuclei were stained by the Hoechst dye (blue).</p

HCV impacts subcellular PI4P distribution.

<p>(A) Huh7.5 cells, JFH-A4 and Con1-SR cells were analyzed by confocal microscopy for the presence of PI4P (green) in the plasma membranes (PM, upper panel) or in the intracellular membrane (IM, lower panel) using the protocols described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002576#s4" target="_blank">Materials and Methods</a>. Nuclei were stained by the Hoechst dye (blue). For internal membrane staining, giantin (red) was used as a specific marker for Golgi membranes. (B) Quantification of PI4P levels by immunofluorescence analysis. Changes in mean fluorescence intensity relative to the control (Huh7.5 cells) are shown. Four randomly picked fields were analyzed per each condition. Normalization was performed as detailed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002576#s4" target="_blank">Materials and Methods</a>. Data are presented as averages ± SEM. *, p<0.05; **, p<0.01; ***, p<0.001.</p

AL-9 inhibits PI4KIIIα in Huh7.5 cells.

<p>(A) Confocal microscopy images of Huh7.5 cells treated for 2 hours with DMSO (left column) or with 1, 2, 4 or 8 µM of AL-9 (columns 2 to 5). PI4P (green) localized to the plasma membrane (PM) was detected using the plasma membrane staining protocol (upper panel) <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002576#ppat.1002576-Hammond1" target="_blank">[37]</a>. Nuclei were stained by the Hoechst dye (blue). PI4P in the intracellular membranes (IM) was revealed using the Golgi staining protocol (lower panel). Together with PI4P, Golgi membranes were stained with the Golgi marker giantin (red). Colocalization of PI4P with Golgi membranes results in yellow color (zoomed sections are indicated by a yellow square). (B) Quantification of PI4P levels by immunofluorescence analysis. Changes in mean fluorescence intensity relative to the control (DMSO) are shown. Four randomly picked fields were analyzed per each condition. Normalization was performed as detailed in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002576#s4" target="_blank">Materials and Methods</a>. Data are presented as averages ± SEM. **, p<0.01; ***, p<0.001.</p

Effect of PIK93 on Golgi or plasma membrane PI4P in Huh7.5 cells.

<p>(A) Confocal microscopy images of Huh7.5 cells incubated with DMSO (left column), 0.5 µM PIK93 (central column) or with 30 µM PIK93 (right column) for 2 hours prior to fixation and staining as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002576#s4" target="_blank">Materials and Methods</a>. PI4P (green) localized to the plasma membrane (PM) was detected using the plasma membrane staining protocol (upper panel) <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002576#ppat.1002576-Hammond1" target="_blank">[37]</a>. Nuclei were stained by the Hoechst dye (blue). PI4P in the intracellular membranes (IM) was revealed using the Golgi staining protocol (lower panel). Together with PI4P, Golgi membranes were stained with the Golgi marker giantin (red). Colocalization of PI4P with Golgi membranes results in yellow color (zoomed sections are indicated by a yellow square). (B) Quantification of PI4P levels by immunofluorescence analysis. Changes in mean fluorescence intensity relative to the control (DMSO) are shown. Four randomly picked fields were analyzed per each condition. Normalization was performed as detailed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002576#s4" target="_blank">Materials and Methods</a>. Data are presented as averages ± SEM. *, p<0.05; **, p<0.01; ***, p<0.001.</p

AL-9 inhibits PI4KIIIα in HCV-replicating cells.

<p>(A) JFH-A4 cells were treated with DMSO or AL-9 for 4 hours and internal membranes were stained for PI4P (green) and the Golgi marker giantin (red) using the Golgi staining protocol, as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002576#s4" target="_blank">Materials and Methods</a>. DMSO or AL-9 concentrations are indicated within the image. Alternatively, cells were stained for NS5A as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002576#s4" target="_blank">Materials and Methods</a> (indicated as NS5A). Nuclear DNA was stained with Hoechst dye (blue). PI4KIIIα, associated with the HCV-associated membranous web is inhibited by AL-9. The decrease of PI4P is not due to inhibition of the HCV replication indicated by unchanged NS5A expression and localization (lower panel). (B) Quantification of PI4P levels by immunofluorescence analysis. Changes in mean fluorescence intensity relative to the control (DMSO) are shown. Four randomly picked fields were analyzed per each condition. Normalization was performed as detailed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002576#s4" target="_blank">Materials and Methods</a>. Data are presented as averages ± SEM. **, p<0.01; ***, p<0.001.</p

Chemical structure of AL-9.

<p>For the synthetic pathway and procedure see Supporting Information.</p