Subcellular association of CK1δ with membrane structures and COPI positive vesicles.

<p>NRK cells stably expressing the fusion protein TGN38-EGFP and untransfected NRK cells were either untreated (<b>A–C</b>), treated with BFA (10 µg/ml) (<b>D–F</b>) or IC261 (50 µM) (<b>G–I</b>) and prepared for analysis by immunofluorescence microscopy. The Golgi apparatus was labeled by using a specific antibody (MG160), COPI positive vesicles were labeled with a β-COP specific antibody, and CK1δ was labeled by using the specific antibody 128A.</p

Microtubule depolymerization by IC261 treatment is reversible.

<p>(<b>A</b>) CV-1 cells expressing EYFP-tubulin were treated at time point “0 min” with 3.2 µM IC261 and observed by time-resolved fluorescence microscopy (see video sequence, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100090#pone.0100090.s009" target="_blank">movie S5</a>). The spindle apparatus of the representative cell shown here was dissolved within 8 min. At time point “10 min” IC261 was removed by exchange of media. Within a few minutes spindle MTs were built up again (“15 min”) and 20 min after removal a morphologically unimpaired spindle apparatus had been developed (“30 min”). After 2 h the cell proceeded into anaphase and cytokinesis (“155 min”). (<b>B</b>) Densitometric analysis of grey values. For quantitative analysis the relative mean intensity of EYFP-tubulin fluorescence signal in a defined region of interest (ROI) around the spindle apparatus and in the cytoplasm was measured by the software CellR. Due to IC261 treatment at time point “0 min” (arrow up) the relative intensity immediately decreased due to MT depolymerization and subsequent removal of IC261 at time point “10 min” (arrow down) lead to a reconstruction of microtubules.</p

Effect of IC261, nocodazole, taxol, taxol/IC261 on TGN morphology in NRK cells. Line 1–5:

<p>NRK cells stably expressing the fusion protein TGN38-EGFP were cultured in a flow-through chamber and observed by time-resolved fluorescence microscopy. At time point “0 min” cell were treated with DMSO (0.1%) <b>(line 1)</b>, 50 µM IC261 <b>(line 2)</b>, 5 µM nocodazole <b>(line 3)</b> or 10 µM taxol <b>(line 4)</b>. Here representative cells are shown for the stated time points (see video sequence, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100090#pone.0100090.s005" target="_blank">movie S1</a>). The solvent DMSO and the treatment with taxol showed no effect on the TGN structure. IC261 as well as nocodazole treatment fragmented the tubular membrane structure of the TGN into vesicles distributed throughout the cell. At time point “−10 min” cells were treated with 10 µM taxol and from time point “0 min” on with 10 µM taxol +50 µM IC261 <b>(line 5)</b>. Additional treatment with taxol could prevent the IC261 induced effects on the TGN.</p

Concentration dependent effects of IC261 on the cell cycle and apoptosis in CV-1 and AC1-M88 cells.

<p>CV-1 and AC1-M88 cells were cultivated for 12, 24 and 48 h with different concentrations of IC261 (0.2–3.2 µM) or DMSO (0.0 µM) and subsequently analyzed by FACS analysis with FACScan (Becton Dickinson) (as described in Material and Methods). IC261 induced a full G2/M arrest (arrow) at a cell type dependent concentration (CV1∶1.6 µM, AC1-M88∶0.8 µM). At half of this concentration IC261 induces an increase of the subG1 population (black triangle: CV-1∶0.8 µM, AC1-M88∶0.4 µM). Interestingly, at higher concentrations the amount of cells in subG1 population is smaller indicating less apoptosis (open triangle).</p

CK1δ phosphorylation state influences the effects of CK1δ- and ε-specific inhibitors.

<p>(<b>A</b>) Kinase assays were performed in the presence or absence of either 5 nM of compound <b>17 </b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068803#pone.0068803-Peifer1" target="_blank">[82]</a> or 20 nM of compound <b>8 </b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068803#pone.0068803-Bischof1" target="_blank">[58]</a> using GST-p53^1–64 (FP267) as substrate and GST-wt CK1δ or GST-CK1δ^{S328A, S370A, T397A} as enzymes. * Observed effects are significant at p<0.05. (<b>B</b>) Kinase assays were performed in the presence or absence of either D4476 (300 nM), compound <b>17</b> (10 nM) or compound <b>8</b> (20 nM) using GST-p53^1–64 (FP267) as substrate and GST-wt CK1δ alone or in combination with Chk1 as enzymes. * Observed effects are significant at p<0.05.</p

CK1δ Kinase Activity Is Modulated by Chk1-Mediated Phosphorylation

<div><p>CK1δ, a member of the casein kinase 1 family, is involved in the regulation of various cellular processes and has been associated with the pathophysiology of neurodegenerative diseases and cancer. Therefore recently, interest in generating highly specific inhibitors for personalized therapy has increased enormously. However, the efficacy of newly developed inhibitors is affected by the phosphorylation state of CK1δ. Cellular kinases phosphorylating CK1δ within its C-terminal domain have been identified but still more information regarding the role of site-specific phosphorylation in modulating the activity of CK1δ is required. Here we show that Chk1 phosphorylates rat CK1δ at serine residues 328, 331, 370, and threonine residue 397 as well as the human CK1δ transcription variants 1 and 2. CK1δ mutant proteins bearing one, two or three mutations at these identified phosphorylation sites exhibited significant differences in their kinetic properties compared to wild-type CK1δ. Additionally, CK1δ co-precipitates with Chk1 from HT1080 cell extracts and activation of cellular Chk1 resulted in a significant decrease in cellular CK1δ kinase activity. Taken together, these data point towards a possible regulatory relationship between Chk1 and CK1δ.</p></div

The CK1δ C-terminal domain contains target sites for Chk1.

<p>(<b>A</b>) Consensus motif for Chk1 as suggested by O’Neill and co-workers <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068803#pone.0068803-ONeill1" target="_blank">[75]</a>. X, no particular amino acid preference; hy, hydrophobic amino acid; ba, basic amino acid. (<b>B</b>) Surrounding sequences of potential phosphorylation sites for Chk1 within the C-terminal domain of CK1δ, determined according to the published consensus sequence <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068803#pone.0068803-ONeill1" target="_blank">[75]</a>. (<b>C</b>) The wild-type GST-CK1δ fusion proteins FP1006, FP1022, and FP1183 were generated according to the positions of the predicted Chk1 phosphorylation sites within the C-terminal domain of rat CK1δ.</p

Phosphopeptide and phosphoamino acid analyses of Chk1-phosphorylated GST-CK1δ fusion proteins.

<p>Fusion proteins GST-CK1δ^305–375 (FP1006) and GST-CK1δ^{305–375 S328A} (FP1269) (<b>A</b>); GST-CK1δ^353–375 (FP1022) and GST-CK1δ^{353–375 S370A} (FP1021) (<b>B</b>); GST-CK1δ^375–428 (FP1183) and GST-CK1δ^{375–428 T397A} (FP1221) (<b>C</b>) were phosphorylated by Chk1 <i>in vitro</i>, processed and analyzed by two-dimensional phosphopeptide analyses as described in the Materials and Methods section. Arrow positions indicate identical phosphopeptide positions. Subsequent phosphoamino acid analysis of the indicated peptide from (A) is shown in panel (<b>D</b>). Mixed analyses confirm the identity of the arrow-marked peptides.</p

CK1δ is phosphorylated by Chk1 <i>in vitro.</i>

<p>Chk1-mediated phosphorylation of three C-terminal CK1δ fusion protein sets containing either wild-type or mutant sequences encompassing amino acids 305–375 (<b>A</b>), 353–375 (<b>B</b>), and 375–428 (<b>C</b>) of the rat CK1δ sequence. The GST-CK1δ fusion proteins were phosphorylated by Chk1 <i>in vitro</i> and separated in SDS-PAGE. Substrate phosphorylation was quantified by Cherenkov counting. Results are shown as normalized bar graphs.</p

Sequences of CK1δ-specific primers used for cloning and site-directed mutagenesis (SDM).

<p>Sequences of CK1δ-specific primers used for cloning and site-directed mutagenesis (SDM).</p