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

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

Generated CK1δ-specific fragments, mutants and nomenclature of fusion proteins.

<p>Generated CK1δ-specific fragments, mutants and nomenclature of fusion proteins.</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

Chk1 target residues influence activity and kinetic parameters of CK1δ.

<p>(<b>A</b>) The kinetic parameters K_m and V_max of GST-wt CK1δ (FP449) and generated phosphorylation-site mutants were determined by <i>in vitro</i> kinase assays using α-casein as substrate. Substrate phosphorylation was quantified by Cherenkov counting and data were fitted to the Michaelis-Menten equation. V_max is expressed as pmol phosphate transferred per minute per mg of recombinant kinase. (<b>B</b>) GST-CK1δ was pre-incubated with activated Chk1 which was precipitated from hydroxyurea-treated HT1080 cells (Chk1(IP)) for 10 min. Subsequently, GST-β-catenin^1–181 was phosphorylated by GST-CK1δ alone or after pre-incubation with Chk1(IP) for additional 30 min. Data are presented as normalized bar graph.</p

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

CK1δ-specific kinase activity in HT1080 cells is decreased after activation of Chk1.

<p>Cellular Chk1 was activated by treating HT1080 cells with 2.5 mM hydroxyurea (HU) for the indicated periods of time. (<b>A</b>) Activation of Chk1 (indicated by phosphorylated Ser-345) and expression levels of Chk1 and CK1δ were determined by immunoblotting. Detection of β-actin served as loading control. (<b>B</b>) CK1 kinase activity in fractionated extracts from HT1080 cells before and after treatment with 2.5 mM HU for 2, 4, 6 and 8 hours, respectively, was determined using GST-p53^1–64 (FP267) as substrate. The detected kinase activity was normalized towards the untreated control. (<b>C</b>) Presence of CK1δ in the kinase peak fractions shown in (B) was confirmed by use of the CK1δ-specific inhibitor compound <b>17</b> at 50 nM <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068803#pone.0068803-Peifer1" target="_blank">[82]</a>. (<b>D</b>) HT1080 cells were treated with 2.5 mM HU and/or the Chk1-specific inhibitor SB-218078 for 2 h. CK1 kinase activity in fractionated extracts was determined using GST-p53^1–64 (FP267) as substrate. The detected kinase activity was normalized towards the untreated control.</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

Nano LC-MS/MS analysis of purified GST-CK1δ^305–428.

<p>Phosphate-labeled GST-CK1δ^305–428 was isolated and digested by trypsin. The resulting phosphopeptides were analyzed by Nano LC-MS/MS analysis as described in Materials and Methods. Mass spectra were analyzed using the software suite MaxQuant, version 1.0.14.3. Potential phosphorylated peptides and residues matching the sequence of rat CK1δ are shown. Ph, phosphorylation; ox, oxidized.</p

Kinetic parameters of wild-type and mutant GST-CK1δ fusion proteins.

<p>Identified Chk1 target residues in GST-wt CK1δ (FP449) were exchanged to alanine and the kinetic parameters of the resulting mutants were determined using α-casein as substrate. Data were fitted to the Michaelis-Menten equation. Obtained V_max and K_m values are reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068803#pone-0068803-t004" target="_blank">table 4</a>. V_max is expressed as pmol phosphate transferred per minute per mg of recombinant kinase. Standard error values are indicated in parentheses.</p