DataSheet1_Comprehensive Characterization of CK1δ-Mediated Tau Phosphorylation in Alzheimer’s Disease.pdf

A main pathological event in Alzheimer’s disease is the generation of neurofibrillary tangles originating from hyperphosphorylated and subsequently aggregated tau proteins. Previous reports demonstrated the critical involvement of members of the protein kinase family CK1 in the pathogenesis of Alzheimer’s disease by hyperphosphorylation of tau. However, precise mechanisms and effects of CK1-mediated tau phosphorylation are still not fully understood. In this study, we analyzed recombinant tau441 phosphorylated by CK1δ in vitro via mass spectrometry and identified ten potential phosphorylation sites, five of them are associated to Alzheimer’s disease. To confirm these results, in vitro kinase assays and two-dimensional phosphopeptide analyses were performed with tau441 phosphomutants confirming Alzheimer’s disease-associated residues Ser68/Thr71 and Ser289 as CK1δ-specific phosphorylation sites. Treatment of differentiated human neural progenitor cells with PF-670462 and Western blot analysis identified Ser214 as CK1δ-targeted phosphorylation site. The use of an in vitro tau aggregation assay demonstrated a possible role of CK1δ in tau aggregation. Results obtained in this study highlight the potential of CK1δ to be a promising target in the treatment of Alzheimer’s disease.</p

DataSheet1_Comprehensive Characterization of CK1δ-Mediated Tau Phosphorylation in Alzheimer’s Disease.docx

A main pathological event in Alzheimer’s disease is the generation of neurofibrillary tangles originating from hyperphosphorylated and subsequently aggregated tau proteins. Previous reports demonstrated the critical involvement of members of the protein kinase family CK1 in the pathogenesis of Alzheimer’s disease by hyperphosphorylation of tau. However, precise mechanisms and effects of CK1-mediated tau phosphorylation are still not fully understood. In this study, we analyzed recombinant tau441 phosphorylated by CK1δ in vitro via mass spectrometry and identified ten potential phosphorylation sites, five of them are associated to Alzheimer’s disease. To confirm these results, in vitro kinase assays and two-dimensional phosphopeptide analyses were performed with tau441 phosphomutants confirming Alzheimer’s disease-associated residues Ser68/Thr71 and Ser289 as CK1δ-specific phosphorylation sites. Treatment of differentiated human neural progenitor cells with PF-670462 and Western blot analysis identified Ser214 as CK1δ-targeted phosphorylation site. The use of an in vitro tau aggregation assay demonstrated a possible role of CK1δ in tau aggregation. Results obtained in this study highlight the potential of CK1δ to be a promising target in the treatment of Alzheimer’s disease.</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

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

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

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