Cell cycle-coupled CK1δ turnover, autoinhibition, and activity
- Heidelberg University Biochemistry Center, Germany
Figures
Figure 1
CK1δ inhibition results in decreased centrosomal localization.
(A) U2OStx cells were treated with a combination of cycloheximide (CHX) and PF670462 for 4 hr to inhibit protein synthesis and kinase activity or left untreated. Endogenous CK1δ and pericentrin (PCNT) were detected by immunofluorescence (IF). Green (AF488): anti-CK1δ, magenta (Cy3): anti-PCNT. Nuclei were identified with DAPI. In untreated cells, CK1δ exhibited clear nuclear and pericentrosomal staining. In CHX+PF670462-treated cells, pericentrosomal localization of CK1δ was decreased and CK1δ accumulated in the nucleus. Scale bar = 50 µm. Right panel: Density above surrounding background of CK1δ at pericentrosomal region. Quantification of n=10 cells, Welch’s t-test. * indicates p-value<0.05. (B) Doxycycline (DOX)-induced U2OStx_CK1δ cells were either left untreated or treated with CHX and PF670462 for 4 hr and analyzed as described above. Green (AF488): CK1δ-FLAG was detected with anti-FLAG antibody. Magenta (CY3): anti-PCNT. In untreated cells, CK1δ-FLAG was concentrated at the pericentrosomal region. In CHX+PF670462-treated cells, pericentrosomal localization of CK1δ-FLAG was decreased and CK1δ-FLAG accumulated in the nucleus. Right panel: Density above surrounding background of CK1δ-FLAG at pericentrosomal region. Quantification of n=10 cells. Welch’s t-test. **** indicates p-value<0.0001. U2OStx cells (C) and DOX-induced U2OStx_CK1δ cells (D) were analyzed as described in (A) and (B), respectively. Fiji-based multi-channel line plot analysis showing the fluorescence intensity profiles of CK1δ (C) and CK1δ-FLAG (D) relative to PCNT along the indicated region of interest (ROI) in representative untreated cells and cells treated with CHX or PF670462. Scale bar = 20 µm.
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Figure 1—source data 1
Data underlying the plot in Figure 1A.
- https://cdn.elifesciences.org/articles/112381/elife-112381-fig1-data1-v1.xlsx
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Figure 1—source data 2
Data underlying the plot in Figure 1B.
- https://cdn.elifesciences.org/articles/112381/elife-112381-fig1-data2-v1.xlsx
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Figure 1—source data 3
Data underlying the plot in Figure 1C.
- https://cdn.elifesciences.org/articles/112381/elife-112381-fig1-data3-v1.xlsx
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Figure 1—source data 4
Data underlying the plot in Figure 1D.
- https://cdn.elifesciences.org/articles/112381/elife-112381-fig1-data4-v1.xlsx
Figure 2
PER2 overexpression displaces CK1δ from the centrosome. U2OStx_mK2-CRY1 cells were transfected with PER2, followed by doxycycline (DOX) induction.
(A) Immunofluorescence (IF) for PER2 shows the formation of nuclear foci containing both overexpressed PER2 and mK2-CRY1 as reported previously (Serrano et al., 2026). Scale bar = 20 µm. (B) IF for CK1δ shows that the kinase expressed from its endogenous locus is recruited to the nuclear PER2-CRY1 foci and depleted from the pericentrosomal region, in agreement with previous observations (Serrano et al., 2026). Four fields of view (FOVs) are shown. Presence and absence of nuclear mK2-CRY1 foci indicate PER2-transfected and untransfected cells, respectively. Pericentrosomal CK1δ localization, accessed by CK1δ-positive (green) and CRY1-negative (magenta) foci, was observed in all untransfected cells examined (50 out of 50 cells evaluated). In PER2 and CRY1-expressing cells, CK1δ accumulated in CRY1-positive (magenta) nuclear foci. No cell (0 out of 15 cells evaluated) displayed pericentrosomal localization of CK1δ (CRY1-negative focus). Fisher’s exact test, p=4.8 × 10–15. Scale bar = 20 µm.
Figure 3
Phosphatase inhibition results in CK1δ phosphorylation.
(A) Immunoblot analysis of calyculin A (CalA)- and okadaic acid (OA)-induced phosphorylation of CK1δ-FLAG. Phosphorylation of CK1δ is strongly induced by CalA and, albeit with markedly lower efficiency, also by OA. n=3. (B) Immunoblot analysis demonstrating the absence of a CalA-induced electrophoretic mobility shift in the CK1δA mutant kinase. n=3. (C) Immunoblot analysis of CalA- and OA-induced phosphorylation of kinase-dead CK1δ-K38R. n=3. (D) Time course of CalA-induced phosphorylation of CK1δ and CK1δ-K38R. n=3. Ponceau S staining of membranes is shown as loading control.
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Figure 3—source data 1
Annotated uncropped immunoblots for Figure 3, indicating lane identities and bands used in analysis.
- https://cdn.elifesciences.org/articles/112381/elife-112381-fig3-data1-v1.docx
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Figure 3—source data 2
Original uncropped immunoblots for Figure 3.
- https://cdn.elifesciences.org/articles/112381/elife-112381-fig3-data2-v1.zip
Figure 4
CK1δ/ε phosphorylation stabilizes overexpressed kinase.
(A) Phosphorylation protects overexpressed CK1δ from degradation. U2OStx_CK1δ cells were incubated with cycloheximide (CHX) (left part) or pre-treated for 1 hr with calyculin A (CalA) (middle part) or with CalA+PF670462 (right part). CK1δ was analyzed at the indicated time points after CHX addition. Overexpressed CK1δ is rapidly degraded. CalA and CalA+PF670462 induce maximal and intermediate phosphorylated CK1δ and stabilize the kinase. n=3. (B) CalA and CalA+PF670462 induce phosphorylation of CK1ε and stabilize the kinase. n=3. (C) Densitometric quantification of n=3 western blots (see A) shown as mean ± SD. Overexpressed unphosphorylated CK1δ is degraded with a half-life of about 15 min. Both CalA and CalA+PF670462 treatments stabilize the kinase. (D) Densitometric quantification of n=3 western blots (see B) shown as mean ± SD. (E) CK1δ is preferentially degraded in the nucleus. U2OStx cells stably expressing NLS-mNG-CK1δ and NES-mNG-CK1δ were incubated with CHX. Samples were analyzed at the indicated time points after CHX addition. Overexpressed NLS-mNG-CK1δ is unstable while NES-mNG-CK1δ is stable. Ponceau S staining of membranes is shown as loading control.
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Figure 4—source data 1
Annotated uncropped immunoblots for Figure 4, indicating lane identities and bands used in analysis.
- https://cdn.elifesciences.org/articles/112381/elife-112381-fig4-data1-v1.docx
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Figure 4—source data 2
Original uncropped immunoblots for Figure 4.
- https://cdn.elifesciences.org/articles/112381/elife-112381-fig4-data2-v1.zip
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Figure 4—source data 3
Data underlying the plot in Figure 4C.
- https://cdn.elifesciences.org/articles/112381/elife-112381-fig4-data3-v1.xlsx
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Figure 4—source data 4
Data underlying the plot in Figure 4D.
- https://cdn.elifesciences.org/articles/112381/elife-112381-fig4-data4-v1.xlsx
Figure 5
CK1δ is stabilized post-S phase and phosphorylated during G2/M.
(A) Schematic of S phase arrest and release of U2OStx_CK1δ cells. (B) Overexpressed CK1δ accumulates post-S phase release. n=3. (C) Schematic of G1/S and G2/M phase arrest of U2OStx_CK1δ or U2OStx cells. (D, E) Overexpressed CK1δ in U2OStx_CK1δ cells (D) and endogenous CK1δ in U2OStx cells (E) are unphosphorylated in unsynchronized and G1/S-arrested cells. G2/M-arrested cells accumulate hyperphosphorylated CK1δ. n=3. Ponceau S staining of membranes is shown as a loading control.
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Figure 5—source data 1
Annotated uncropped immunoblots for Figure 5, indicating lane identities and bands used in analysis.
- https://cdn.elifesciences.org/articles/112381/elife-112381-fig5-data1-v1.docx
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Figure 5—source data 2
Original uncropped immunoblots for Figure 5.
- https://cdn.elifesciences.org/articles/112381/elife-112381-fig5-data2-v1.zip
Figure 6
CK1δ localization during mitosis.
(A) Upper panels: DAPI staining of U2OStx_CK1δ cells was used to identify stages of the cell cycle. Middle panels. Immunofluorescence (IF) analysis of overexpressed CK1δ with FLAG antibody. Lower panels: Merged DAPI and FLAG image. Scale bar = 20 μm. (B) Upper panels: DAPI staining of U2OStx cells was used to identify stages of the cell cycle. Middle panels. IF analysis of endogenous CK1δ with anti-CK1δ antibody. Lower panels: Merged DAPI and CK1δ image. Scale bar = 20 μm. Pro: prophase, Meta-Ana: metaphase-anaphase, Telo-Cytokinesis: telophase-cytokinesis.
Figure 7
Model of CK1δ regulation over the course of the cell cycle.
For details, see main text (discussion section).
Author response image 1
Protein sequence alignments between CK1δ, CK1ε, and CK1α highlighting high sequence conservation in the structured kinase domain and sequence divergence at the C-terminal tail.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Homo sapiens) | U2OStx | Life Technologies | R712-07 | |
| Cell line (Homo sapiens) | HEK293T | ATCC | CRL-3216 | |
| Antibody | Anti-CK1δ, mouse monoclonal | abcam | ab85320, AF12G4, RRID:AB_1860174 | IF: 1:1000 IB: 1:5000 |
| Antibody | Anti-PCNT, rabbit polyclonal | abcam | ab4448, RRID:AB_304461, generous gift from Dr. Cornelia Sala and Prof. Elmar Schiebel | IF: 1:500 |
| Antibody | Anti-FLAG, mouse monoclonal | Sigma-Aldrich | F3165, RRID:AB_259529 | IB: 1:5000 IF: 1:200 |
| Antibody | Anti-PER2, rabbit polyclonal | Pineda Antikörper-Service | Rabbit sera purified in-house | 1:50 |
| Antibody | Anti-V5, mouse monoclonal | Thermo Fisher Scientific | R960. RRID:AB_2556564 | 1:5000 |
| Antibody | Anti-mouse IgG (H+L) conjugated to AlexaFluor 488, goat polyclonal | Thermo Fisher Scientific | A11001, RRID:AB_2534069 | 1:500 |
| Antibody | Anti-rabbit IgG (H+L) conjugated to Cy3, goat polyclonal | Thermo Fisher Scientific | A10520, RRID:AB_10563288 | 1:500 |
| Antibody | Anti-mouse IgG (H+L)-HRP conjugate, goat polyclonal | Bio-Rad | 170–6516, RRID:AB_11125547 | 1:10000 |
| Chemical compound, drug | Hygromycin B | Roth | CP13.4 | |
| Chemical compound, drug | Zeocin | Life Technologies | R25001 | |
| Chemical compound, drug | Doxycycline (DOX) | Clontech | 631311 | |
| Chemical compound, drug | Calyculin A (CalA) | LC Laboratories | C-3987 | |
| Chemical compound, drug | Okadaic acid (OA) | Merck | O9381 | |
| Chemical compound, drug | PF670462 | Tocris Bio-Techne | 3316 | |
| Chemical compound, drug | Cycloheximide (CHX) | PanReac AppliChem | A0879 | |
| Chemical compound, drug | Thymidine | Merck | T9250 | |
| Chemical compound, drug | Nocodazole | Merck | SML1665 | |
| Recombinant DNA reagent | pcDNA4/TO | Life Technologies | V1020-20 | |
| Commercial assay or kit | PCR Cleanup Kit | Macherey-Nagel | 740609 | |
| Commercial assay or kit | GeneJET Plasmid Extraction Kit | Thermo Fisher Scientific | K0503 | |
| Commercial assay or kit | Xfect Transfection Reagent | Takara Bio | 631317 | |
| Commercial assay or kit | ProLong Glass Antifade Mountant with NucBlue | Thermo Fisher Scientific | P36983 | |
| Software, algorithm | Fiji (ImageJ) | NIH | https://imagej.nih.gov/ij/ | |
| Software, algorithm | GraphPad Prism 10 | GraphPad Software, Inc, La Jolla, CA, USA | http://www.graphpad.com/ | |
| Software, algorithm | Nikon Imaging | Nikon Europe B.V., Amstelveen, Netherlands | https://www.microscope.healthcare.nikon.com/en_EU/products/software/nis-elements |
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Cell cycle-coupled CK1δ turnover, autoinhibition, and activity
eLife 15:RP112381.
https://doi.org/10.7554/eLife.112381.3
