CK1δ inhibition results in decreased centrosomal localization.

(A) U2OStx cells were treated with a combination of CHX and PF670462 for 4 h to inhibit protein synthesis and kinase activity or left untreated. Endogenous CK1δ and pericentrin (PCNT) were detected by 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) DOX-induced U2OStx_CK1δ cells were either left untreated or treated with CHX and PF670462 for 4 h 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.

PER2 overexpression displaces CK1δ from the centrosome

U2OStx_mK2-CRY1 cells were transfected with PER2, followed by DOX induction. (A) IF for PER2 shows the formation of nuclear foci containing both overexpressed PER2 and mK2-CRY1 as reported previously28. 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 observations28. Four 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.

Phosphatase inhibition results in CK1δ phosphorylation

(A) Immunoblot analysis of CalA- and 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 are shown as loading control.

CK1δ/ε phosphorylation stabilizes overexpressed kinase.

(A) Phosphorylation protects overexpressed CK1δ from degradation. U2OStx_CK1δ cells were incubated with cycloheximide (CHX) (left part) or pretreated for 1 h with 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 are shown as loading control.

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 are shown as loading control.

CK1δ localization during mitosis

(A) Upper panels: DAPI-staining of U2OStx_CK1δ cells was used to identify stages of the cell cycle. Middle panels. 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.

Model of CK1δ regulation over the course of the cell cycle

For details see main text (discussion section).