Casein Kinase 1δ-dependent Wee1 Protein Degradation*

Background: Wee1 is an essential gene in mammals that encodes the cell cycle and cancer associated protein Wee1 kinase. Results: Inhibition of CK1δ kinase increases the levels of Wee1 protein kinase. Conclusion: Casein kinase 1δ is required for Wee1 degradation in HeLa cells and mouse embryonic fibroblasts. Significance: This is a previously unappreciated role for CK1δ in controlling Wee1 degradation and cell cycle progression. Eukaryotic mitotic entry is controlled by Cdk1, which is activated by the Cdc25 phosphatase and inhibited by Wee1 tyrosine kinase, a target of the ubiquitin proteasome pathway. Here we use a reporter of Wee1 degradation, K328M-Wee1-luciferase, to screen a kinase-directed chemical library. Hit profiling identified CK1δ-dependent Wee1 degradation. Small-molecule CK1δ inhibitors specifically disrupted Wee1 destruction and arrested HeLa cell proliferation. Pharmacological inhibition, siRNA knockdown, or conditional deletion of CK1δ also reduced Wee1 turnover. Thus, these studies define a previously unappreciated role for CK1δ in controlling the cell cycle.

Ubiquitin-mediated proteolytic pathways regulate critical cell cycle transitions by targeting specific inhibitors for degradation by the proteasome. Substrate targeting to the proteasome requires recognition by a specific E3 ubiquitin ligase that works in conjunction with an E2 ubiquitin-conjugating enzyme to transfer ubiquitin to the substrate (1). When a sufficient number of ubiquitins have been added to the substrate, it is recognized by the proteasome, and degradation ensues. The SCF ubiquitin ligases represent one of the largest classes of E3 ubiquitin ligases. These ligases contain the proteins Skp-1, Cul-1, and a substrate recognition molecule termed the F-box protein. F-box proteins contain a 40 amino acid F-box domain that is required for interaction with Skp-1 and ϳ70 F-box proteins are present in the vertebrate genome (2). SCF substrate degradation is generally initiated by posttranslational phosphorylations that are necessary for initiating ubiquitylation. This allows binding by the F-box protein component of the SCF complex. When recognition occurs, the substrate is brought into close proximity to the E2 enzyme, which initiates ubiquitin transfer.
One of the most important SCF substrates degraded in a phosphorylation-dependent manner is Wee1, an inhibitor of mitotic entry (3). Wee1 is a highly conserved kinase that inactivates the mitosis-specific kinase Cdk1-cyclin B1 complex during the S and G 2 phases of the cell cycle by phosphorylating Cdk1 at tyrosine 15. Wee1 activity is antagonized by the phosphatase Cdc25, which removes Cdk1 phosphorylation of tyrosine 15 involved in activation of Cdk1-cyclin B1 (4). Not surprisingly, the control of Wee1 degradation is an important regulator of mitotic entry (5)(6)(7)(8)(9). Degradation of nuclear Wee1 (5, 10) during G 2 phase of the cell cycle increases the activity of Cdk1, leading to mitotic entry (7,8,11,12). To identify kinases that control Wee1 phosphorylation and subsequent degradation, we screened a 16,000member, kinase-directed, small molecule library available at Scripps Florida and a reporter of Wee1 turnover, K328M-Wee1-luciferase (13). Highly specific and potent CK1␦ small molecule inhibitors were identified, which uncovered an essential role for casein kinase 1␦ in controlling Wee1 degradation and cell cycle progression.
In Vitro Kinase Assays-An in vitro kinase assay to detect CK1 or FLT3 activity, CK1 and FLT3 assays, as well as a complete kinase profile of 296 kinases was performed by Reaction Biology Corp. For FLT3, 20 M final Abltide was used (sequence EAIYAAPFAKKK). For CK1 (all isoforms), 20 M CK1 final Abltide was used (sequence KRRRAL(pS)VASLPGL) in a standard kinase assay with [ 32 P]ATP (PerkinElmer Life Sciences, 3000 Ci/mmol, 5mCi/ml) and purified kinase. Incorporation of [ 32 P]ATP into the peptide was measured after a filterbinding assay.
In Vitro Phosphorylation of Wee1 with CK1␦-293T cells were transfected with pCS2ϩ-FLAG-Wee1 K328M in a 10-cm tissue culture dish and incubated for 48 h in a tissue culture incubator (37°C, 10% CO 2 ). Cells were collected, washed in PBS, and resuspended in lysis buffer (PBS containing 0.1% IGEPAL CA-630, 10% glycerol, 5 mM NaF, microcystin LR, and protease inhibitor mixture). Lysates were clarified by centrifugation and incubated with one-tenth the volume of packed EZview Red anti-FLAG M2 affinity gel beads (Sigma-Aldrich, catalog no. F2426) overnight at 4°C. Beads were isolated by centrifugation and washed three times in wash buffer (PBS, 150 mM NaCl, 10% glycerol, 0.1% IGEPAL CA-630). Beads were then incubated with 1ϫ kinase buffer (25 mM Tris (pH 8.5), 0.01% Brj-35, 10 mM MgCl 2 , 1 mM EGTA, and 10 mM ATP) and 20 units of CSNK1D (Invitrogen, catalog no. PV3665) for 20 min at 30°C. Laemmli sample buffer was added to terminate the reactions, and the samples were boiled and resolved by SDS-PAGE. Bands corresponding to FLAG-Wee1 K328M were excised and processed for mass spectrometry. FLAG-Wee1 K328M incubated with CK1␦ or buffer was analyzed by mass spectrometry, and phosphorylated peptides obtained in each case were observed. An identical protocol was utilized for in vitro phosphorylation of FLAG-Wee1 K328M or FLAG-Wee1 K328M ⌬214 by CK1␦ using 5 Ci [␥-32 P]ATP (PerkinElmer, catalog no. BLU002H250UC). A CycLex Wee1 kinase activity assay was performed according to the instructions of the manufacturer (MBL).
In-gel Digestion of Wee1 Bands-Protein bands were excised and cut into 1 ϫ 1 mm cubes. Protein reduction, alkylation, and digestion were performed using standard in-gel digestion protocols. All reagents were prepared in 100 mM ammonium bicarbonate buffer. Gel pieces were dehydrated with acetonitrile prior to the addition of reduction, alkylation, and digestion solutions. Briefly, the resulting gel cubes were reduced with 10 mM DTT for 45 min at 56°C and then alkylated with 55 mM iodoacetamide for 30 min at room temperature. The alkylated proteins were digested by incubating the gel pieces with 12.5 ng/l trypsin for 12-16 h at 37°C. The resulting peptides were then extracted with subsequent ammonium bicarbonate and acetonitrile washes. The resulting peptide mixture was dried in a speed-vac and then resuspended in 5% formic acid and acetonitrile for mass spectrometry analysis.
LC-MS Analysis and Data Processing-All MS/MS spectra were acquired on a LTQ ion trap mass spectrometer linked to a Surveyor HPLC system (Thermo Scientific, San Jose, CA). Chromatography was performed on an in-house column that was packed with C18 Magic (3 mm, 200 Å, Michrom Biosciences). A 40-min linear ramp from 5% acetonitrile/0.1% formic acid to 35% acetonitrile/0.1% formic acid was used for peptide elution. A normalized collision energy of 32.0 was used for peptide fragmentation in MS2 and MS3. Neutral loss scanning was performed by monitoring the MS2 spectra for neutral losses that were indicative of phosphorylation (i.e. 32.70, 38.70, 49.00, 58.00, 98.00, and 116.00 Da). When identified, the correspond-ing neutral loss peaks were fragmented further in MS3 for subsequent sequencing.
The raw data files were first converted to mgf files and then queried against the Uniprot human protein database with Mascot (Matrix Science, London, UK) protein identification software. For this query, the number of allowed missed cleavages was set to 3, and carbamidomethyl cysteine was set as a fixed modification. Oxidation (Met), NQ deamidation, and phospho (STY) were all assigned as variable modifications. An additional query was performed against the same database using the Protein Pilot software and the Paragon algorithm. For this algorithm, a thorough search was performed with an emphasis on gel-based identification, biological modifications, and phosphorylation.
Cell Synchronization-Mitotic entry assays were performed essentially as described previously (11). Briefly, HeLa cells were treated with 2 mM thymidine for 18 h, and then they were released from the thymidine block for 8 h. 2 mM thymidine was then incubated again on the cells for an additional 8 h. In the case of mitotic entry in the presence of compounds, thymidine was washed away, and then compound or DMSO 3 along with 330 nM nocodazole was added to the cells. Cells were processed for phospho-histone H3 (catalog no. sc-8656-R, Santa Cruz Biotechnology) and Skp1 (catalog no. sc-7163, Santa Cruz Biotechnology) immunoreactivity. Cells were also isolated at 2-h intervals and processed for FACS analysis (catalog no. P3566, Invitrogen).
Cycloheximide Degradation Assay-100 g/ml cycloheximide or DMSO was added to HeLa cells 2 days after they were transfected with plasmids or siRNAs or treated with compounds. Cells were harvested at specific time points, and extracts were prepared as described above, followed by SDS-PAGE and Western blotting.
CK1␦/⑀ Deletion in Mouse Fibroblasts-Double-floxed mutant fibroblasts were grown in DMEM supplemented with 10% FBS. The fibroblasts were infected with adenoviral gfp or adenoviral cre in 100-mm dishes when the cells reached ϳ70% confluency, and the cultures were then split twice over ϳ5 days. At the third split, the cells were transferred to 60-mm dishes, (15) infected with wild-type CK1␦ or no virus as indicated, and harvested 24 h later.
Cell Extract Preparation, Antibodies, and Coimmunoprecipitation Assay-Cells were homogenized and extracts were prepared using lysis buffer (50 mM Tris, 150 mM NaCl, 1% Triton X-100, 1ϫ protease inhibitor mixture, and 1 M Microcystin LR). Cells were lysed by the freeze/thaw method (liquid nitrogen/37°C water bath) followed by two passages through a 20.5gauge needle. The soluble fraction was recovered by centrifugation at 14,000 rpm for 20 min at 4°C. Protein concentration was measured with a BCA protein assay kit (Pierce), and 30 g of protein from each sample was resolved by SDS-PAGE. The resolved bands were transferred onto a nitrocellulose membrane by Western blotting and then probed with relevant antibodies.
For the coimmunoprecipitation assay, cell lysates were incubated with one-tenth the volume of packed EZview Red anti-FLAG M2 affinity gel beads (Sigma-Aldrich, catalog no. F2426) on a rotary shaker overnight at 4°C. The next day, beads were washed with TBS, and the bound FLAG fusion proteins were eluted by boiling at 92°C for 5 min in Laemmli sample buffer. The supernatants were collected and analyzed by SDS-PAGE and Western blotting.

SR-653234
Selectively Stabilizes Wee1-Wee1 degradation during mitosis is dependent upon Plk1, Cdk1, and CK2 activity. They phosphorylate multiple N-terminal residues to initiate recognition by an SCF ubiquitin ligase containing the F-box protein ␤-TrCP (7,8). Wee1 is turned over in cell extracts isolated from HeLa cells in S/G 2 phase cells (8,12). Furthermore, overexpression of nonphosphorylatable versions of Wee1 limits mitotic entry and arrests cells in G 2 phase (8,12). Thus, there must be regulatory mechanisms and specifically kinases controlling Wee1 turnover during S and G 2 phase. We utilized a chemical biology approach to identify kinases responsible for Wee1 degradation during interphase. We expressed K328M Wee1-luciferase (a kinase-inactive version of Wee1-luciferase) in HeLa cells and subsequently incubated these cells with compounds from a 16,000-member, kinase-directed chemical library. Using this strategy, we isolated a class of compounds that specifically induced Wee1 stabilization relative to another protein turned over via the ubiquitin proteasome pathway, N-cyclin B1-luciferase (16) (Fig. 1, A and B). Of these screening hits, SR-653234 was the most potent in stabilizing K328M-Wee1-luciferase as well as endogenous Wee1 (Fig. 1, B-D). Importantly, SR-653234 specifically inhibited turnover of K328M-Wee1-luciferase, suggesting that it limits Wee1 turnover and not expression (Fig. 1, E and F).
SR-1277 Is a Potent and Selective Inhibitor of Wee1 Degradation and Ck1␦-Because similar purine compounds (17) (Fig. 1,  A and B) inhibit kinase activity, we profiled SR-653234 kinase activity against a panel of kinases. The results of these assays demonstrated that SR-653234 is a potent inhibitor of casein kinase 1␦, casein kinase 1⑀, LKB1, and FLT3 relative to the DMSO control or to the highly promiscuous kinase inhibitor staurosporine (Fig. 2, A and B). Importantly, kinases implicated previously in Wee1 turnover, such as Cdk1-cyclin B1, CK2, PLK1, or Cdk2-cyclin A2, were not inhibited by SR-653234 (7,8,12,18). Dose response measurements of kinases that were inhibited by SR-653234 by 65% or more in single-dose measurements provided evidence that the IC 50 s for inhibiting LKB1, FLT3, CK1␦, and CK1⑀ were 92, 100, 161, and 540 nM, respectively (Fig. 2, A and B) (14). Notably, SR-653234 is a more active inhibitor of CK1␦ and CK1⑀ than the broad-spectrum kinase inhibitor staurosporine (14). By contrast, staurosporine was a more potent inhibitor of FLT3 activity than SR-653234 (14). These studies suggest that SR-653234-mediated stabilization of Wee1 is due to inhibition of LKB1, FLT3, and CK1␦ and/or CK1⑀ activities.
The stabilization of Wee1 activity by SR-653234, SR-1277, and D4476 suggests that CK1␦ and/or CK1⑀ is required for Wee1 turnover. To test this possibility, we first used two siRNAs that target CK1␦ and assayed Wee1 turnover. CK1␦ siRNA overexpression depleted CK1␦ levels and specifically reduced Wee1 turnover relative to GFP siRNA (Fig. 4A). By contrast, depletion of CK1⑀ did not affect Wee1 proteolysis (Fig. 4B). Consistent with these findings, conditional deletion of CK1␦ and ⑀ in mouse embryonic fibroblasts increased Wee1 levels, which could be reversed by CK1␦ overexpression (Fig.  4C). Collectively, these studies suggest that CK1␦ is required for Wee1 degradation in both HeLa cells and mouse embryonic fibroblasts.
CK1␦ Phosphorylates Wee1 in an N-terminal Region Required for Turnover-Our pharmacological and genetic studies suggested that CK1␦ may directly control Wee1 destruction. We hypothesized that CK1␦ controls Wee1 turnover via phosphorylation because several CK1␦ consensus motifs can be found in human Wee1 (NetPhos 2.0). To determine whether CK1␦ directly phosphorylates Wee1, we incubated recombinant CK1␦ with immunopurified FLAG-Wee1 K328M or a version of Wee1 lacking its N terminus, FLAG-Wee1 K328M ⌬214, in an in vitro phosphorylation assay containing [␥-32 P]ATP. As shown in Fig. 5A, CK1␦ phosphorylated FLAG-Wee1 K328M, but not FLAG-Wee1 K328M ⌬214, suggesting that the N terminus of Wee1 is required for CK1␦-dependent phosphorylation. This was confirmed by mass spectrometry, wherein we identified the Wee1 site serine 212 as specifically phosphorylated in the presence of recombinant CK1␦ (Fig. 5, B and C). Collectively, these results suggest that CK1␦ phosphorylates Wee1 on its N terminus.
Prior studies demonstrated that multiple N-terminal phosphorylations initiate Wee1 turnover (7,20). Thus, we predicted that serine 212 phosphorylation is part of a motif that induces Wee1 recognition by ubiquitin ligases. We mutated serine 211 and serine 212 to alanine (FLAG-Wee1 K328M GAAL) and measured the degradation of the mutated version relative to FLAG-Wee1 K328M. Serine 211 phosphorylation occurred under control conditions in the absence of CK1␦ and in the presence of GSK3-␤ (Fig. 5, D and E). As shown in Fig. 5F, wild-type Wee1 was turned over within 2 h. However, FLAG-Wee1 K328M GAAL was not appreciably degraded within that time frame (the single site K328M GSAL and K328M GASL mutants were degraded with similar kinetics as FLAG-Wee1 K328M). This stabilization did not affect the levels of other cell cycle markers, such as cdc27, cyclin B1, and phosphohistone 3 (serine 10 phosphorylation). By contrast, reduced degradation of Wee1 is likely related to binding to ␤-TrCP because FLAG-Wee1 K328M GAAL did not associate as well as FLAG-Wee1K328M with ␤-TrCP-V5 (Fig. 5G). These studies suggest that CK1␦-dependent phosphorylation is required for Wee1 turnover via SCF-␤-TrCP. It is possible that, in addition to inhibiting degradation, CK1␦ may affect Wee1 kinase activity. However, CK1␦ phosphorylation of Wee1 does not affect Wee1 activity, as determined by an in vitro Wee1 activity assay containing recombinant Wee1 and CK1␦ (Fig. 5H).
Casein Kinase 1 ␦ Activity Is Required for Cell Cycle Progression-Our pharmacological, genetic, and biochemical studies suggest that CK1␦ targets Wee1 for destruction to initiate progression through S or G 2 /M phase because Wee1 has been implicated in these cell cycle phases (6,(23)(24)(25)(26). To test a possible role for CK1␦ in S to G 2 /M phase progression, we examined the levels of CK1␦ in cells progressing from S phase into mitosis (Fig. 6, A and B). We synchronized HeLa cells using a double thymidine block, which arrests them at the G 1 /S phase transition, released them into nocodazolecontaining medium, and measured CK1␦ levels via Western blot analysis as cells progressed into mitosis. CK1␦ levels increased as cells progressed into mitosis (Fig. 6, A and B). By contrast, CK1␣, CK1⑀, or CK ␥2 levels either remained constant or decreased as cells proceeded from S to G 2 /M phase (Fig. 6, A and B).
Because CK1␦ levels increased during S and G 2 phase, we hypothesized that CK1␦ may play a role in progression through these phases. Inhibition of CK1␦ activity by SR-653234 or SR-1277 treatment in asynchronous HeLa cells induced an accumulation of cells in both S and G 2 /M phase, suggesting that CK1␦ mediates S and G 2 /M phase progression, as judged by PI-FACS analysis (Fig. 6C). SR-653234 or SR-1277 treatment also increased the levels of cyclinB1 and phospho-Tyr-cdc2, which are known to increase during S and G 2 /M phase (Fig.  6D). To further analyze a possible role in S and G 2 /M phase progression, we synchronized HeLa cells at the G 1 /S phase transition using a double thymidine protocol and subsequently added DMSO, SR-653234, or SR-1277 before shifting the cells into nocodazole-containing media (Fig. 6E). Subsequently, we collected cells at 2-h intervals and measured the effect of SR-653234 or SR-1277 treatment upon mitotic entry using phospho-histone H3 Western blot analysis. As shown in Fig.  6D, mitotic entry was decreased sharply in cells treated with either SR-653234 or SR-1277, which was accompanied by Wee1 stabilization. DMSO-treated cells entered mitosis within 6 h after release from the double thymidine release. By contrast, SR-653234-or SR-1277-treated cells entered mitosis within 10 or 12 h after release, respectively. However, inhibition of mitotic entry could be due to an inability of cells to progress through S phase in the presence of SR-653234 or SR-1277. Thus, these studies suggest that CK1␦ inhibition via SR-653234 or SR-1277 treatment significantly inhibits progression through S and G 2 /M phase.

DISCUSSION
This study demonstrates a novel role for CK1␦ kinase in Wee1 regulation. Several lines of evidence suggest that CK1␦ controls Wee1 destruction. First, novel small molecule inhibitors of CK1␦ regulate Wee1 kinase levels. Second, the well characterized Ck1␦/⑀ inhibitor D4476 stabilized Wee1. Third, CK1␦ depletion by siRNA-mediated transfection in HeLa cells or conditional deletion of CK1␦/⑀ in mouse embryonic fibroblasts stabilizes Wee1. Fourth, CK1␦ phosphorylated Wee1 in vitro in the N-terminal region required for turnover. Finally, CK1␦ levels increase as cells progress through S and G 2 /M phase, suggesting a role for CK1␦ in controlling Wee1 levels when its activity must be regulated.
Because CK1␦ phosphorylates and promotes degradation of Wee 1, CK1␦ control of the cell cycle could be linked to different processes controlled by Wee1, including the G 2 /M transition, S phase progression, and epigenetic regulation (22)(23)(24)27). Wee1 was first reported to function as an inhibitory kinase for Cdk1 (26). However, subsequent studies demonstrated that it also antagonizes a driver of S phase, the cyclin A1-Cdk2 complex, through inhibitory phosphorylation of Cdk2 at Tyr-15 (22)(23)(24)(25). A recent study also suggested a role for Wee1 in S  HeLa cells were treated with the indicated amounts of either SR-653234 or SR-1277 and processed for PI-FACS. The plot represents the mean Ϯ S.E. of three independent experiments. D, asynchronous HeLa cells were treated with the indicated amounts of either SR-653234 or SR-1277, and levels of cyclin B1, phospho-tyrosine-15-cdc2, or the loading control Skp1 were determined. One representative Western blot analysis performed in triplicate is shown. E, SR-653234 and SR-1277 inhibit S/G 2 /M phase progression in synchronized cells. HeLa cells were synchronized at the G 1 /S phase transition using a double thymidine synchronization procedure. Subsequently, cells were released from the block by washing away thymidine. Cells were then incubated in the presence of DMSO, 100 nM SR-653234, or 50 nM SR-1277 along with 330 nM nocodazole for 14 h. Cells were collected every 2 h and processed for Western blot or FACS analysis. Western blot analyses were performed for Wee1, phosoho-histone H3 (Ser-10), or Skp1 (loading control).
phase as an epigenetic modifier that phosphorylates histone 2b at Tyr-37 and inhibits transcription of the histone cluster Hist1 (27). Thus, these complex roles for Wee1 may explain why we observed an increase in S and G 2 /M phase cells after CK1␦ inhibition. For instance, CK1␦ could promote the degradation of Wee1 and, therefore, induce cyclin A-Cdk2 and cyclin B-Cdk1 activation as well as histone 2b-dependent transcription. In turn, higher cyclin A-Cdk2 activity could potentially promote S phase progression, whereas Cdk1-cyclin B1 activation might induce the G 2 /M transition. By contrast, increased Wee1 after CK1␦ inhibition or depletion may stall cells in S phase because Wee1 functions in the S phase checkpoint (28) or in G 2 because Wee1 degradation is required for the G 2 -to-M transition (10,11). However, further studies are required to delineate these complex roles for CK1␦ and Wee1.
Our novel small molecule CK1␦ inhibitors are useful tools to uncover these roles. Although other CK1 inhibitors have been shown to have off-target effects via p38 MAPK or tubulin inhibition (29,30), SR-653234 and SR-1277 are not likely to modulate these targets. Our in vitro kinase profiling using multiple methods demonstrate specificity for CK1␦, and we did not observe p38 MAPK inhibition at any SR-653234 and SR-1277 concentrations tested (14). Further, SR-653234 and SR-1277 treatment did not arrest cells at prometaphase or metaphase, as determined by live cell imaging, 4 and, thus, SR-653234-and SR-1277-mediated inhibition of tubulin is unlikely. It is possible that the effects of SR-653234 and SR-1277 on the cell cycle and Wee1 destruction are due to inhibition of both CK1␦ and CK1⑀. However, we observed effects on cell cycle progression at a concentration of SR-1277 of 50 nM, which is similar to the in vitro IC 50 of CK1␦ (49 nM) but not CK1⑀ (258 nM). Thus, we favor a model where SR-653234 and SR-1277 inhibit cell cycle progression and Wee1 destruction via CK1␦ inhibition.
We find that CK1␦ is a major kinase controlling Wee1. Although several mammalian isoforms of CK1 have been reported (␣, ␥1, ␥2, ␥3, ␦, ⑀1, ⑀2, and ⑀3), our inhibitors specifically target the CK1␦ and CK1⑀ isoforms and are more selective for CK1␦ (31) (Fig. 2, A and B). Further, CK1␦ is unique among CK1 isoforms because it increases from S to G 2 /M phase. Thus, CK1␦ may be one of the only CK1 isoforms that controls Wee1 degradation, although depletion of all CK1 isoforms is required to compare their relative contribution to Wee1 destruction. At least under our experimental conditions, depletion of CK1⑀ did not affect Wee1 turnover, 4 suggesting that CK1␦, and not CK1⑀, is responsible for Wee1 proteolysis.
Our studies are the first to implicate CK1␦ in control of the core mammalian cell cycle machinery. Our results suggest that CK1␦ controls Wee1 during S and G 2 phase, although we cannot rule out regulation during other cell cycle phases. Treatment of cells synchronized at G 1 /S transition with highly specific small molecules reduced mitotic entry of cells progressing through S and G 2 phase, as judged by phospho-histone H3 analysis (Fig. 6). By contrast, PI-FACS analysis of SR-653234-, SR-1277-, or DMSO-treated cells progressing into mitosis from G 1 /S phase showed a similar profile for all cells. 4 This may suggest that SR-653234 or SR-1277 treatment reduces G 2 /M transition, which can be observed by phospho-histone H3 analysis but not PI-FACS because the latter cannot distinguish G 2 from mitosis. We were unable to deplete CK1␦ by siRNA and subsequently synchronize cells at G 1 /S transition because of cytotoxicity upon synchronization. 4 However, CK1␦ depletion in asynchronous cells increased the number of cells in the S and G 2 /M phases of the cell cycle, as judged by PI-FACS analysis.
CK1␦'s control of the cell cycle is due, in part, to the ability of CK1 to target Wee1 for destruction, although other CK1␦ substrates involved in cell cycle progression likely exist. This is supported by our observation that inhibition of Wee1 activity does not overcome mitotic entry defects observed after SR-653234 and SR-1277 treatment, although this may be due to toxicities observed with Wee1 inhibition alone. Thus, further studies including conditional inhibition of Wee1 in vitro and in vivo will be required to examine the relative contribution of the CK1␦-Wee1 pathway to cell cycle progression. Nonetheless, our studies strongly implicate CK1␦ in controlling eukaryotic cell cycle transitions in part through regulating Wee1 turnover.