Published OnlineFirst November 18, 2013; DOI: 10.1158/0008-5472.CAN-13-1175

Casein Kinase 1ε Promotes Cell Proliferation by Regulating mRNA Translation Sejeong Shin, Laura Wolgamott, Philippe P. Roux, et al. Cancer Res 2014;74:201-211. Published OnlineFirst November 18, 2013.

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Cancer Research

Molecular and Cellular Pathobiology

Casein Kinase 1« Promotes Cell Proliferation by Regulating mRNA Translation Sejeong Shin1, Laura Wolgamott1, Philippe P. Roux2,3, and Sang-Oh Yoon1

Abstract Deregulation of translation initiation factors contributes to many pathogenic conditions, including cancer. Here, we report the definition of a novel regulatory pathway for translational initiation with possible therapeutic import in cancer. Specifically, we found that casein kinase 1e (CK1e) is highly expressed in breast tumors and plays a critical role in cancer cell proliferation by controlling mRNA translation. Eukaryotic translation initiation factor eIF4E, an essential component of the translation initiation complex eIF4F, is downregulated by binding the negative-acting factor 4E-BP1. We found that genetic or pharmacologic inhibition of CK1e attenuated 4E-BP1 phosphorylation, thereby increasing 4E-BP1 binding to eIF4E and inhibiting mRNA translation. Mechanistic investigations showed that CK1e interacted with and phosphorylated 4E-BP1 at two novel sites T41 and T50, which were essential for 4E-BP1 inactivation along with increased mRNA translation and cell proliferation. In summary, our work identified CK1e as a pivotal regulator of mRNA translation and cell proliferation that acts by inhibiting 4E-BP1 function. As CK1e is highly expressed in breast tumors, these findings offer an initial rationale to explore CK1e blockade as a therapeutic strategy to treat cancers driven by deregulated mRNA translation. Cancer Res; 74(1); 201–11. 2013 AACR.

Introduction mRNA translation plays a critical role in the control of cellular processes to modulate cell growth, cell size, and cell proliferation (1, 2). In eukaryotic cells, the majority of mRNA translation occurs in a cap-dependent manner, meaning that it relies on a complex of proteins that assemble at the 7-methylguanosine (m7G) cap at the 50 end of mRNA (3). mRNA translation is principally regulated at the initiation stage rather than the elongation or termination step, which enables rapid, reversible, and spatial control of gene expression (2, 4, 5). Therefore, translation initiation in eukaryotes is highly regulated (4). The best established mechanism of controlling translation initiation is through regulation of the eIF4F complex that comprises eIF4E, eIF4A, and eIF4G (5). Translation initiation starts when the eIF4F complex binds to the m7G cap of mRNA and recruits the 40S ribosome (3, 6). Formation of the

Authors' Affiliations: 1Department of Cancer and Cell Biology, University of Cincinnati College of Medicine, Cincinnati, Ohio; 2Department of Pathology and Cell Biology; and 3Institute for Research in Immunology and  de Montre al, Montreal, Quebec, Canada Cancer, Universite Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Current address for S. Shin and S.-O. Yoon: Department of Cell Biology, Harvard Medical School, Boston, Massachusetts. Corresponding Author: Sang-Oh Yoon, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115. Phone: 617-432-1281; Fax: 617-432-1144; E-mail: [email protected] doi: 10.1158/0008-5472.CAN-13-1175 2013 American Association for Cancer Research.

eIF4F complex is dependent on the bioavailability of eIF4E, a major cap-binding protein. eIF4E is considered a key factor in mRNA translation because the eIF4F complex is the major regulator of translation initiation, and initiation is a major rate-limiting step of translation (2, 3). eIF4E is known to be mainly regulated by the eIF4E inhibitory binding proteins (4E-BP). 4E-BP1 is the major form of the three isoforms of 4E-BPs. 4E-BP1 negatively regulates protein synthesis by binding to and inhibiting eIF4E. The unphosphorylated or hypophosphorylated form of 4E-BP1 is active and inhibits eIF4E function. Phosphorylation of 4E-BP1 relieves its binding to eIF4E, allowing eIF4E to start mRNA translation (6, 7). Therefore, 4E-BP1 is a critical negative regulator of protein synthesis (6). The best characterized signaling pathway that regulates mRNA translation is the mammalian (mechanistic) target of rapamycin complex 1 (mTORC1) pathway. Activation of mTORC1 plays a significant role in translational control and cell proliferation by regulating 4E-BP1 phosphorylation (1). However, recent studies show that allosteric inhibitors of mTORC1 (e.g., rapamycin) and ATP-competitive mTOR inhibitors (e.g., PP242 and WYE354) do not substantially inhibit 4EBP1 phosphorylation and cell proliferation in many cell types (8–10). Because 4E-BP1 has many phosphorylation sites, but mTORC1 is known to regulate only a few of them, it is plausible to assume that there may be kinases other than mTORC1 that regulate mRNA translation and cell proliferation through regulation of 4E-BP1 phosphorylation. Identification of these kinases is essential for further understanding of signalingmediated protein synthesis. We were interested in kinases involved in the regulation of circadian rhythm that is regulated by oscillations of

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transcription/translation. Circadian rhythm and protein synthesis regulate cellular metabolism, cell cycle progression, and cell proliferation. Aberrance of these processes leads to metabolism-related diseases such as cancer and diabetes, and change of life span (1, 11–13). A recent study revealed that circadian rhythm coordinates mRNA translation through regulation of ribosome biogenesis (14). Therefore, we were curious if the kinases that regulate circadian rhythm could control mRNA translation. Casein kinase 1 (CK1) is the key kinase that regulates circadian rhythm (11, 12, 15). The CK1 family consists of several isoforms, such as CK1a, CK1d, CK1e, and CK1g. These isoforms share highly conserved kinase domains, but differ significantly in noncatalytic domains, suggesting that each isoform may play a specific role in regulating biologic processes (16). Growing evidence suggests that circadian rhythm is closely related to cancer, although the exact mechanisms of this relationship are not established yet. It is known that deregulation of circadian rhythm promotes tumor development and progression (11, 12). In addition, the genetic and/or functional disruption of the molecular circadian rhythm is found in various cancers (17). Because of the essential roles of circadian rhythm on cell metabolism and cell proliferation, the key molecules that regulate circadian rhythm may also play critical roles in tumor progression (11, 12, 18). In fact, some CK1 isoforms were recently identified as tumor promoters (19). However, the molecular mechanisms by which these CK1s promote tumor progression have not been fully understood. Here, we provide strong evidence that CK1e is a key regulator of cell proliferation by modulating protein synthesis. Our data show that CK1e plays a key role in regulating 4E-BP1 phosphorylation, thereby regulating cap-dependent translation.

Materials and Methods Cells and reagents HEK293 and HCC1806 cells were purchased from American Type Culture Collection. 293TD cells were kindly provided by Dr. Andrew L. Kung (Dana-Farber Cancer Institute, Boston, MA). 4E-BP1/2 wild-type (WT) and knockout mouse embryonic fibroblasts (MEF) were generously provided by Dr. Nahum Sonenberg (McGill University, Canada). All antibodies except CK1e antibodies were purchased from Cell Signaling Technology. Anti-CK1e antibodies were obtained from BD Biosciences and Abcam. PF-670462 and recombinant CK1e were obtained from Tocris and Sigma, respectively. Cancer tissue microarrays were purchased from Origene. Plasmids and generation of stable cells pCMV-myc-CK1 isoform plasmids were generously provided by Dr. Wenyi Wei (Beth Israel Deaconess Medical Center, Boston, MA). The bicistronic reporter plasmid pREMCVF and pGEX-4EBP1 were kindly provided by Dr. Anne E. Willis (University of Leicester, Leicester, UK) and Dr. Ping-Kun Zhou (University of South China, Hunan, China), respectively. Lentiviral short hairpin RNA (shRNA) plasmids were obtained from Sigma. Stable cells were generated as described previ-

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ously (20). 4E-BP1 plasmids were prepared as described previously (21). The point mutation of 4E-BP1 was generated by using the Expand Long Template PCR System (Roche). Polysomal fractionation Polysome fractionation was performed as described previously (22). Cell death assays The sub-G1 cell population was measured using flow cytometry as described previously (23, 24). Protein synthesis assay To determine nascent protein synthesis, Click-iT AHA (Lazidohomoalaine), Alexa Fluor 488 alkyne, and Click-iT cell reaction buffer kit were purchased from Invitrogen and used according to the manufacturer's protocol. Animal studies Animal studies were performed according to Institutional Animal Care and Use Committee-approved protocols and institutional guidelines. To determine the effect of CK1e knockdown or 4E-BP1 mutant T41A/T50A on tumor growth, stable HCC1806 cells expressing shRNA, mutant, or control were injected subcutaneously into 5- to 6-week-old female nu/nu mice and tumor diameter was measured. Protein phosphotransferase assay For the CK1e kinase assay using 4E-BP1 as the substrate, HEK293 cells were transfected with pcDNA3-HA-4E-BP1 WT or T41A/T50A mutant for 2 days and treated for 2 hours with LY294002, a PI3-kinase pathway inhibitor. Cells were lysed with buffer A (20 mmol/L HEPES (pH 7.5), 150 mmol/L NaCl, 10 mmol/L EDTA, 1 mmol/L EGTA, 10 mmol/L b-glycerophosphate, 1 mmol/L sodium orthovanadate, 5 mmol/L NaF, 2 mmol/L phenylmethylsulfonyl fluoride, 2 mg/mL aprotinin, 2 mg/mL leupeptin, and 1 mg/mL pepstatin) containing 1% Triton X-100, and immunoprecipitation was performed using an HA antibody immobilized onto sepharose matrix (Covance). Beads were washed twice in buffer A and three times in l-phosphatase buffer (NEB), and incubated with l-phosphatase (NEB) for 1.5 hours at 30 C. Beads were then washed three times with kinase reaction buffer (30 mmol/L HEPES, pH 7.5, 10 mmol/L MgCl2, 1 mmol/L DTT, and 5 mmol/L b-glycerophosphate). Recombinant CK1e expressed by baculovirus in Sf9 insect cells (Sigma) was incubated with purified HA-4E-BP1 WT or T41A/T50A mutant for 20 minutes at 30 C in kinase reaction buffer containing 5 mCi [g-32P] ATP (Perkin–Elmer) and 50 mmol/L ATP. The reaction products were subjected to SDS–PAGE, and dried gel was autoradiographed. For the CK1e kinase assay using recombinant His-4E-BP1 purified from Escherichia coli strain SG13009, the same reaction buffer, time, and temperature were used. Cap-binding assay Cap-binding assay and bicistronic luciferase assay were performed as described previously (22).

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Casein Kinase 1e Regulates mRNA Translation

Figure 1. CK1e is required for cell proliferation. Data are representative of at least three independent experiments. Where applicable, data are the means  SEM of three separate experiments performed in triplicate. Results were statistically significant ( , P < 0.01) as assessed by using the Student t test. A, stable HEK293 cells with CK1 isoform knockdowns were generated. The rate of proliferation was measured by counting cell numbers. B, the cell death rate was assessed as described in Materials and Methods using stable CK1e knockdown cells. C, HEK293 cells growing in the presence of serum were treated with the CK1e inhibitor PF-670462 (25 mmol/L) for 2 days, after which cell numbers were counted. D, cell proliferation rate was measured in stable HCC1806 cells with control or CK1e knockdown. E, cell proliferation rate was measured in HCC1806 cells treated with dimethyl sulfoxide control or PF-670462 (25 mmol/L; PF) for 2 days. F, CK1e expression was detected in breast cancer tissue microarrays. G and H, stable HCC1806 control or CK1e knockdown cells were injected into mice. G and H, tumor growth was monitored ( , P < 0.01; G), and images of tumors isolated from the mice (H). Scale bar, 1 cm.

Immunoprecipitation Breast cancer tissue was homogenized and lysed with radioimmunoprecipitation assay buffer. After centrifugation, the supernatants were incubated with anti-4E-BP1 antibody at 4 C for 2 hours, and then incubated with MagnaBind Protein G beads for an additional 1 hour. Beads were washed four times with a buffer and eluted in 2 reducing sample buffer. Immunohistochemistry Immunohistochemistry with the breast cancer tissue microarrays was performed at the Pathology Research Core in Cincinnati Children's Hospital.

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Results CK1« plays an essential role in cell proliferation We were interested in determining the effect of different CK1 isoforms on the proliferation of HEK293 cells that are known to express CK1a, CK1d, CK1e, and CK1g (25, 26). Using shRNA to knockdown these isoforms (Supplementary Fig. S1), we found that CK1e is the most potent regulator of cell proliferation (Fig. 1A). We then determined if the CK1e knockdown effect on cell proliferation was due to increased cell death. As shown in Fig. 1B, there was no significant difference in cell death between knockdown and control cells. We next treated the cells with a CK1e inhibitor, PF-670462, and found

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that this inhibitor dramatically decreased cell proliferation (Fig. 1C) without inducing significant cell death (Supplementary Fig. S2). We also used the HCC1806 breast cancer cell line to confirm that CK1e is a critical regulator of cell proliferation. As shown in Fig. 1D and E, knockdown of CK1e or CK1e inhibitor treatment significantly decreased cell proliferation. We next determined whether CK1e is overexpressed in cancers using the Human Protein Atlas (www.proteinatlas. org), a database of protein expression profiles based on immunohistochemistry of a large number of human cancer tissues. As shown in Supplementary Fig. S3, CK1e is more highly expressed in breast cancer than in normal tissues. In addition, we performed immunohistochemistry on microarrays of normal and breast tumor tissues, and similarly found that CK1e is overexpressed in breast cancer (Fig. 1F). To determine the function of CK1e in tumor growth, we used a mouse xenograft model with stable control or CK1e knockdown HCC1806 breast cancer cell lines. As shown in Fig. 1G and H, CK1e knockdown cells showed a dramatic reduction in tumor growth. Taken together, these results suggest that CK1e is a critical regulator of breast cancer cell growth. CK1« regulates protein synthesis To determine the mechanism by which CK1e regulates cell proliferation, we assessed whether CK1e could modulate global protein synthesis. We first examined polysome profiles and found that knockdown of CK1e (Fig. 2A) or treatment of cells with CK1e inhibitor (Fig. 2B) significantly decreased polysome assembly. To further confirm this, we determined the effect of CK1e on nascent protein synthesis. As shown in Fig. 2C, inhibition of CK1e profoundly decreased nascent protein synthesis. These results suggest that CK1e plays a major role in regulating mRNA translation. CK1« is a key player in cap-dependent translation To determine whether CK1e was involved in cap-dependent translation, we used a dual-luciferase reporter system that monitors the ratio between cap-dependent (Renilla luciferase) and cap-independent internal ribosome entry site (firefly luciferase) translation initiation (9). Knockdown of CK1e (Fig. 3A) or treatment of cells with CK1e inhibitor (Fig. 3B) decreased the relative cap-dependent translation rate. Cap-dependent translation is mainly regulated by the availability of eIF4E that is controlled by its phosphorylation at Ser209 and by its inhibitory binding protein (4E-BPs). We first determined the phosphorylation status of eIF4E. Neither CK1e knockdown (Fig. 3C) nor inhibitor treatment (Fig. 3D) changed its phosphorylation. We next explored the function of CK1e in binding of 4E-BP1 to eIF4E on the cap. For this, we used 7-methyl GTP Sepharose beads in which 7-methyl GTP mimics the cap at the 50 end of mRNA. Although CK1e knockdown (Fig. 3E) or treatment with CK1e inhibitor (Fig. 3F) did not appreciably change the amount of 7-methyl GTP-bound eIF4E, it dramatically increased binding of 4E-BP1 to 7-methyl GTP. It is known that stimulation of cells with growth factors or hormones induces eIF4E and 4E-BP1 dissociation, thereby increasing eIF4E activity. To determine whether inhibition of CK1e blocks dissociation of 4E-BP1 from eIF4E, cells were maintained in the absence of serum and treated

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Figure 2. CK1e regulates protein synthesis. Data are representative of at least three independent experiments. A and B, polysomal fractionation was performed using stable control or CK1e knockdown HEK293 cells (A) and using HEK293 cells treated with dimethyl sulfoxide (control) or PF670462 (PF; B). C, HEK293 cells were treated with CK1e inhibitor PF670462 for 24 hours and nascent protein synthesis rate was measured.

with insulin. As shown in Fig. 3G, 4E-BP1 and eIF4E remained associated when stimulated with insulin in the presence of the CK1e inhibitor. Taken together, our results suggest that CK1e knockdown or inhibitor treatment decreases cap-dependent translation by increasing the binding of 4E-BP1 and eIF4E. CK1« regulates 4E-BP1 activity through phosphorylation of 4E-BP1 The binding of 4E-BP1 to eIF4E depends on 4E-BP1 phosphorylation. It is known that Thr37/Thr46 must be

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Casein Kinase 1e Regulates mRNA Translation

Figure 3. CK1e is a key player of cap-dependent translation. Data are representative of at least three independent experiments. Where applicable, data are the means  SEM of three separate experiments performed in triplicate. Results were statistically significant ( , P < 0.01) as assessed by using the Student t test. A, stable control or CK1e knockdown HEK293 cells were transfected with the bicistronic reporter plasmid pREMCVF. After 48 hours, luciferase activity was measured. B, HEK293 cells were transfected with the pREMCVF for 24 hours, treated with dimethyl sulfoxide or CK1e inhibitor PF-670462 for 24 hours, and luciferase activity was measured. C and D, immunoblot analysis was performed on stable control or CK1e knockdown HEK293 cells (C) and HEK293 cells treated with dimethyl sulfoxide or PF-670462 (25 mmol/L; D) for 6 hours. E and F, 4E-BP1 capbinding activity was measured using 7-methyl GTP Sepharose with stable control or CK1e knockdown HEK293 cells growing in the presence of serum (E) and with HEK29 cells treated with dimethyl sulfoxide or PF-670462 (25 mmol/L; F) for 6 hours. G, HEK293 cells growing in the absence of serum for 24 hours were treated with dimethyl sulfoxide or PF-670462 (25 mmol/L) for 6 hours. Cells were then treated with insulin for 30 minutes and cap-binding activity was measured.

phosphorylated first, followed by Thr70 phosphorylation. Finally, Ser65 is phosphorylated, and then 4E-BP1 becomes inactive and dissociates from eIF4E (27, 28). On the basis of our findings that CK1e regulates binding of 4E-BP1 to eIF4E,

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we were curious if CK1e regulated phosphorylation of 4EBP1. To determine this, we examined 4E-BP1 phosphorylation at S65, the last phosphorylation site essential for 4E-BP1 activity. As shown in Fig. 4A, only the CK1e knockdown

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Figure 4. CK1e regulates 4E-BP1 activity through phosphorylation of 4E-BP1. Data are representative of at least three independent experiments. A and B, immunoblot analysis was performed on stable control or CK1 isoform knockdown (A) or CK1e knockdown (B) HEK293 cells. C, immunoblot analysis was performed on HEK293 cells treated with dimethyl sulfoxide or CK1e inhibitor PF-670462 (25 mmol/L) for 6 hours. D, HEK293 cells growing in the absence of serum for 24 hours were treated with dimethyl sulfoxide or PF-670462 (25 mmol/L) for 6 hours. Cells were then treated with insulin for 30 minutes, lysed, and immunoblot analysis was performed. E, immunoblot analysis was performed on stable control or CK1e knockdown HCC1806 cells. F, HCC1806 cells growing in the presence of serum were treated with PF-670462 (25 mmol/L) for 6 hours. Cells were then lysed and immunoblot analysis was performed.

decreased 4E-BP1 phosphorylation at S65. Using CK1e knockdown cells and inhibitor-treated cells, we determined phosphorylation of the other well-known phosphorylation sites. As shown in Fig. 4B and C, inhibition of CK1e decreased 4E-BP1 phosphorylation at all sites examined, suggesting that 4E-BP1 is active and binds to eIF4E under these conditions as shown in Fig. 3E–G. We next examined the effect of CK1e inhibition on the phosphorylation of 4EBP1 after insulin treatment. As shown in Fig. 4D, CK1e inhibitor treatment blocked S65 phosphorylation. We examined the same phenomena in HCC1806 cells. CK1e knockdown (Fig. 4E) or inhibitor treatment (Fig. 4F) decreased 4EBP1 phosphorylation. CK1« regulates 4E-BP1 activity by phosphorylation at T41/T50 We were interested in the mechanism by which CK1e regulates 4E-BP1 phosphorylation. The major regulator of

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4E-BP1 phosphorylation is mTORC1. To determine whether CK1e could regulate mTORC1, we examined phosphorylation of ribosomal protein S6 kinase 1 (S6K1), a direct downstream target of mTORC1, which is used as a readout of mTORC1 activity. As shown in Fig. 5A and B, neither knockdown of CK1e nor CK1e inhibitor treatment changed S6K1 phosphorylation. We also measured phosphorylation of Akt and ERK1/2, the main upstream regulators of mTORC1, and PRAS40 and Raptor, the direct regulators of mTORC1 activity by binding to mTORC1. Knockdown of CK1e (Fig. 5C) or CK1e inhibitor treatment (Fig. 5D) did not show appreciable changes in the phosphorylation of these upstream and direct regulators of mTORC1, which suggests that other mechanisms may exist for CK1e to regulate 4E-BP1 phosphorylation. To determine if CK1e could be a direct kinase for 4E-BP1, we first used protein kinase specificity prediction programs. Interestingly, they predicted that CK1 could phosphorylate 4E-BP1 at T41 (TPGGT41) and T50 (TPGGT50), which have the same sequence and are

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Casein Kinase 1e Regulates mRNA Translation

Figure 5. CK1e regulates 4E-BP1 activity by phosphorylating at T41/T50. Data are representative of at least three independent experiments. A–D, immunoblot analysis was performed on stable control or CK1e knockdown HEK293 cells (A and C) and HEK293 cells treated with dimethyl sulfoxide or CK1e inhibitor PF670462 (25 mmol/L; B and D) for 6 hours. E, sequence alignment of 4E-BP1, 4E-BP2, and 4E-BP3. F, HEK293 cells were transfected with myc-CK1e and HA-4E-BP1 plasmids. After 48 hours, cells were lysed and immunoprecipitation was performed with anti-HA antibodies. G, breast cancer tissue was lysed and immunoprecipitation was performed with 4E-BP1 antibody. H, in vitro phosphotransferase assays were performed using recombinant CK1e and immunopurified HA-4E-BP1 or HA-4E-BP1 T41A/T50A mutant.

conserved in 4E-BP2 and 4E-BP3 (Fig. 5E). To determine if CK1e could bind to 4E-BP1 in cells, we performed immunoprecipitation. As shown in Fig. 5F, CK1e interacted with 4EBP1. We also used purified 4E-BP1 from E. coli and confirmed that CK1e interacted with 4E-BP1 (Supplementary Fig. S4). We next performed in vivo interaction assay using breast cancer tissue. As shown in Fig. 5G, CK1e coimmunoprecipitated with 4E-BP1. These results suggest that CK1e interacts with 4E-BP1 in vitro and in vivo. We next performed a kinase assay to

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determine whether CK1e could phosphorylate 4E-BP1 at T41/ T50. As shown in Fig. 5H, CK1e significantly increased phosphorylation of WT 4E-BP1, while it slightly increased phosphorylation of T41A/T50A mutant 4E-BP1. We also used purified recombinant 4E-BP1 from E. coli and found that CK1e dramatically increased phosphorylation of WT 4E-BP1 compared with T41A/T50A 4E-BP1 (Supplementary Fig. S5). All together, these results suggest that CK1e is a direct kinase that phosphorylates 4E-BP1 at T41/T50.

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Figure 6. 4E-BP1 phosphorylation at T41/T50 is required for 4E-BP1 activity. Data are representative of at least three independent experiments. A and B, stable 4E-BP1 WT or T41/50A mutant expressing HEK293 growing in the presence of serum were lysed and immunoblot analysis (A) and capbinding assay (B) were performed. C and D, HEK293 cells expressing 4E-BP1 WT or T41/50A mutant were grown in the absence of serum for 24 hours and treated with insulin for 30 minutes. Immunoblot analysis (C) and cap-binding assay (D) were performed. E, rate of cell proliferation was measured using HEK293 cells stably expressing control or 4E-BP1 T41A/T50A mutant. F and G, stable HCC1806 control or 4E-BP1 T41/50A mutant cells were injected into mice. F and G, tumor growth was monitored ( , P < 0.01; F) and images with tumors isolated from the mice (G). Scale bar, 1 cm.

4E-BP1 phosphorylation at T41/T50 is essential for 4EBP1 activity Little is known about the function and regulation of 4EBP1 phosphorylation at T41/T50. By stable isotope labeling in cell culture, we recently found that T41 and T50 are phosphorylation sites in 4E-BP1 (29, 30). Other reports have also confirmed the phosphorylation of 4E-BP1 at T41 and T50 under various conditions (31–35), which is also listed in the Cell Signaling Technology PhosphoSitePlus (www.phosphosite.org), a database of phosphorylation sites. Therefore, we were interested in performing further studies on the

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function of T41/T50 phosphorylation. When expressed in HEK293 cells, the T41A/T50A mutant showed a dramatic decrease of phosphorylation at T37/46, T70, and S65 (Fig. 6A), suggesting that T41/50 phosphorylation is required for subsequent stepwise phosphorylation. To determine if T41/ T50 phosphorylation is necessary for 4E-BP1 activity, we first performed a cap-binding assay. As shown in Fig. 6B, the T41A/T50A mutant showed more cap-binding compared with WT 4E-BP1, suggesting that T41A/T50A is more active in inhibiting eIF4E activity compared with WT 4E-BP1 in serum-growing cells. We observed the same phenomena in

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Casein Kinase 1e Regulates mRNA Translation

Figure 7. CK1e regulates capdependent translation and cell proliferation by regulating 4E-BP1. Data are representative of at least three independent experiments. Where applicable, data are the means  SEM of three separate experiments performed in triplicate. Results were statistically significant ( , P < 0.01) as assessed by using the Student t test. A, stable HEK293 cells with 4E-BP1 knockdown were generated. Cells were lysed and immunoblot analysis was performed. B, stable control or 4E-BP1 knockdown cells were treated with CK1e inhibitor PF-670462 for 24 hours and nascent protein synthesis rate was measured. C, stable control or 4E-BP1 knockdown HEK293 cells were transfected with pREMCVF for 24 hours. Cells were treated with dimethyl sulfoxide or CK1e inhibitor PF-670462 for 24 hours, and luciferase activity was measured. D, stable control or 4E-BP1 knockdown HEK293 cells were treated with PF-670462 for 2 days and cell proliferation rate was measured. E, cell proliferation rate was measured using stable 4E-BP1/2 WT or knockout MEFs infected with control or CK1e shRNAs.

different conditions. As shown in Fig. 6C, insulin treatment did not increase S65 phosphorylation in the T41A/T50A mutant. We also performed a cap-binding assay. As shown in Fig. 6D, WT 4E-BP1 became inactivated and dissociated from eIF4E after insulin treatment. However, the T41A/T50A mutant was still bound to eIF4E after stimulation with insulin, suggesting that the T41A/T50A mutant is active even in the presence of insulin (Fig. 6D). These results suggest that 4E-BP1 phosphorylation at T41/T50 is critical for phosphorylation at S65 and 4E-BP1 activity. We next examined proliferation of cells expressing the T41A/T50A mutant and found that this mutation inhibited cell proliferation in HEK293 cells (Fig. 6E). To find if this also affects tumor growth, we injected stable HCC1806 cells expressing the T41A/T50A mutant into mice. As shown in Fig. 6F and G, this mutant significantly affected tumor growth in mice. CK1« regulates cap-dependent translation and cell proliferation by regulating 4E-BP1 Because CK1e inhibition activated 4E-BP1 and prevented dissociation of 4E-BP1 from eIF4E, we hypothesized that CK1e

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inhibition would not affect 4E-BP1-dependent eIF4E activity, translation, and cell proliferation in the presence of very low 4E-BP1 levels. To determine whether CK1e regulates protein synthesis by regulating 4E-BP1, we used stable 4E-BP1 knockdown cells (Fig. 7A). Unlike the control cells, CK1e inhibitor treatment did not profoundly inhibit nascent protein synthesis in 4E-BP1 knockdown cells (Fig. 7B). We also examined whether the CK1e effect on cap-dependent translation and cell proliferation is dependent on 4E-BP1. For this, stable control or 4E-BP1 knockdown cells were treated with CK1e inhibitor. As shown in Fig. 7C and D, CK1e inhibitor decreased capdependent translation and proliferation of control cells. However, CK1e inhibitor treatment did not significantly inhibit capdependent translation and proliferation of 4E-BP1 knockdown cells (Fig. 7C and D). Next, we used 4E-BP1/2 WT and knockout MEFs to further determine whether CK1e regulates cell proliferation through 4E-BPs. As shown in Fig. 7E, knockdown of CK1e dramatically inhibited proliferation of WT MEFs compared with 4E-BP1/2 knockout MEFs. Taken together, our results suggest that CK1e is required for protein synthesis and cell proliferation by regulating 4E-BP1.

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Discussion Deregulation of mRNA translation is directly associated with many pathologic conditions such as cancer and neurologic disorders, suggesting that a better understanding of the mechanism involved in this process is of great physiologic and pathologic importance. Because eIF4E is a rate-limiting factor for cap-dependent mRNA translation, its inhibitor, 4E-BP1, is a critical negative regulator of cancer progression. Phosphorylation status and amount of 4E-BP1 strongly correlate with the survival of patients with cancer (36). Patients with cancer in whom 4E-BP1 is highly phosphorylated or expressed in low amounts exhibit poor survival rates (37). In addition to its role in cancer progression, 4E-BP1 is also a critical regulator of life span and aging (13, 38). In light of the roles that 4E-BPs play as tumor suppressors and aging modulators, it is of great importance to identify signaling pathways that regulate 4E-BP phosphorylation (37). Here, we showed that CK1e is a key regulator of 4E-BP1 function. CK1e had previously been implicated in the regulation of developmental pathways and circadian rhythms (39, 40). Recently, CK1e has gained attention for its oncogenic role, and CK1e was identified as a therapeutic target in MYC-driven cancers (19). Along with this, it is known that CK1e is highly expressed in many cancer types and that it promotes tumorigenesis and tumor progression by regulating circadian rhythm proteins and Wnt signaling (16, 39). Considering the roles of 4EBP1 and mRNA translation in cancer progression, our results that CK1e is a major regulator of 4E-BP1 and mRNA translation suggest that targeting CK1e could benefit patients with cancer characterized by aberrant eIF4E/4E-BP1 regulation. It will be of interest to test whether CK1e targeting alone or in combination with mTOR inhibitors could overcome the resistance of cancer cells against mTOR inhibitors. It will also be interesting to examine if CK1e-mediated 4E-BP1 regulation and its targeting lead to modulation of aging and age-related diseases because 4E-BP1, circadian rhythm, and mRNA translation are already known to significantly modulate life span and aging. Because of the critical role of 4E-BP1 in mRNA translation and cell proliferation, its phosphorylation is highly regulated. It is known that S65 is highly dependent on the mTORC1 pathway (28). The phosphorylation of T70 is complicated and controversial (41, 42). The currently accepted mechanism of 4E-BP1 inactivation is that T37/T46 are phosphorylated first, and T70 and S65 are phosphorylated in a stepwise fashion upon stimulation with growth factors and hormones (27). Here, we showed that T41/T50 are phosphorylated by CK1e, which is required for the phosphorylation of T37/T46, T70, and S65, thereby regulating 4E-BP1 activity and cap-binding translation. Interestingly, CK2, another casein kinase, was shown to

phosphorylate a site in the C-terminal region of 4E-BP1 (43). Taken together, it seems that casein kinases and mTORC1 cooperate to fully inactivate 4E-BP1 and initiate mRNA translation. Although most protein kinases are generally tightly controlled signaling molecules that are switched on in response to specific stimuli (44), a few kinases are constitutively active irrespective of cellular context. These constitutively active kinases are often deeply involved in the activation of many regulatory molecules or they cooperate with many regulatory kinases for the full function of their substrates. CK1 is one of the few constitutively active unregulated kinases, although it was recently suggested that CK1 could be regulated by its binding partners (45). Here, we show that CK1e is a critical factor that regulates mRNA translation and cancer progression. It is known that CK1e is highly overexpressed in many cancers. Therefore, our results suggest that highly overexpressed constitutively active CK1e in cancers could accelerate mRNA translation and tumor progression. Our study underscores the importance of CK1e as a promising target to treat cancer and other diseases characterized by deregulated protein synthesis. Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed.

Authors' Contributions Conception and design: S. Shin, S.-O. Yoon Development of methodology: S. Shin, S.-O. Yoon Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): S. Shin, L. Wolgamott, S.-O. Yoon Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): S. Shin, P.P. Roux, S.-O. Yoon Writing, review, and/or revision of the manuscript: S. Shin, L. Wolgamott, P.P. Roux, S.-O. Yoon Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): S. Shin, S.-O. Yoon

Acknowledgments The authors thank Drs. Wenyi Wei, Andrew L. Kung, David Baltimore, PingKun Zhou, Nahum Sonenberg, and Anne E. Willis for generously providing reagents. The authors also thank Dr. Belinda Peace for editing this article.

Grant Support This work was supported by a start-up fund from the University of Cincinnati College of Medicine and the Marlene Harris-Ride Cincinnati Breast Cancer Pilot Grant Program (S.-O. Yoon). P.P. Roux holds a Canada Research Chair in Signal Transduction and Proteomics. Work in P.P. Roux's laboratory was supported by grants from the Canadian Cancer Society Research Institute, the Cancer Research Society, and the Canadian Institutes for Health Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received April 27, 2013; revised October 25, 2013; accepted October 31, 2013; published OnlineFirst November 18, 2013.

References 1. 2. 3.

210

Silvera D, Formenti SC, Schneider RJ. Translational control in cancer. Nat Rev Cancer 2010;10:254–66. Ruggero D. Translational control in cancer etiology. Cold Spring Harb Perspect Biol 2012;5:a012336. Blagden SP, Willis AE. The biological and therapeutic relevance of mRNA translation in cancer. Nat Rev Clin Oncol 2011;8:280–91.

Cancer Res; 74(1) January 1, 2014

4.

5.

Parsyan A, Svitkin Y, Shahbazian D, Gkogkas C, Lasko P, Merrick WC, et al. mRNA helicases: the tacticians of translational control. Nat Rev Mol Cell Biol 2011;12:235–45. Jackson RJ, Hellen CU, Pestova TV. The mechanism of eukaryotic translation initiation and principles of its regulation. Nat Rev Mol Cell Biol 2010;11:113–27.

Cancer Research

Downloaded from cancerres.aacrjournals.org on February 4, 2014. © 2014 American Association for Cancer Research.

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6. 7. 8.

9.

10.

11. 12.

13.

14.

15. 16.

17. 18. 19.

20.

21.

22.

23.

24.

25.

26.

Kong J, Lasko P. Translational control in cellular and developmental processes. Nat Rev Genet 2012;13:383–94. Roux PP, Topisirovic I. Regulation of mRNA translation by signaling pathways. Cold Spring Harb Perspect Biol 2012;4:a012252. Ducker GS, Atreya CE, Simko JP, Hom YK, Matli MR, Benes CH, et al. Incomplete inhibition of phosphorylation of 4E-BP1 as a mechanism of primary resistance to ATP-competitive mTOR inhibitors. Oncogene 2013 Apr 1. [Epub ahead of print]. Choo AY, Yoon SO, Kim SG, Roux PP, Blenis J. Rapamycin differentially inhibits S6Ks and 4E-BP1 to mediate cell-type-specific repression of mRNA translation. Proc Natl Acad Sci U S A 2008;105:17414–9. Zhang Y, Zheng XF. mTOR-independent 4E-BP1 phosphorylation is associated with cancer resistance to mTOR kinase inhibitors. Cell Cycle 2012;11:594–603. Sahar S, Sassone-Corsi P. Metabolism and cancer: the circadian clock connection. Nat Rev Cancer 2009;9:886–96. Takahashi JS, Hong HK, Ko CH, McDearmon EL. The genetics of mammalian circadian order and disorder: implications for physiology and disease. Nat Rev Genet 2008;9:764–75. Zid BM, Rogers AN, Katewa SD, Vargas MA, Kolipinski MC, Lu TA, et al. 4E-BP extends lifespan upon dietary restriction by enhancing mitochondrial activity in Drosophila. Cell 2009;139:149–60. Jouffe C, Cretenet G, Symul L, Martin E, Atger F, Naef F, et al. The circadian clock coordinates ribosome biogenesis. PLoS Biol 2013;11: e1001455. Reischl S, Kramer A. Kinases and phosphatases in the mammalian circadian clock. FEBS Lett 2011;585:1393–9. Yang WS, Stockwell BR. Inhibition of casein kinase 1-epsilon induces cancer-cell-selective, PERIOD2-dependent growth arrest. Genome Biol 2008;9:R92. Rana S, Mahmood S. Circadian rhythm and its role in malignancy. J Circadian Rhythms 2010;8:3. Greene MW. Circadian rhythms and tumor growth. Cancer Lett 2012; 318:115–23. Toyoshima M, Howie HL, Imakura M, Walsh RM, Annis JE, Chang AN, et al. Functional genomics identifies therapeutic targets for MYCdriven cancer. Proc Natl Acad Sci U S A 2012;109:9545–50. Shin S, Wolgamott L, Yu Y, Blenis J, Yoon SO. Glycogen synthase kinase (GSK)-3 promotes p70 ribosomal protein S6 kinase (p70S6K) activity and cell proliferation. Proc Natl Acad Sci U S A 2011;108: E1204–13. Shin S, Wolgamott L, Tcherkezian J, Vallabhapurapu S, Yu Y, Roux PP, et al. Glycogen synthase kinase-3b positively regulates protein synthesis and cell proliferation through the regulation of translation initiation factor 4E-binding protein 1. Oncogene 2013 Apr 15. [Epub ahead of print]. Roux PP, Shahbazian D, Vu H, Holz MK, Cohen MS, Taunton J, et al. RAS/ERK signaling promotes site-specific ribosomal protein S6 phosphorylation via RSK and stimulates cap-dependent translation. J Biol Chem 2007;282:14056–64. Chen Y, Azad MB, Gibson SB. Methods for detecting autophagy and determining autophagy-induced cell death. Can J Physiol Pharmacol 2010;88:285–95. Shin S, Wolgamott L, Yoon SO. Regulation of endothelial cell morphogenesis by the protein kinase D (PKD)/glycogen synthase kinase 3 (GSK3)b pathway. Am J Physiol Cell Physiol 2012;303:C743–56. Del Valle-Perez B, Arques O, Vinyoles M, de Herreros AG, Dunach M. Coordinated action of CK1 isoforms in canonical Wnt signaling. Mol Cell Biol 2011;31:2877–88. Cheong JK, Nguyen TH, Wang H, Tan P, Voorhoeve PM, Lee SH, et al. IC261 induces cell cycle arrest and apoptosis of human cancer cells via

www.aacrjournals.org

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38. 39. 40. 41.

42. 43.

44. 45.

CK1d/e and Wnt/b-catenin independent inhibition of mitotic spindle formation. Oncogene 2011;30:2558–69. Gingras AC, Gygi SP, Raught B, Polakiewicz RD, Abraham RT, Hoekstra MF, et al. Regulation of 4E-BP1 phosphorylation: a novel two-step mechanism. Genes Dev 1999;13:1422–37. Gingras AC, Raught B, Gygi SP, Niedzwiecka A, Miron M, Burley SK, et al. Hierarchical phosphorylation of the translation inhibitor 4E-BP1. Genes Dev 2001;15:2852–64. Yu Y, Yoon SO, Poulogiannis G, Yang Q, Ma XM, Villen J, et al. Phosphoproteomic analysis identifies Grb10 as an mTORC1 substrate that negatively regulates insulin signaling. Science 2011;332:1322–6. Shin S, Wolgamott L, Tcherkezian J, Vallabhapurapu S, Yu Y, Roux PP, et al. Glycogen synthase kinase-3b positively regulates protein synthesis and cell proliferation through the regulation of translation initiation factor 4E-binding protein 1. Oncogene 2013 Apr 15. [Epub ahead of print]. Wilson-Grady JT, Haas W, Gygi SP. Quantitative comparison of the fasted and re-fed mouse liver phosphoproteomes using lower pH reductive dimethylation. Methods 2013;61:277–86. Stokes MP, Farnsworth CL, Moritz A, Silva JC, Jia X, Lee KA, et al. PTMScan direct: identification and quantification of peptides from critical signaling proteins by immunoaffinity enrichment coupled with LC-MS/MS. Mol Cell Proteomics 2012;11:187–201. Hsu PP, Kang SA, Rameseder J, Zhang Y, Ottina KA, Lim D, et al. The mTOR-regulated phosphoproteome reveals a mechanism of mTORC1-mediated inhibition of growth factor signaling. Science 2011;332:1317–22. Huttlin EL, Jedrychowski MP, Elias JE, Goswami T, Rad R, Beausoleil SA, et al. A tissue-specific atlas of mouse protein phosphorylation and expression. Cell 2010;143:1174–89. Manes NP, Dong L, Zhou W, Du X, Reghu N, Kool AC, et al. Discovery of mouse spleen signaling responses to anthrax using label-free quantitative phosphoproteomics via mass spectrometry. Mol Cell Proteomics 2011;10:M110.000927. Armengol G, Rojo F, Castellvi J, Iglesias C, Cuatrecasas M, Pons B, et al. 4E-binding protein 1: a key molecular "funnel factor" in human cancer with clinical implications. Cancer Res 2007;67:7551–5. Sonenberg N. eIF4E, the mRNA cap-binding protein: from basic discovery to translational research. Biochem Cell Biol 2008;86: 178–83. Kennedy BK, Mackay VL. Translate this . . . during dietary restriction. Cell Metab 2009;10:247–8. Cheong JK, Virshup DM. Casein kinase 1: complexity in the family. Int J Biochem Cell Biol 2011;43:465–9. Price MA. CKI, there's more than one: casein kinase I family members in Wnt and Hedgehog signaling. Genes Dev 2006;20:399–410. Ayuso MI, Hernandez-Jimenez M, Martin ME, Salinas M, Alcazar A. New hierarchical phosphorylation pathway of the translational repressor eIF4E-binding protein 1 (4E-BP1) in ischemia-reperfusion stress. J Biol Chem 2010;285:34355–63. Wang X, Proud CG. Methods for studying signal-dependent regulation of translation factor activity. Methods Enzymol 2007;431:113–42. Lawrence JC Jr, Fadden P, Haystead TA, Lin TA. PHAS proteins as mediators of the actions of insulin, growth factors and cAMP on protein synthesis and cell proliferation. Adv Enzyme Regul 1997; 37:239–67. Pinna LA. The raison d'etre of constitutively active protein kinases: the lesson of CK2. Acc Chem Res 2003;36:378–84. Cruciat CM, Dolde C, de Groot RE, Ohkawara B, Reinhard C, Korswagen HC, et al. RNA helicase DDX3 is a regulatory subunit of casein kinase 1 in Wnt-b-catenin signaling. Science 2013;339:1436–41.

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Casein kinase 1ε promotes cell proliferation by regulating mRNA translation.

Deregulation of translation initiation factors contributes to many pathogenic conditions, including cancer. Here, we report the definition of a novel ...
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