Exploring the role of casein kinase 1α splice variants across cancer cell lines.

Casein kinase 1α (CK1α) is a serine/threonine protein kinase that acts in various cellular processes affecting cell division and signal transduction. CK1α is present as multiple splice variants that are distinguished by the presence or absence of a long insert (L-insert) and a short carboxyl-terminal insert (S-insert). When overexpressed, zebrafish CK1α splice variants exhibit different biological properties, such as subcellular localization and catalytic activity. However, whether endogenous, alternatively spliced CK1α gene products also differ in their biological functions has yet to be elucidated. Here, we identify a panel of splice variant specific CK1α antibodies and use them to show that four CK1α splice variants are expressed in mammals. We subsequently show that the relative abundance of CK1α splice variants varies across distinct mouse tissues and between various cancer cell lines. Furthermore, we identify pathways whose expression is noticeably altered in cell lines enriched with select splice variants of CK1α. Finally, we show that the S-insert of CK1α promotes the growth of HCT 116 cells as cells engineered to lack the S-insert display decreased cell growth. Together, we provide tools and methods to identify individual CK1α splice variants, which we use to begin to uncover the differential biological properties driven by specific splice variants of mammalian CK1α.

CK1 and PP1 regulate Rift Valley fever virus genome replication through L protein phosphorylation.

Rift Valley fever virus (RVFV) is an arbovirus in the Phenuiviridae family identified initially by the large 'abortion storms' observed among ruminants; RVFV can also infect humans. In humans, there is a wide variation of clinical symptoms ranging from subclinical to mild febrile illness to hepatitis, retinitis, delayed-onset encephalitis, or even hemorrhagic fever. The RVFV is a tri-segmented negative-sense RNA virus consisting of S, M, and L segments. The L segment encodes the RNA-dependent RNA polymerase (RdRp), termed the L protein, which is responsible for both viral mRNA synthesis and genome replication. Phosphorylation of viral RdRps is known to regulate viral replication. This study shows that RVFV L protein is serine phosphorylated and identified Casein Kinase 1 alpha (CK1α) and protein phosphatase 1 alpha (PP1α) as L protein binding partners. Inhibition of CK1 and PP1 through small molecule inhibitor treatment, D4476 and 1E7-03, respectively, caused a change in the phosphorylated status of the L protein. Inhibition of PP1α resulted in increased L protein phosphorylation whereas inhibition of CK1α decreased L protein phosphorylation. It was also found that in RVFV infected cells, PP1α localized to the cytoplasmic compartment. Treatment of RVFV infected cells with CK1 inhibitors reduced virus production in both mammalian and mosquito cells. Lastly, inhibition of either CK1 or PP1 reduced viral genomic RNA levels. These data indicate that L protein is phosphorylated and that CK1 and PP1 play a crucial role in regulating the L protein phosphorylation cycle, which is critical to viral RNA production and viral replication.

Discovery of novel co-degradation CK1α and CDK7/9 PROTACs with p53 activation for treating acute myeloid leukemia.

Reactivating p53 activity to restore its anticancer function is an attractive cancer treatment strategy. In this study, we designed and synthesized a series of novel PROTACs to reactivate p53 via the co-degradation of CK1α and CDK7/9 proteins. Bioactivity studies showed that the selected PROTAC 13i exhibited potency antiproliferative activity in MV4-11 (IC50 = 0.096 ± 0.012 µM) and MOLM-13 (IC50 = 0.072 ± 0.014 µM) cells, and induced apoptosis of MV4-11 cells. Western-blot analysis showed that PROTAC 13i triple CK1α and CDK7/9 protein degradation resulted in the significantly increased expression of p53. At the same time, the transcriptional repression due to the degradation significantly reduced downstream gene expression of MYC, MDM2, BCL-2 and MCL-1, and reduced the inflammatory cytokine levels of TNF-α, IL-1ß and IL-6 in PMBCs. These results indicate the beneficial impact of simultaneous CK1α and CDK7/9 degradation for acute myeloid leukemia therapy.

Design and Development of IKZF2 and CK1α Dual Degraders.

Lenalidomide achieves its therapeutic efficacy by recruiting and removing proteins of therapeutic interest through the E3 ligase substrate adapter cereblon. Here, we report the design and characterization of 81 cereblon ligands for their ability to degrade the transcription factor Helios (IKZF2) and casein kinase 1 alpha (CK1α). We identified a key naphthamide scaffold that depleted both intended targets in acute myeloid leukemia MOLM-13 cells. Structure-activity relationship studies for degradation of the desired targets over other targets (IKZF1, GSPT1) afforded an initial lead compound DEG-35. A subsequent scaffold replacement campaign identified DEG-77, which selectively degrades IKZF2 and CK1α, and possesses suitable pharmacokinetic properties, solubility, and selectivity for in vivo studies. Finally, we show that DEG-77 has antiproliferative activity in the diffuse large B cell lymphoma cell line OCI-LY3 and the ovarian cancer cell line A2780 indicating that the dual degrader strategy may have efficacy against additional types of cancer.

Metabolic exhaustion and casein kinase 1α drive deguelin-induced premature red blood cell death.

1. Deguelin (DGN), a retinoid isolated from many plants, exhibits a potent anticancer activity against a wide spectrum of tumour cells. There is a dearth of evidence, however, regarding the toxicity of DGN to red blood cells (RBCs). This is relevant given the prevalent chemotherapy-associated anaemia observed in cancer patients.2. RBCs were exposed to 1-100 µM of DGN for 24 h at 37 °C. Haemolysis and related markers were photometrically measured while flow cytometry was employed to detect phosphatidylserine exposure through Annexin-V-FITC binding and light scatter properties. Additionally, cytosolic Ca2+ and reactive oxygen species were quantified using Fluo4/AM and H2DCFDA, respectively. DGN was also tested against specific signalling inhibitors in addition to vitamin C and ATP.3. DGN caused a significant increase in Annexin-V-positive cells which was accompanied by cell shrinkage without Ca2+ elevation or oxidative stress. DGN also elicited dose-responsive haemolysis which was ameliorated by preventing KCl efflux and in the presence of sucrose, D4476, and ATP. In whole blood, DGN significantly reduced the reticulocyte count and increased platelet distribution width and large cell count.4. DGN triggers premature RBC eryptosis and haemolysis through casein kinase 1α and ATP depletion, and exhibits a specific toxicity towards reticulocytes and platelets.

A kinome-wide CRISPR screen identifies CK1α as a target to overcome enzalutamide resistance of prostate cancer.

Enzalutamide (ENZA), a second-generation androgen receptor antagonist, has significantly increased progression-free and overall survival of patients with metastatic prostate cancer (PCa). However, resistance remains a prominent obstacle in treatment. Utilizing a kinome-wide CRISPR-Cas9 knockout screen, we identified casein kinase 1α (CK1α) as a therapeutic target to overcome ENZA resistance. Depletion or pharmacologic inhibition of CK1α enhanced ENZA efficacy in ENZA-resistant cells and patient-derived xenografts. Mechanistically, CK1α phosphorylates the serine residue S1270 and modulates the protein abundance of ataxia telangiectasia mutated (ATM), a primary initiator of DNA double-strand break (DSB)-response signaling, which is compromised in ENZA-resistant cells and patients. Inhibition of CK1α stabilizes ATM, resulting in the restoration of DSB signaling, and thus increases ENZA-induced cell death and growth arrest. Our study details a therapeutic approach for ENZA-resistant PCa and characterizes a particular perspective for the function of CK1α in the regulation of DNA-damage response.

Dual IKZF2 and CK1α degrader targets acute myeloid leukemia cells.

Acute myeloid leukemia (AML) is a hematologic malignancy for which several epigenetic regulators have been identified as therapeutic targets. Here we report the development of cereblon-dependent degraders of IKZF2 and casein kinase 1α (CK1α), termed DEG-35 and DEG-77. We utilized a structure-guided approach to develop DEG-35 as a nanomolar degrader of IKZF2, a hematopoietic-specific transcription factor that contributes to myeloid leukemogenesis. DEG-35 possesses additional substrate specificity for the therapeutically relevant target CK1α, which was identified through unbiased proteomics and a PRISM screen assay. Degradation of IKZF2 and CK1α blocks cell growth and induces myeloid differentiation in AML cells through CK1α-p53- and IKZF2-dependent pathways. Target degradation by DEG-35 or a more soluble analog, DEG-77, delays leukemia progression in murine and human AML mouse models. Overall, we provide a strategy for multitargeted degradation of IKZF2 and CK1α to enhance efficacy against AML that may be expanded to additional targets and indications.

Casein Kinase 1α Regulates Testosterone Synthesis and Testis Development in Adult Mice.

Casein kinase 1α (CK1α) is a main component of the Wnt/ß-catenin signaling pathway, which participates in multiple biological processes. Our recent study demonstrated that CK1α is expressed in both germ cells and somatic cells of mouse testes and regulates spermatogenesis. However, little information is known about the role of CK1α in regulating the development of somatic cells in mouse testes. Our results demonstrated that conditional disruption of CK1α in murine Leydig cells sharply decreased testosterone levels; markedly affected testis development, sperm motility, and sperm morphology; and caused subfertility. The germ cell population was partially decreased in CK1α conditional knockout (cKO) mice, while the proliferation of Leydig cells and Sertoli cells was not affected. Furthermore, in vitro results verified that luteinizing hormone upregulates CK1α through the luteinizing hormone/protein kinase/Epidermal Growth Factor Receptor/extracellular regulated protein kinases/2 signaling pathway and that CK1α interacts with and phosphorylates EGFR, which subsequently activates the phosphorylation of ERK1/2, thereby promoting testosterone synthesis. In addition, high-dose testosterone propionate partially rescued the phenotype observed in cKO mice. This study provides new insights into the role of CK1α in steroidogenesis and male reproduction.

Casein kinase 1α mediates eryptosis: a review.

Eryptosis is a coordinated non-lytic cell death of erythrocytes characterized by cell shrinkage, cell membrane scrambling, Ca2+ influx, ceramide accumulation, oxidative stress, activation of calpain and caspases. Physiologically, it aims at removing damaged or aged erythrocytes from circulation. A plethora of diseases are associated with enhanced eryptosis, including metabolic diseases, cardiovascular pathology, renal and hepatic diseases, hematological disorders, systemic autoimmune pathology, and cancer. This makes eryptosis and eryptosis-regulating signaling pathways a target for therapeutic interventions. This review highlights the eryptotic signaling machinery containing several protein kinases and its small molecular inhibitors with a special emphasis on casein kinase 1α (CK1α), a serine/threonine protein kinase with a broad spectrum of activity. In this review article, we provide a critical analysis of the regulatory role of CK1α in eryptosis, highlight triggers of CK1α-mediated suicidal death of red blood cells, cover the knowledge gaps in understanding CK1α-driven eryptosis and discover the opportunity of CK1α-targeted pharmacological modulation of eryptosis. Moreover, we discuss the directions of future research focusing on uncovering crosstalks between CK1α and other eryptosis-regulating kinases and pathways.

Knockdown of Csnk1a1 results in preimplantation developmental arrest in mice.

Casein kinase 1, alpha 1 (CSNK1A1), is a member of the highly conserved serine/threonine protein kinase family. This study was established to analyze the expression and localization of CSNK1A1 and its function in early embryonic development in mice. Csnk1a1 mRNA and protein are expressed in multiple mouse tissues including the ovary. After ovulation and fertilization, Csnk1a1 mRNA and protein were detected in preimplantation embryos and their expression was highest in two-cell-stage embryos. CSNK1A1 protein was also mainly localized in the cytoplasm of preimplantation embryos. Moreover, knockdown of Csnk1a1 in zygotes led to a significant decrease in the rate of blastocyst formation. Furthermore, treatment of zygotes with the CSNK1A1-specific inhibitor D4476 also resulted in embryonic developmental arrest. These results provide the first evidence for a novel function of CSNK1A1 in early embryonic development in mice.

CK1α upregulates the IFNAR1 expression to prompt the anti-HBV effect of type I IFN in hepatoma carcinoma cells.

Casein kinase 1α (CK1α) mediates the phosphorylation and degradation of interferon-α/ß receptor 1 (IFNAR1) in response to viral infection. However, how CK1α regulates hepatitis B virus (HBV) replication and the anti-HBV effects of IFN-α are less reported. Here we show that CK1α can interact with IFNAR1 in hepatoma carcinoma cells and increased the abundance of IFNAR1 by reducing the ubiquitination levels in the presence of HBV. Furthermore, CK1α promotes the IFN-α triggered JAK-STAT signaling pathway and consequently enhances the antiviral effects of IFN-α against HBV replication. Our results collectively provide evidence that CK1α positively regulates the anti-HBV activity of IFN-α in hepatoma carcinoma cells, which would be a promising therapeutic target to improve the effectiveness of IFN-α therapy to cure CHB.

The Casein kinase 1α agonist pyrvinium attenuates Wnt-mediated CK1α degradation via interaction with the E3 ubiquitin ligase component Cereblon.

The Cullin-RING ligase 4 E3 ubiquitin ligase component Cereblon (CRBN) is a well-established target for a class of small molecules termed immunomodulatory drugs (IMiDs). These drugs drive CRBN to modulate the degradation of a number of neosubstrates required for the growth of multiple cancers. Whereas the mechanism underlying the activation of CRBN by IMiDs is well described, the normal physiological regulation of CRBN is poorly understood. We recently showed that CRBN is activated following exposure to Wnt ligands and subsequently mediates the degradation of a subset of physiological substrates. Among the Wnt-dependent substrates of CRBN is Casein kinase 1α (CK1α), a known negative regulator of Wnt signaling. Wnt-mediated degradation of CK1α occurs via its association with CRBN at a known IMiD binding pocket. Herein, we demonstrate that a small-molecule CK1α agonist, pyrvinium, directly prevents the Wnt-dependent interaction of CRBN with CK1α, attenuating the consequent CK1α degradation. We further show that pyrvinium disrupts the ability of CRBN to interact with CK1α at the IMiD binding pocket within the CRBN-CK1α complex. Of note, this function of pyrvinium is independent of its previously reported ability to enhance CK1α kinase activity. Furthermore, we also demonstrate that pyrvinium attenuates CRBN-induced Wnt pathway activation in vivo. Collectively, these results reveal a novel dual mechanism through which pyrvinium inhibits Wnt signaling by both attenuating the CRBN-mediated destabilization of CK1α and activating CK1α kinase activity.

Casein kinase 1α regulates murine spermatogenesis via p53-Sox3 signaling.

Casein kinase 1α (CK1α), acting as one member of the ß-catenin degradation complex, negatively regulates the Wnt/ß-catenin signaling pathway. CK1α knockout usually causes both Wnt/ß-catenin and p53 activation. Our results demonstrated that conditional disruption of CK1α in spermatogonia impaired spermatogenesis and resulted in male mouse infertility. The progenitor cell population was dramatically decreased in CK1α conditional knockout (cKO) mice, while the proliferation of spermatogonial stem cells (SSCs) was not affected. Furthermore, our molecular analyses identified that CK1α loss was accompanied by nuclear stability of p53 protein in mouse spermatogonia, and dual-luciferase reporter and chromatin immunoprecipitation assays revealed that p53 directly targeted the Sox3 gene. In addition, the p53 inhibitor pifithrin α (PFTα) partially rescued the phenotype observed in cKO mice. Collectively, our data suggest that CK1α regulates spermatogenesis and male fertility through p53-Sox3 signaling, and they deepen our understanding of the regulatory mechanism underlying the male reproductive system.

Decreased Expressions of CK1α and PTEN in Sinonasal Inverted Papilloma.

To investigate the diagnostic value of casein kinase 1α (CK1α) and phosphatase and tensin homolog (PTEN) in sinonasal inverted papilloma (SNIP), 42 control subjects and 56 SNIP patients were recruited in this study. Demographic and clinical characteristics, computerized tomography scans and endoscopic examinations were analyzed according to the Krouse staging system. Real-time quantitative-polymerase chain reaction and Western blotting were performed to detect CK1α and PTEN expression levels in different subgroups. Receiver operating characteristic and correlation analyses were conducted to assess their clinical significance in SNIP diagnosis. The expression levels of CK1α and PTEN were decreased in SNIP patients. Interestingly, the declined mRNA levels were consistent with the elevated Krouse staging and closely associated with the pathophysiological characteristics. Their expression levels also negatively correlated with neutrophil counts and positively correlated with lymphocyte counts in the blood of SNIP patients. This study suggests that CK1α and PTEN might be useful biomarkers for the occurrence and recurrence diagnosis of SNIP.

S100A16 promotes acute kidney injury by activating HRD1-induced ubiquitination and degradation of GSK3ß and CK1α.

The pathogenesis of acute kidney injury (AKI) is associated with the activation of multiple signaling pathways, including Wnt/ß-catenin signaling. However, the mechanism of Wnt/ß-catenin pathway activation in renal interstitial fibroblasts during AKI is unclear. S100 calcium-binding protein A16 (S100A16), a new member of calcium-binding protein S100 family, is a multi-functional signaling factor involved in various pathogenies, including tumors, glycolipid metabolism disorder, and chronic kidney disease (CKD). We investigated the potential participation of S100A16 in Wnt/ß-catenin pathway activation during AKI by subjecting wild-type (WT) and S100A16 knockout (S100A16+/-) mice to the ischemia-reperfusion injury (IRI), and revealed S100A16 upregulation in this model, in which knockout of S100A16 impeded the Wnt/ß-catenin signaling pathway activation and recovered the expression of downstream hepatocyte growth factor (HGF). We also found that S100A16 was highly expressed in Platelet-derived growth factor receptor beta (PDGFRß) positive renal fibroblasts in vivo. Consistently, in rat renal interstitial fibroblasts (NRK-49F cells), both hypoxia/reoxygenation and S100A16 overexpression exacerbated fibroblasts apoptosis and inhibited HGF secretion; whereas S100A16 knockdown or Wnt/ß-catenin pathway inhibitor ICG-001 reversed these changes. Mechanistically, we showed that S100A16 promoted Wnt/ß-catenin signaling activation via the ubiquitylation and degradation of ß-catenin complex members, glycogen synthase kinase 3ß (GSK3ß) and casein kinase 1α (CK1α), mediated by E3 ubiquitin ligase, the HMG-CoA reductase degradation protein 1 (HRD1). Our study identified the S100A16 as a key regulator in the activation of Wnt/ß-catenin signaling pathway in AKI.

Sequential Phosphorylation of Hepatitis C Virus NS5A Protein Requires the ATP-Binding Domain of NS3 Helicase.

The propagation of the hepatitis C virus (HCV) is regulated in part by the phosphorylation of its nonstructural protein NS5A that undergoes sequential phosphorylation on several highly conserved serine residues and switches from a hypo- to a hyperphosphorylated state. Previous studies have shown that NS5A sequential phosphorylation requires NS3 encoded on the same NS3-NS4A-NS4B-NS5A polyprotein. Subtle mutations in NS3 without affecting its protease activity could affect NS5A phosphorylation. Given the ATPase domain in the NS3 COOH terminus, we tested whether NS3 participates in NS5A phosphorylation similarly to the nucleoside diphosphate kinase-like activity of the rotavirus NSP2 nucleoside triphosphatase (NTPase). Mutations in the NS3 ATP-binding motifs blunted NS5A hyperphosphorylation and phosphorylation at serines 225, 232, and 235, whereas a mutation in the RNA-binding domain did not. The phosphorylation events were not rescued with wild-type NS3 provided in trans. When provided with an NS3 ATPase-compatible ATP analog, N6-benzyl-ATP-γ-S, thiophosphorylated NS5A was detected in the cells expressing the wild-type NS3-NS5B polyprotein. The thiophosphorylation level was lower in the cells expressing NS3-NS5B with a mutation in the NS3 ATP-binding domain. In vitro assays with a synthetic peptide and purified wild-type NS3 followed by dot blotting and mass spectrometry found weak NS5A phosphorylation at serines 222 and 225 that was sensitive to an inhibitor of casein kinase Iα but not helicase. When casein kinase Iα was included in the assay, much stronger phosphorylation was observed at serines 225, 232, and 235. We concluded that NS5A sequential phosphorylation requires the ATP-binding domain of the NS3 helicase and that casein kinase Iα is a potent NS5A kinase. IMPORTANCE For more than 20 years, NS3 was known to participate in NS5A sequential phosphorylation. In the present study, we show for the first time that the ATP-binding domain of NS3 is involved in NS5A phosphorylation. In vitro assays showed that casein kinase Iα is a very potent kinase responsible for NS5A phosphorylation at serines 225, 232, and 235. Our data suggest that ATP binding by NS3 probably results in conformational changes that recruit casein kinase Iα to phosphorylate NS5A, initially at S225 and subsequently at S232 and S235. Our discovery reveals intricate requirements of the structural integrity of NS3 for NS5A hyperphosphorylation and HCV replication.

Defining molecular glues with a dual-nanobody cannabidiol sensor.

"Molecular glue" (MG) is a term coined to describe the mechanism of action of the plant hormone auxin and subsequently used to characterize synthetic small molecule protein degraders exemplified by immune-modulatory imide drugs (IMiDs). Prospective development of MGs, however, has been hampered by its elusive definition and thermodynamic characteristics. Here, we report the crystal structure of a dual-nanobody cannabidiol-sensing system, in which the ligand promotes protein-protein interaction in a manner analogous to auxin. Through quantitative analyses, we draw close parallels among the dual-nanobody cannabidiol sensor, the auxin perception complex, and the IMiDs-bound CRL4CRBN E3, which can bind and ubiquitinate "neo-substrates". All three systems, including the recruitment of IKZF1 and CK1α to CRBN, are characterized by the lack of ligand binding activity in at least one protein partner and an under-appreciated preexisting low micromolar affinity between the two proteinaceous subunits that is enhanced by the ligand to reach the nanomolar range. These two unifying features define MGs as a special class of proximity inducers distinct from bifunctional compounds and can be used as criteria to guide target selection for future rational discovery of MGs.

Development of PDE6D and CK1α Degraders through Chemical Derivatization of FPFT-2216.

Immunomodulatory drugs are a class of drugs approved for the treatment of multiple myeloma. These compounds exert their clinical effects by inducing interactions between the CRL4CRBN E3 ubiquitin ligase and a C2H2 zinc finger degron motif, resulting in degradation of degron-containing targets. However, although many cellular proteins feature the degron motif, only a subset of those are degradable via this strategy. Here, we demonstrated that FPFT-2216, a previously reported "molecular glue" compound, degrades PDE6D, in addition to IKZF1, IKZF3, and CK1α. We used FPFT-2216 as a starting point for a focused medicinal chemistry campaign and developed TMX-4100 and TMX-4116, which exhibit greater selectivity for degrading PDE6D and CK1α, respectively. We also showed that the region in PDE6D that interacts with the FPFT-2216 derivatives is not the previously pursued prenyl-binding pocket. Moreover, we found that PDE6D depletion by FPFT-2216 does not impede the growth of KRASG12C-dependent MIA PaCa-2 cells, highlighting the challenges of drugging PDE6D-KRAS. Taken together, the approach we described here represents a general scheme to rapidly develop selective degraders by reprogramming E3 ubiquitin ligase substrate specificity.

Calcium-oxidative stress signaling axis and casein kinase 1α mediate eryptosis and hemolysis elicited by novel p53 agonist inauhzin.

Inauhzin (INZ) is a novel p53 agonist with antitumor activity. Anemia is a common side effect of chemotherapy and may arise from red blood cell (RBC) hemolysis or eryptosis. In this study, we investigate the mechanisms of INZ toxicity in human RBCs. RBCs were isolated from healthy donors and treated with antitumor concentrations of INZ (5-500 µM) for 24 h at 37 °C. Hemoglobin was photometrically measured, and cells were stained with Annexin-V-FITC for phosphatidylserine (PS), Fluo4/AM for calcium, and 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) for oxidative stress. INZ caused significant dose-responsive, calcium-dependent hemolysis starting at 40 µM. Furthermore, INZ significantly increased Annexin-positive cells and Fluo4 and DCF fluorescence. The cytotoxicity of INZ was also significantly mitigated in presence of D4476. INZ possesses hemolytic and eryptotic potential characterized by cell membrane scrambling, intracellular calcium overload, cell shrinkage, and oxidative stress secondary to calcium influx from the extracellular space.

CK1α protects WAVE from degradation to regulate cell shape and motility in the immune response.

The WAVE regulatory complex (WRC) is the main activator of the Arp2/3 complex, promoting lamellipodial protrusions in migrating cells. The WRC is basally inactive but can be activated by Rac1 and phospholipids, and through phosphorylation. However, the in vivo relevance of the phosphorylation of WAVE proteins remains largely unknown. Here, we identified casein kinase I alpha (CK1α) as a regulator of WAVE, thereby controlling cell shape and cell motility in Drosophila macrophages. CK1α binds and phosphorylates WAVE in vitro. Phosphorylation of WAVE by CK1α appears not to be required for activation but, rather, regulates its stability. Pharmacologic inhibition of CK1α promotes ubiquitin-dependent degradation of WAVE. Consistently, loss of Ck1α but not ck2 function phenocopies the depletion of WAVE. Phosphorylation-deficient mutations in the CK1α consensus sequences within the VCA domain of WAVE can neither rescue mutant lethality nor lamellipodium defects. By contrast, phosphomimetic mutations rescue all cellular and developmental defects. Finally, RNAi-mediated suppression of 26S proteasome or E3 ligase complexes substantially rescues lamellipodia defects in CK1α-depleted macrophages. Therefore, we conclude that basal phosphorylation of WAVE by CK1α protects it from premature ubiquitin-dependent degradation, thus promoting WAVE function in vivo. This article has an associated First Person interview with the first author of the paper.