Gingival proteomics reveals the role of TGF beta and YAP/TAZ signaling in Raine syndrome fibrosis.

Raine syndrome (RNS) is a rare autosomal recessive osteosclerotic dysplasia. RNS is caused by loss-of-function disease-causative variants of the FAM20C gene that encodes a kinase that phosphorylates most of the secreted proteins found in the body fluids and extracellular matrix. The most common RNS clinical features are generalized osteosclerosis, facial dysmorphism, intracerebral calcifications and respiratory defects. In non-lethal RNS forms, oral traits include a well-studied hypoplastic amelogenesis imperfecta (AI) and a much less characterized gingival phenotype. We used immunomorphological, biochemical, and siRNA approaches to analyze gingival tissues and primary cultures of gingival fibroblasts of two unrelated, previously reported RNS patients. We showed that fibrosis, pathological gingival calcifications and increased expression of various profibrotic and pro-osteogenic proteins such as POSTN, SPARC and VIM were common findings. Proteomic analysis of differentially expressed proteins demonstrated that proteins involved in extracellular matrix (ECM) regulation and related to the TGFß/SMAD signaling pathway were increased. Functional analyses confirmed the upregulation of TGFß/SMAD signaling and subsequently uncovered the involvement of two closely related transcription cofactors important in fibrogenesis, Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ). Knocking down of FAM20C confirmed the TGFß-YAP/TAZ interplay indicating that a profibrotic loop enabled gingival fibrosis in RNS patients. In summary, our in vivo and in vitro data provide a detailed description of the RNS gingival phenotype. They show that gingival fibrosis and calcifications are associated with, and most likely caused by excessed ECM production and disorganization. They furthermore uncover the contribution of increased TGFß-YAP/TAZ signaling in the pathogenesis of the gingival fibrosis.

Knocking out FAM20C in pre-osteoblasts leads to up-regulation of osteoclast differentiation to affect long bone development.

Family with sequence similarity 20 member C (FAM20C) is a Golgi casein kinase that phosphorylates extracellularly-secreted regulatory proteins involved in bone development and mineralization, but its specific role in bone development is still largely unknown. In this study, to examine the specific mechanisms that FAM20C influences bone development, we cross-bred Osx-Cre with FAM20Cflox/flox mice to establish a Osx-Cre; FAM20Cflox/flox knockout (oKO) mouse model; FAM20C was KO in pre-osteoblasts. oKO development was examined at 1-10 weeks, in which compared to control FAM20Cflox/flox, they had lower body weights and bone tissue mineralization. Furthermore, oKO had lower bone volume fractions, thickness, and trabecular numbers, along with higher degrees of trabecular separation. These mice also had decreased femoral metaphyseal cartilage proliferation layer, along with thickened hypertrophic layer and increased apoptotic cell counts. Transcriptomic analysis found that differentially-expressed genes in oKO were concentrated in the osteoclast differentiation pathway, in line with increased osteoclast presence. Additionally, up-regulation of osteoclast-related, and down-regulation of osteogenesis-related genes, were identified, in which the most up-regulated genes were signal regulatory protein ß-1 family (Sirpb1a-c) and mitogen-activated protein kinase 13. Overall, FAM20C KO in pre-osteoblasts leads to abnormal long bone development, likely due to subsequent up-regulation of osteoclast differentiation-associated genes.

Raine syndrome: Prenatally identified severe craniofacial phenotype with multisuture synostosis and brain abnormalities associated with variants in FAM20C.

Raine syndrome (MIM 259775) is a rare autosomal recessive disorder, first described by Raine et al. in 1989, with an estimated prevalence of <1/1,000,000. This is due to pathogenic variants in FAM20C characterized by osteosclerosis, typical craniofacial features, and brain calcifications. Here, we report a novel variant in FAM20C, describe a uniquely severe craniofacial and CNS phenotype of Raine syndrome, and correlate it with prenatal findings. Fetal phenotyping was based on ultrasound and MRI. Solo exome sequencing was performed from DNA extracted from postmortem skin biopsy. Targeted parental variant testing was subsequently performed. A homozygous missense variant NM_020223.4 (c.1445 G > A (p.Gly482Glu)) was identified in FAM20C associated with Raine syndrome. The infant had the characteristic dysmorphic features seen in Raine syndrome. He had particularly significant CNS manifestations consisting of multisuture craniosynostosis with protrusion of the brain parenchyma through fontanelles and cranial lacunae. Histological sections of the brain showed marked periventricular gliosis with regions of infarction, hemorrhage, and cavitation with global periventricular leukomalacia. Numerous dystrophic calcifications were diffusely present. Here, we demonstrate the identification of a novel variant in FAM20C in an infant with the characteristic features seen in Raine syndrome. The patient expands the characteristic phenotype of Raine syndrome to include a uniquely severe CNS phenotype, first identified on prenatal imaging.

Casein Kinase 1 Phosphomimetic Mutations Negatively Impact Connexin-43 Gap Junctions in Human Pluripotent Stem Cell-Derived Cardiomyocytes.

The transplantation of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) has shown promise in preclinical models of myocardial infarction, but graft myocardium exhibits incomplete host-graft electromechanical integration and a propensity for pro-arrhythmic behavior. Perhaps contributing to this situation, hPSC-CM grafts show low expression of connexin 43 (Cx43), the major gap junction (GJ) protein, in ventricular myocardia. We hypothesized that Cx43 expression and function could be rescued by engineering Cx43 in hPSC-CMs with a series of phosphatase-resistant mutations at three casein kinase 1 phosphorylation sites (Cx43-S3E) that have been previously reported to stabilize Cx43 GJs and reduce arrhythmias in transgenic mice. However, contrary to our predictions, transgenic Cx43-S3E hPSC-CMs exhibited reduced Cx43 expression relative to wild-type cells, both at baseline and following ischemic challenge. Cx43-S3E hPSC-CMs showed correspondingly slower conduction velocities, increased automaticity, and differential expression of other connexin isoforms and various genes involved in cardiac excitation-contraction coupling. Cx43-S3E hPSC-CMs also had phosphorylation marks associated with Cx43 GJ internalization, a finding that may account for their impaired GJ localization. Taken collectively, our data indicate that the Cx43-S3E mutation behaves differently in hPSC-CMs than in adult mouse ventricular myocytes and that multiple biological factors likely need to be addressed synchronously to ensure proper Cx43 expression, localization, and function.

Parallel Nonfunctionalization of CK1δ/ε Kinase Ohnologs Following a Whole-Genome Duplication Event.

Whole-genome duplication (WGD) followed by speciation allows us to examine the parallel evolution of ohnolog pairs. In the yeast family Saccharomycetaceae, HRR25 is a rare case of repeated ohnolog maintenance. This gene has reverted to a single copy in Saccharomyces cerevisiae where it is now essential, but has been maintained as pairs in at least 7 species post-WGD. In S. cerevisiae, HRR25 encodes the casein kinase 1δ/ε and plays a role in a variety of functions through its kinase activity and protein-protein interactions (PPIs). We hypothesized that the maintenance of duplicated HRR25 ohnologs could be a result of repeated subfunctionalization. We tested this hypothesis through a functional complementation assay in S. cerevisiae, testing all pairwise combinations of 25 orthologs (including 7 ohnolog pairs). Contrary to our expectations, we observed no cases of pair-dependent complementation, which would have supported the subfunctionalization hypothesis. Instead, most post-WGD species have one ohnolog that failed to complement, suggesting their nonfunctionalization or neofunctionalization. The ohnologs incapable of complementation have undergone more rapid protein evolution, lost most PPIs that were observed for their functional counterparts and singletons from post-WGD and non-WGD species, and have nonconserved cellular localization, consistent with their ongoing loss of function. The analysis in Naumovozyma castellii shows that the noncomplementing ohnolog is expressed at a lower level and has become nonessential. Taken together, our results indicate that HRR25 orthologs are undergoing gradual nonfunctionalization.

Casein kinase 1 and 2 phosphorylate Argonaute proteins to regulate miRNA-mediated gene silencing.

MicroRNAs (miRNAs) together with Argonaute (AGO) proteins form the core of the RNA-induced silencing complex (RISC) to regulate gene expression of their target RNAs post-transcriptionally. Argonaute proteins are subjected to intensive regulation via various post-translational modifications that can affect their stability, silencing efficacy and specificity for targeted gene regulation. We report here that in Caenorhabditis elegans, two conserved serine/threonine kinases - casein kinase 1 alpha 1 (CK1A1) and casein kinase 2 (CK2) - regulate a highly conserved phosphorylation cluster of 4 Serine residues (S988:S998) on the miRNA-specific AGO protein ALG-1. We show that CK1A1 phosphorylates ALG-1 at sites S992 and S995, while CK2 phosphorylates ALG-1 at sites S988 and S998. Furthermore, we demonstrate that phospho-mimicking mutants of the entire S988:S998 cluster rescue the various developmental defects observed upon depleting CK1A1 and CK2. In humans, we show that CK1A1 also acts as a priming kinase of this cluster on AGO2. Altogether, our data suggest that phosphorylation of AGO within the cluster by CK1A1 and CK2 is required for efficient miRISC-target RNA binding and silencing.

Data-independent acquisition-based global phosphoproteomics reveal the diverse roles of casein kinase 1 in plant development.

Casein kinase 1 (CK1) is serine/threonine protein kinase highly conserved among eukaryotes, and regulates multiple developmental and signaling events through phosphorylation of target proteins. Arabidopsis early flowering 1 (EL1)-like (AELs) are plant-specific CK1s with varied functions, but identification and validation of their substrates is a major bottleneck in elucidating their physiological roles. Here, we conducted a quantitative phosphoproteomic analysis in data-independent acquisition mode to systematically identify CK1 substrates. We extracted proteins from seedlings overexpressing individual AEL genes (AEL1/2/3/4-OE) or lacking AEL function (all ael single mutants and two triple mutants) to identify the high-confidence phosphopeptides with significantly altered abundance compared to wild-type Col-0. Among these, we selected 3985 phosphopeptides with higher abundance in AEL-OE lines or lower abundance in ael mutants compared with Col-0 as AEL-upregulated phosphopeptides, and defined 1032 phosphoproteins. Eight CK1s substrate motifs were enriched among AEL-upregulated phosphopeptides and verified, which allowed us to predict additional candidate substrates and functions of CK1s. We functionally characterized a newly identified substrate C3H17, a CCCH-type zinc finger transcription factor, through biochemical and genetic analyses, revealing a role for AEL-promoted C3H17 protein stability and transactivation activity in regulating embryogenesis. As CK1s are highly conserved across eukaryotes, we searched the rice, mouse, and human protein databases using newly identified CK1 substrate motifs, yielding many more candidate substrates than currently known, largely expanding our understanding of the common and distinct functions exerted by CK1s in Arabidopsis and humans, facilitating future mechanistic studies of CK1-mediated phosphorylation in different species.

Potential Role of Protein Kinase FAM20C on the Brain in Raine Syndrome, an In Silico Analysis.

FAM20C (family with sequence similarity 20, member C) is a serine/threonine-specific protein kinase that is ubiquitously expressed and mainly associated with biomineralization and phosphatemia regulation. It is mostly known due to pathogenic variants causing its deficiency, which results in Raine syndrome (RNS), a sclerosing bone dysplasia with hypophosphatemia. The phenotype is recognized by the skeletal features, which are related to hypophosphorylation of different FAM20C bone-target proteins. However, FAM20C has many targets, including brain proteins and the cerebrospinal fluid phosphoproteome. Individuals with RNS can have developmental delay, intellectual disability, seizures, and structural brain defects, but little is known about FAM20C brain-target-protein dysregulation or about a potential pathogenesis associated with neurologic features. In order to identify the potential FAM20C actions on the brain, an in silico analysis was conducted. Structural and functional defects reported in RNS were described; FAM20C targets and interactors were identified, including their brain expression. Gene ontology of molecular processes, function, and components was completed for these targets, as well as for potential involved signaling pathways and diseases. The BioGRID and Human Protein Atlas databases, the Gorilla tool, and the PANTHER and DisGeNET databases were used. Results show that genes with high expression in the brain are involved in cholesterol and lipoprotein processes, plus axo-dendritic transport and the neuron part. These results could highlight some proteins involved in the neurologic pathogenesis of RNS.

PERIOD phosphorylation leads to feedback inhibition of CK1 activity to control circadian period.

PERIOD (PER) and Casein Kinase 1δ regulate circadian rhythms through a phosphoswitch that controls PER stability and repressive activity in the molecular clock. CK1δ phosphorylation of the familial advanced sleep phase (FASP) serine cluster embedded within the Casein Kinase 1 binding domain (CK1BD) of mammalian PER1/2 inhibits its activity on phosphodegrons to stabilize PER and extend circadian period. Here, we show that the phosphorylated FASP region (pFASP) of PER2 directly interacts with and inhibits CK1δ. Co-crystal structures in conjunction with molecular dynamics simulations reveal how pFASP phosphoserines dock into conserved anion binding sites near the active site of CK1δ. Limiting phosphorylation of the FASP serine cluster reduces product inhibition, decreasing PER2 stability and shortening circadian period in human cells. We found that Drosophila PER also regulates CK1δ via feedback inhibition through the phosphorylated PER-Short domain, revealing a conserved mechanism by which PER phosphorylation near the CK1BD regulates CK1 kinase activity.

Casein kinase 1 gamma regulates oxidative stress response via interacting with the NADPH dual oxidase complex.

Oxidative stress response is a fundamental biological process mediated by conserved mechanisms. The identities and functions of some key regulators remain unknown. Here, we report a novel role of C. elegans casein kinase 1 gamma CSNK-1 (also known as CK1γ or CSNK1G) in regulating oxidative stress response and ROS levels. csnk-1 interacted with the bli-3/tsp-15/doxa-1 NADPH dual oxidase genes via genetic nonallelic noncomplementation to affect C. elegans survival in oxidative stress. The genetic interaction was supported by specific biochemical interactions between DOXA-1 and CSNK-1 and potentially between their human orthologs DUOXA2 and CSNK1G2. Consistently, CSNK-1 was required for normal ROS levels in C. elegans. CSNK1G2 and DUOXA2 each can promote ROS levels in human cells, effects that were suppressed by a small molecule casein kinase 1 inhibitor. We also detected genetic interactions between csnk-1 and skn-1 Nrf2 in oxidative stress response. Together, we propose that CSNK-1 CSNK1G defines a novel conserved regulatory mechanism for ROS homeostasis.

Intracranial calcification in Fam20c-deficient mice recapitulates human Raine syndrome.

FAM20C (family with sequence similarity 20-member C) is a protein kinase that phosphorylates secretory proteins, including the proteins that are essential to the formation and mineralization of calcified tissues. FAM20C loss-of-function mutations cause Raine syndrome in humans, characterized by generalized osteosclerosis, distinctive craniofacial dysmorphism, along with extensive intracranial calcification. Our previous studies revealed that inactivation of Fam20c in mice led to hypophosphatemic rickets. In this study, we examined the expression of Fam20c in the mouse brain and investigated brain calcification in Fam20c-deficient mice. Reverse transcription polymerase chain reaction (RT-PCR), Western-blotting and in situ hybridization analyses demonstrated the broad expression of Fam20c in the mouse brain tissue. X-ray and histological analyses showed that the global deletion of Fam20c (mediated by Sox2-cre) resulted in brain calcification in mice after postnatal 3 months and that the calcifications were bilaterally distributed within the brain. There was mild perifocal microgliosis as well as astrogliosis around calcospherites. The calcifications were first observed in the thalamus, and later in the forebrain and hindbrain. Furthermore, brain-specific deletion (mediated by Nestin-cre) of Fam20c in mice also led to cerebral calcification at an older age (postnatal 6 months), but no obvious skeletal or dental defects. Our results suggest that the local loss of FAM20C function in the brain may directly account for intracranial calcification. We propose that FAM20C plays an essential role in maintaining normal brain homeostasis and preventing ectopic brain calcification.

Epigenetic and transcriptional activation of the secretory kinase FAM20C as an oncogene in glioma.

Gliomas are the most prevalent and aggressive malignancies of the nervous system. Previous bioinformatic studies have revealed the crucial role of the secretory pathway kinase FAM20C in the prediction of glioma invasion and malignancy. However, little is known about the pathogenesis of FAM20C in the regulation of glioma. Here, we construct the full-length transcriptome atlas in paired gliomas and observe that 22 genes are upregulated by full-length transcriptome and differential APA analysis. Analysis of ATAC-seq data reveals that both FAM20C and NPTN are the hub genes with chromatin openness and differential expression. Further, in vitro and in vivo studies suggest that FAM20C stimulates the proliferation and metastasis of glioma cells. Meanwhile, NPTN, a novel cancer suppressor gene, counteracts the function of FAM20C by inhibiting both the proliferation and migration of glioma. The blockade of FAM20C by neutralizing antibodies results in the regression of xenograft tumors. Moreover, MAX, BRD4, MYC, and REST are found to be the potential trans-active factors for the regulation of FAM20C. Taken together, our results uncover the oncogenic role of FAM20C in glioma and shed new light on the treatment of glioma by abolishing FAM20C.

Altered sulfation status of FAM20C-dependent chondroitin sulfate is associated with osteosclerotic bone dysplasia.

Raine syndrome, a lethal osteosclerotic bone dysplasia in humans, is caused by loss-of-function mutations in FAM20C; however, Fam20c deficiency in mice does not recapitulate the human disorder, so the underlying pathoetiological mechanisms remain poorly understood. Here we show that FAM20C, in addition to the reported casein kinase activity, also fine-tunes the biosynthesis of chondroitin sulfate (CS) chains to impact bone homeostasis. Specifically, FAM20C with Raine-originated mutations loses the ability to interact with chondroitin 4-O-sulfotransferase-1, and is associated with reduced 4-sulfation/6-sulfation (4S/6S) ratio of CS chains and upregulated biomineralization in human osteosarcoma cells. By contrast, overexpressing chondroitin 6-O-sulfotransferase-1 reduces CS 4S/6S ratio, and induces osteoblast differentiation in vitro and higher bone mineral density in transgenic mice. Meanwhile, a potential xylose kinase activity of FAM20C does not impact CS 4S/6S ratio, and is not associated with Raine syndrome mutations. Our results thus implicate CS 4S/6S ratio imbalances caused by FAM20C mutations as a contributor of Raine syndrome etiology.

circCsnk1g3- and circAnkib1-regulated interferon responses in sarcoma promote tumorigenesis by shaping the immune microenvironment.

Exonic circular RNAs (circRNAs) produce predominantly non-coding RNA species that have been recently profiled in many tumors. However, their functional contribution to cancer progression is still poorly understood. Here, we identify the circRNAs expressed in soft tissue sarcoma cells and explore how the circRNAs regulate sarcoma growth in vivo. We show that circCsnk1g3 and circAnkib1 promote tumor growth by shaping a pro-tumorigenic microenvironment, possibly due to their capabilities to regulate tumor-promoting elements extrinsic to the tumor cells. Accordingly, circCsnk1g3 and circAnkib1 can control the expression of interferon-related genes and pro-inflammatory factors in the sarcoma cells, thus directing immune cell recruitment into the tumor mass, and hence their activation. Mechanistically, circRNAs may repress pro-inflammatory elements by buffering activation of the pathways mediated by RIG-I, the cytosolic viral RNA sensor. The current findings suggest that the targeting of specific circRNAs could augment the efficacy of tumor and immune response to mainstay therapies.

The conserved C-terminal residues of FAM83H are required for the recruitment of casein kinase 1 to the keratin cytoskeleton.

The casein kinase 1 (CK1) family of serine/threonine protein kinases is involved in diverse cellular events at discrete subcellular compartments. FAM83H acts as a scaffold protein that recruits CK1 to the keratin cytoskeleton or to the nuclear speckles, which are storage sites for splicing factors. We determined the amino acid region of FAM83H required for recruiting CK1 to the keratin cytoskeleton. The subcellular localization of mutant FAM83H proteins with deletions of amino acid residues at different positions was evaluated via immunofluorescence. FAM83H mutants with deleted C-terminal residues 1134-1139, which are conserved among vertebrates, lost the ability to localize and recruit CK1 to the keratin cytoskeleton, suggesting that these residues are required for recruiting CK1 to the keratin cytoskeleton. The deletion of these residues (1134-1139) translocated FAM83H and CK1 to the nuclear speckles. Amino acid residues 1 to 603 of FAM83H were determined to contain the region responsible for the recruitment of CK1 to the nuclear speckles. Our results indicated that FAM83H recruits CK1 preferentially to the keratin cytoskeleton and alternatively to the nuclear speckles.

High temperature induces male sterility via MYB66-MYB4-Casein kinase I signaling in cotton.

High temperature (HT) causes male sterility and decreases crop yields. Our previous works have demonstrated that sugar and auxin signaling pathways, Gossypium hirsutum Casein kinase I (GhCKI), and DNA methylation are all involved in HT-induced male sterility in cotton. However, the signaling mechanisms leading to distinct GhCKI expression patterns induced by HT between HT-tolerant and HT-sensitive cotton anthers remain largely unknown. Here, we identified a GhCKI promoter (ProGhCKI) region that functions in response to HT in anthers and found the transcription factor GhMYB4 binds to this region to act as an upstream positive regulator of GhCKI. In the tapetum of early-stage cotton anthers, upregulated expression of GhMYB4 under HT and overexpressed GhMYB4 under normal temperature both led to severe male sterility phenotypes, coupled with enhanced expression of GhCKI. We also found that GhMYB4 interacts with GhMYB66 to form a heterodimer to enhance its binding to ProGhCKI. However, GhMYB66 showed an expression pattern similar to GhMYB4 under HT but did not directly bind to ProGhCKI. Furthermore, HT reduced siRNA-mediated CHH DNA methylations in the GhMYB4 promoter, which enhanced the expression of GhMYB4 in tetrad stage anthers and promoted the formation of the GhMYB4/GhMYB66 heterodimer, which in turn elevated the transcription of GhCKI in the tapetum, leading to male sterility. Overall, we shed light on the GhMYB66-GhMYB4-GhCKI regulatory pathway in response to HT in cotton anthers.

Raine syndrome: Report of a novel mutation and review of the different antenatal imaging modalities used to diagnose this disease.

INTRODUCTION: Raine syndrome is an autosomal recessive disorder characterized mainly by the presence of exophthalmos, choanal atresia or stenosis, osteosclerosis, and cerebral calcifications. There are around 50 cases described in the literature with a prevalence of less than 1/1,000,000. It is secondary to pathogenic variants in the FAM20 C gene, located on chromosome 7p22.3. CASE REPORT: We report a consanguineous family with three affected pregnancies. In the first two, exophthalmos and bone abnormalities were noted, ending in one intra-uterine demise and one neonatal death, without identifying any genetic disorder. During the couple's most recent pregnancy, fetal anomaly sonogram and fetal CT scan revealed microcephaly, intracranial calcifications, exophthalmos, hypertelorism, depressed nasal bridge, midface hypoplasia and thoracic hypoplasia. Fetal blood sampling for whole exome sequencing revealed a novel pathogenic homozygous variant c.1363+1G > A in the FAM20 C gene associated with Raine syndrome. Delivery occurred at 26 weeks of gestation after rupture of membranes followed by neonatal death due to respiratory failure. REVIEW: A review of the distinctive features of Raine syndrome, the contribution of different prenatal imaging modalities (Ultrasound, Computed Tomography and Magnetic Resonance Imaging) in making the diagnosis and the molecular characterization of this disorder is provided.

Protein proximity networks and functional evaluation of the casein kinase 1 gamma family reveal unique roles for CK1γ3 in WNT signaling.

Aberrant activation or suppression of WNT/ß-catenin signaling contributes to cancer initiation and progression, neurodegeneration, and bone disease. However, despite great need and more than 40 years of research, targeted therapies for the WNT pathway have yet to be fully realized. Kinases are considered exceptionally druggable and occupy key nodes within the WNT signaling network, but several pathway-relevant kinases remain understudied and "dark." Here, we studied the function of the casein kinase 1γ (CSNK1γ) subfamily of human kinases and their roles in WNT signaling. miniTurbo-based proximity biotinylation and mass spectrometry analysis of CSNK1γ1, CSNK1γ2, and CSNK1γ3 revealed numerous components of the ß-catenin-dependent and ß-catenin-independent WNT pathways. In gain-of-function experiments, we found that CSNK1γ3 but not CSNK1γ1 or CSNK1γ2 activated ß-catenin-dependent WNT signaling, with minimal effect on other signaling pathways. We also show that within the family, CSNK1γ3 expression uniquely induced low-density lipoprotein receptor-related protein 6 phosphorylation, which mediates downstream WNT signaling transduction. Conversely, siRNA-mediated silencing of CSNK1γ3 alone had no impact on WNT signaling, though cosilencing of all three family members decreased WNT pathway activity. Finally, we characterized two moderately selective and potent small-molecule inhibitors of the CSNK1γ family. We show that these inhibitors and a CSNK1γ3 kinase-dead mutant suppressed but did not eliminate WNT-driven low-density lipoprotein receptor-related protein 6 phosphorylation and ß-catenin stabilization. Our data suggest that while CSNK1γ3 expression uniquely drives pathway activity, potential functional redundancy within the family necessitates loss of all three family members to suppress the WNT signaling pathway.

Ci/Gli Phosphorylation by the Fused/Ulk Family Kinases.

Hedgehog (Hh) signaling culminates in the conversion of the latent transcription factor Cubitus interruptus (Ci)/Gli from a repressor form (CiR/GliR) into an activator form (CiA/GliA). While sequential phosphorylation of Ci/Gli by protein kinase A(PKA), glycogen synthase kinase 3 (GSK3), and casein kinase 1 (CK1) is essential for its proteolytic processing that generates CiR/GliR, sequential phosphorylation of Ci/Gli by the Fused (Fu)/Unc-51 like kinase (Ulk) family kinases Fu/Ulk3/Stk36 and CK1 contributes to the formation of CiA/GliA. Fu/Ulk3/Stk36-mediated phosphorylation of Ci/Gli is stimulated by Hh, leading to altered interaction between Ci/Gli and the Hh pathway repressor Sufu. Here we describe both in vitro and in vivo assays that determine Ci/Gli phosphorylation by the Fu/Ulk family kinases and its regulation by Hh.