Members of the casein kinase 1 (CK1) family are important regulators of multiple signaling pathways. CK1α is a well-known negative regulator of the Wnt/β-catenin pathway, which promotes the degradation of β-catenin via its phosphorylation of Ser45. In contrast, the closest paralog of CK1α, CK1α-like, is a poorly characterized kinase of unknown function. In this study, we show that the deletion of CK1α, but not CK1α-like, resulted in a strong activation of the Wnt/β-catenin pathway. Wnt-3a treatment further enhanced the activation, which suggests there are at least two modes, a CK1α-dependent and Wnt-dependent, of β-catenin regulation. Rescue experiments showed that only two out of ten naturally occurring splice CK1α/α-like variants were able to rescue the augmented Wnt/β-catenin signaling caused by CK1α deficiency in cells. Importantly, the ability to phosphorylate β-catenin on Ser45 in the in vitro kinase assay was required but not sufficient for such rescue. Our compound CK1α and GSK3α/β KO models suggest that the additional nonredundant function of CK1α in the Wnt pathway beyond Ser45-β-catenin phosphorylation includes Axin phosphorylation. Finally, we established NanoBRET assays for the three most common CK1α splice variants as well as CK1α-like. Target engagement data revealed comparable potency of known CK1α inhibitors for all CK1α variants but not for CK1α-like. In summary, our work brings important novel insights into the biology of CK1α, including evidence for the lack of redundancy with other CK1 kinases in the negative regulation of the Wnt/β-catenin pathway at the level of β-catenin and Axin.

• MeSH
• Alternative Splicing MeSH
• beta Catenin * metabolism   genetics   MeSH
• Phosphorylation MeSH
• HEK293 Cells MeSH
• Casein Kinase Ialpha * metabolism   genetics   MeSH
• Glycogen Synthase Kinase 3 metabolism   genetics   MeSH
• Glycogen Synthase Kinase 3 beta metabolism   genetics   MeSH
• Humans MeSH
• Wnt3A Protein metabolism   genetics   MeSH
• Wnt Signaling Pathway * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Comparative Study MeSH

Abnormal accumulation of lymphoblasts in the blood and bone marrow is the main characteristic of acute lymphoblastic leukaemia (ALL). Glucocorticoids are effective drugs for ALL, while glucocorticoid resistance is an obstacle to ALL therapy. MicroRNAs (miRNAs) are implicated in the drug resistance and modulate the response of ALL to glucocorticoids. The role of miR-503 in glucocorticoid sensitivity of ALL was investigated in this study. Firstly, T-leukaemic cells were isolated from patients with ALL. The human ALL cell line (CCRF/CEM) was incubated with dexamethasone to establish a glucocorticoid- resistant ALL cell line (CCRF/CEM-R). Data from MTT showed that IC50 (50% inhibitory concentration) of dexamethasone in T-leukaemic cells isolated from glucocorticoid-resistant ALL patients or CCRF/CEM-R was increased compared with IC50 in T-leukaemic cells isolated from glucocorticoid- sensitive ALL patients or CCRF/CEM. MiR- 503 was down-regulated in glucocorticoid-resistant leukaemic cells and CCRF/CEM-R. Secondly, overexpression of miR-503 sensitized CCRF/CEM-R to dexamethasone. Moreover, over-expression of miR- 503 also promoted the sensitivity of ALL cells to dexamethasone. Thirdly, miR-503 bound to WNT3A mRNA and negatively regulated the expression of WNT3A. Over-expression of miR-503 reduced protein expression of nuclear β-catenin, and over-expression of WNT3A attenuated the miR-503 overexpression- induced decrease in nuclear β-catenin. Lastly, the over-expression of miR-503-induced increased sensitivity of ALL-resistant cells and CCRF/ CEM-R to dexamethasone was attenuated by overexpression of WNT3A. In conclusion, miR-503 targeted WNT3A mRNA to sensitize ALL cells to glucocorticoids through inactivation of the Wnt/β-catenin pathway.

• MeSH
• Precursor Cell Lymphoblastic Leukemia-Lymphoma * drug therapy   genetics   metabolism   MeSH
• beta Catenin MeSH
• Dexamethasone pharmacology   therapeutic use   MeSH
• Glucocorticoids pharmacology   therapeutic use   MeSH
• Humans MeSH
• RNA, Messenger MeSH
• MicroRNAs * genetics   metabolism   MeSH
• Wnt3A Protein therapeutic use   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Regulation of phosphatidylinositol phosphates plays a crucial role in signal transduction, membrane trafficking or autophagy. Members of the myotubularin family of lipid phosphatases contribute to phosphoinositide metabolism by counteracting the activity of phosphoinositide kinases. The mechanisms determining their subcellular localization and targeting to specific membrane compartments are still poorly understood. We show here that the inactive phosphatase MTMR9 localizes to the intermediate compartment and to the Golgi apparatus and is able to recruit its active phosphatase partners MTMR6 and MTMR8 to these locations. Furthermore, MTMR8 and MTMR9 co-localize with the small GTPase RAB1A and regulate its localization. Loss of MTMR9 expression compromises the integrity of the Golgi apparatus and results in altered distribution of RAB1A and actin nucleation-promoting factor WHAMM. Loss or overexpression of MTMR9 leads to decreased rate of protein secretion. We demonstrate that secretion of physiologically relevant cargo exemplified by the WNT3A protein is affected after perturbation of MTMR9 levels.

• MeSH
• Endoplasmic Reticulum metabolism   MeSH
• Exocytosis MeSH
• Golgi Apparatus metabolism   MeSH
• HEK293 Cells MeSH
• HeLa Cells MeSH
• Humans MeSH
• Protein Tyrosine Phosphatases, Non-Receptor genetics   metabolism   MeSH
• Wnt3A Protein metabolism   MeSH
• rab1 GTP-Binding Proteins metabolism   MeSH
• Wnt Signaling Pathway MeSH
• Protein Transport MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Aberrant fibroblast growth factor (FGF) signaling disturbs chondrocyte differentiation in skeletal dysplasia, but the mechanisms underlying this process remain unclear. Recently, FGF was found to activate canonical WNT/β-catenin pathway in chondrocytes via Erk MAP kinase-mediated phosphorylation of WNT co-receptor Lrp6. Here, we explore the cellular consequences of such a signaling interaction. WNT enhanced the FGF-mediated suppression of chondrocyte differentiation in mouse limb bud micromass and limb organ cultures, leading to inhibition of cartilage nodule formation in micromass cultures, and suppression of growth in cultured limbs. Simultaneous activation of the FGF and WNT/β-catenin pathways resulted in loss of chondrocyte extracellular matrix, expression of genes typical for mineralized tissues and alteration of cellular shape. WNT enhanced the FGF-mediated downregulation of chondrocyte proteoglycan and collagen extracellular matrix via inhibition of matrix synthesis and induction of proteinases involved in matrix degradation. Expression of genes regulating RhoA GTPase pathway was induced by FGF in cooperation with WNT, and inhibition of the RhoA signaling rescued the FGF/WNT-mediated changes in chondrocyte cellular shape. Our results suggest that aberrant FGF signaling cooperates with WNT/β-catenin in suppression of chondrocyte differentiation.

• MeSH
• beta Catenin genetics   metabolism   MeSH
• Models, Biological MeSH
• Cell Differentiation drug effects   genetics   MeSH
• Chondrocytes drug effects   metabolism   MeSH
• Cartilage cytology   drug effects   metabolism   MeSH
• Fibroblast Growth Factors pharmacology   MeSH
• Fibroblast Growth Factor 2 pharmacology   MeSH
• HEK293 Cells MeSH
• Limb Buds drug effects   embryology   metabolism   MeSH
• Microscopy, Confocal MeSH
• Rats MeSH
• Cells, Cultured MeSH
• Low Density Lipoprotein Receptor-Related Protein-6 genetics   metabolism   MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• Reverse Transcriptase Polymerase Chain Reaction MeSH
• Wnt3A Protein pharmacology   MeSH
• Wnt Proteins genetics   metabolism   pharmacology   MeSH
• Receptors, Fibroblast Growth Factor genetics   metabolism   MeSH
• Signal Transduction drug effects   genetics   MeSH
• Drug Synergism MeSH
• Transcriptome drug effects   genetics   MeSH
• Blotting, Western MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

β-Arrestin is a scaffold protein that regulates signal transduction by seven transmembrane-spanning receptors. Among other functions it is also critically required for Wnt/β-catenin signal transduction. In the present study we provide for the first time a mechanistic basis for the β-arrestin function in Wnt/β-catenin signaling. We demonstrate that β-arrestin is required for efficient Wnt3a-induced Lrp6 phosphorylation, a key event in downstream signaling. β-Arrestin regulates Lrp6 phosphorylation via a novel interaction with phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)-binding protein Amer1/WTX/Fam123b. Amer1 has been shown very recently to bridge Wnt-induced and Dishevelled-associated PtdIns(4,5)P2 production to the phosphorylation of Lrp6. Using fluorescence recovery after photobleaching we show here that β-arrestin is required for the Wnt3a-induced Amer1 membrane dynamics and downstream signaling. Finally, we show that β-arrestin interacts with PtdIns kinases PI4KIIα and PIP5KIβ. Importantly, cells lacking β-arrestin showed higher steady-state levels of the relevant PtdInsP and were unable to increase levels of these PtdInsP in response to Wnt3a. In summary, our data show that β-arrestins regulate Wnt3a-induced Lrp6 phosphorylation by the regulation of the membrane dynamics of Amer1. We propose that β-arrestins via their scaffolding function facilitate Amer1 interaction with PtdIns(4,5)P2, which is produced locally upon Wnt3a stimulation by β-arrestin- and Dishevelled-associated kinases.

• MeSH
• Adaptor Proteins, Signal Transducing genetics   metabolism   MeSH
• Arrestins genetics   metabolism   MeSH
• Cell Membrane metabolism   MeSH
• Embryo, Mammalian cytology   MeSH
• Fibroblasts cytology   metabolism   MeSH
• Phosphatidylinositol 4,5-Diphosphate metabolism   MeSH
• Phosphoproteins genetics   metabolism   MeSH
• Phosphorylation MeSH
• Phosphotransferases (Alcohol Group Acceptor) genetics   metabolism   MeSH
• HEK293 Cells MeSH
• Microscopy, Confocal MeSH
• Cells, Cultured MeSH
• Low Density Lipoprotein Receptor-Related Protein-6 genetics   metabolism   MeSH
• Humans MeSH
• Mice, Knockout MeSH
• Mice MeSH
• Tumor Suppressor Proteins genetics   metabolism   MeSH
• Wnt3A Protein genetics   metabolism   MeSH
• RNA Interference MeSH
• Protein Binding MeSH
• Blotting, Western MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

In mammalian dentate gyrus subgranular zone, the addition of new neurons throughout adulthood is a remarkable form of structural plasticity. Yet, the molecular controls over subgranular zone neural stem cell proliferation, survival, and differentiation are poorly understood. In this study we analysed the expression of Wnt 3a, β-catenin, cyclin D1 and proliferating cell nuclear antigen in mouse subgranular zone to elucidate the involvement of Wnt pathway in subgranular zone neural stem cell proliferation. We performed immunohistochemistry and RT-PCR for the above molecules on adult and postnatal developing hippocampal tissues of mice, respectively. RT-PCR analysis showed a gradual increase in expression of mRNA of Wnt 3a, β-catenin, cyclin D1 and proliferating cell nuclear antigen as the postnatal hippocampus developed, and immunohistochemical analysis showed a highly positive immunoreactive expression for Wnt 3a, β-catenin, cyclin D1 and proliferating cell nuclear antigen in the subgranular zone cells. Together, our data suggested that the Wnt pathway is activated in subgranular zone and could play an important role in regulating subgranular zone neural stem cell proliferation in mouse hippocampus.

• MeSH
• beta Catenin genetics   metabolism   MeSH
• Cyclin D1 genetics   metabolism   MeSH
• Dentate Gyrus metabolism   MeSH
• Hippocampus metabolism   MeSH
• Immunohistochemistry MeSH
• Mice MeSH
• Neural Stem Cells cytology   metabolism   MeSH
• Cell Proliferation MeSH
• Proliferating Cell Nuclear Antigen genetics   metabolism   MeSH
• Wnt3A Protein genetics   metabolism   MeSH
• Wnt Signaling Pathway physiology   genetics   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Research Support, Non-U.S. Gov't MeSH

The Wnt family of proteins is a group of extracellular signalling molecules that regulate cell-fate decisions in developing and adult tissues. It is presumed that all 19 mammalian Wnt family members contain two types of post-translational modification: the covalent attachment of fatty acids at two distinct positions, and the N-glycosylation of multiple asparagines. We examined how these modifications contribute to the secretion, extracellular movement and signalling activity of mouse Wnt1 and Wnt3a ligands. We revealed that O-linked acylation of serine is required for the subsequent S-palmitoylation of cysteine. As such, mutant proteins that lack the crucial serine residue are not lipidated. Interestingly, although double-acylation of Wnt1 was indispensable for signalling in mammalian cells, in Xenopus embryos the S-palmitoyl-deficient form retained the signalling activity. In the case of Wnt3a, the functional duality of the attached acyls was less prominent, since the ligand lacking S-linked palmitate was still capable of signalling in various cellular contexts. Finally, we show that the signalling competency of both Wnt1 and Wnt3a is related to their ability to associate with the extracellular matrix.

• MeSH
• Cell Line MeSH
• Cysteine metabolism   MeSH
• Embryonic Development MeSH
• Rats MeSH
• Humans MeSH
• Lipoylation MeSH
• Molecular Sequence Data MeSH
• Mutation MeSH
• Mice MeSH
• Wnt1 Protein genetics   metabolism   MeSH
• Wnt3 Protein MeSH
• Wnt3A Protein MeSH
• Wnt Proteins genetics   metabolism   MeSH
• Xenopus Proteins MeSH
• Amino Acid Sequence MeSH
• Serine metabolism   MeSH
• Amino Acid Substitution MeSH
• Xenopus embryology   metabolism   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH