Phosphorylation of CEP83 by TTBK2 is necessary for cilia initiation

© 2019 Lo et al. Primary cilia are microtubule-based organelles that play important roles in development and tissue homeostasis. Tau-tubulin kinase-2 (TTBK2) is genetically linked to spinocerebellar ataxia type 11, and its kinase activity is crucial for ciliogenesis. Although it has been shown that TTBK2 is recruited to the centriole by distal appendage protein CEP164, little is known about TTBK2 substrates associated with its role in ciliogenesis. Here, we perform superresolution microscopy and discover that serum starvation results in TTBK2 redistribution from the periphery toward the root of distal appendages. Our biochemical analyses uncover CEP83 as a bona fide TTBK2 substrate with four phosphorylation sites characterized. We also demonstrate that CEP164-dependent TTBK2 recruitment to distal appendages is required for subsequent CEP83 phosphorylation. Specifically, TTBK2-dependent CEP83 phosphorylation is important for early ciliogenesis steps, including ciliary vesicle docking and CP110 removal. In summary, our results reveal a molecular mechanism of kinase regulation in ciliogenesis and identify CEP83 as a key substrate of TTBK2 during cilia initiation.Ministry of Science and Technology, Taiwan (MOST 105-2628-B-010-004-MY3, MOST 107-2313-B-010-001, MOST 108-2628-B-010-007, MOST 107- 2633-B-009-003, and Shackleton Program Grant); Yen Tjing Ling Medical Foundation (CI-107-17 and CI-108-12); Ministry of Education, Taiwan, Higher Education Sprout Project (107AC-D920); National Core Facility for Biopharmaceuticals, Taiwan, Clinical and Industrial Genomic Application Development Service (MOST 107-2319-B-010-002)

Searching urinary tumor-associated proteins for bladder transitional cell carcinoma in southwestern Taiwan using gel-based proteomics

Background and purpose: We try to search for specific serum or urinary biomarkers for the early detection, follow-up, and prediction of tumor recurrence, progression, and clinical outcome is a difficult task in individuals with bladder cancer. Materials and methods: In this study, urinary samples were dialyzed to remove any interfering molecules and concentration by lyophilization. The urinary proteome maps of 10 healthy volunteers and 10 bladder transitional cell carcinoma (BTTC) patients were explored through two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) coupled with mass spectrometry. With no fractionation, the proteome maps acquired in this study likely represented the total urinary proteins. Results: Comparative proteomics indicated that six proteins were down-regulated and five proteins were up- regulated in BTCC patients as compared with normal. The down-regulated spots were identified as human haptoglobin precursor, human heparan sulfate proteoglycan perlecan, inter-alpha-trypsin inhibitor heavy chain H4 precursor, and AMBP protein precursor. The up-regulated spots were identified as peroxiredoxin 2, heparan sulfate proteoglycan perlecan, protease serine 1 fragment and AMBP protein precursor. Most of these de-regulated proteins were extracellular matrix–associated proteins, which may play roles in regulating the immune response, signal transduction and tumor invasions. Conclusion: In this paper, 11 de-regulated proteins were observed in the urinary specimens of BTTC patients from the southwestern coast of Taiwan where Blackfoot disease is endemic and the unusually high incidence of BTTC in this area might attribute to high arsenic content in the drinking water. It is possible that long-term arsenic-induced alteration of these de-regulated proteins, most of which were extracellularmatrix – (ECM) related proteins which may play roles in regulating the immune response, signal transduction and tumor invasions, might be involved in BTTC development in southwestern Taiwan

ERK1/2-Mediated Phosphorylation of Small Hepatitis Delta Antigen at Serine 177 Enhances Hepatitis Delta Virus Antigenomic RNA Replication ▿

The small hepatitis delta virus (HDV) antigen (SHDAg) plays an essential role in HDV RNA double-rolling-circle replication. Several posttranslational modifications (PTMs) of HDAgs, including phosphorylation, acetylation, and methylation, have been characterized. Among the PTMs, the serine 177 residue of SHDAg is a phosphorylation site, and its mutation preferentially abolishes HDV RNA replication from antigenomic RNA to genomic RNA. Using coimmunoprecipitation analysis, the cellular kinases extracellular signal-related kinases 1 and 2 (ERK1/2) are found to be associated with the Flag-tagged SHDAg mutant (Ser-177 replaced with Cys-177). In an in vitro kinase assay, serine 177 of SHDAg was phosphorylated directly by either Flag-ERK1 or Flag-ERK2. Activation of endogenous ERK1/2 by a constitutively active MEK1 (hemagglutinin-AcMEK1) increased phosphorylation of SHDAg at Ser-177; this phosphorylation was confirmed by immunoblotting using an antibody against phosphorylated S177 and mass spectrometric analysis. Interestingly, we found an increase in the HDV replication from antigenomic RNA to genomic RNA but not in that from genomic RNA to antigenomic RNA. The Ser-177 residue was critical for SHDAg interaction with RNA polymerase II (RNAPII), the enzyme proposed to regulate antigenomic RNA replication. These results demonstrate the role of ERK1/2-mediated Ser-177 phosphorylation in modulating HDV antigenomic RNA replication, possibly through RNAPII regulation. The results may shed light on the mechanisms of HDV RNA replication

Large Hepatitis Delta Antigen Is a Novel Clathrin Adaptor-Like Protein▿

Clathrin-mediated endocytosis is a common pathway for viral entry, but little is known about the direct association of viral protein with clathrin in the cytoplasm. In this study, a putative clathrin box known to be conserved in clathrin adaptors was identified at the C terminus of the large hepatitis delta antigen (HDAg-L). Similar to clathrin adaptors, HDAg-L directly interacted with the N terminus of the clathrin heavy chain through the clathrin box. HDAg-L is a nucleocytoplasmic shuttle protein important for the assembly of hepatitis delta virus (HDV). Here, we demonstrated that brefeldin A and wortmannin, inhibitors of clathrin-mediated exocytosis and endosomal trafficking, respectively, specifically blocked HDV assembly but had no effect on the assembly of the small surface antigen of hepatitis B virus. In addition, cytoplasm-localized HDAg-L inhibited the clathrin-mediated endocytosis of transferrin and the degradation of epidermal growth factor receptor. These results indicate that HDAg-L is a new clathrin adaptor-like protein, and it may be involved in the maturation and pathogenesis of HDV coinfection or superinfection with hepatitis B virus through interaction with clathrin