title: The molecular chaperone TRiC/CCT binds to the Trp-Asp 40 (WD40) repeat protein WDR68 and promotes its folding, protein kinase DYRK1A binding, and nuclear accumulation.
abstract: Trp-Asp (WD) repeat protein 68 (WDR68) is an evolutionarily conserved WD40 repeat protein that binds to several proteins, including dual specificity tyrosine phosphorylation-regulated protein kinase (DYRK1A), MAPK/ERK kinase kinase 1 (MEKK1), and Cullin4-damage-specific DNA-binding protein 1 (CUL4-DDB1). WDR68 affects multiple and diverse physiological functions, such as controlling anthocyanin synthesis in plants, tissue growth in insects, and craniofacial development in vertebrates. However, the biochemical basis and the regulatory mechanism of WDR68 activity remain largely unknown. To better understand the cellular function of WDR68, here we have isolated and identified cellular WDR68 binding partners using a phosphoproteomic approach. More than 200 cellular proteins with wide varieties of biochemical functions were identified as WDR68-binding protein candidates. Eight T-complex protein 1 (TCP1) subunits comprising the molecular chaperone TCP1 ring complex/chaperonin-containing TCP1 (TRiC/CCT) were identified as major WDR68-binding proteins, and phosphorylation sites in both WDR68 and TRiC/CCT were identified. Co-immunoprecipitation experiments confirmed the binding between TRiC/CCT and WDR68. Computer-aided structural analysis suggested that WDR68 forms a seven-bladed β-propeller ring. Experiments with a series of deletion mutants in combination with the structural modeling showed that three of the seven β-propeller blades of WDR68 are essential and sufficient for TRiC/CCT binding. Knockdown of cellular TRiC/CCT by siRNA caused an abnormal WDR68 structure and led to reduction of its DYRK1A-binding activity. Concomitantly, nuclear accumulation of WDR68 was suppressed by the knockdown of TRiC/CCT, and WDR68 formed cellular aggregates when overexpressed in the TRiC/CCT-deficient cells. Altogether, our results demonstrate that the molecular chaperone TRiC/CCT is essential for correct protein folding, DYRK1A binding, and nuclear accumulation of WDR68.
authors.affiliation: From the Department of Cell and Developmental Biology, Graduate School of Biostudies, Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan and ymiyata@lif.kyoto-u.ac.jp.
authors.forename: Yoshihiko
authors.initials: Y
authors.lastname: Miyata
authors.affiliation: K.K. AB Sciex Japan, Tokyo 140-0001, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Shibata
authors.affiliation: K.K. AB Sciex Japan, Tokyo 140-0001, Japan.
authors.forename: Masato
authors.initials: M
authors.lastname: Aoshima
authors.affiliation: K.K. AB Sciex Japan, Tokyo 140-0001, Japan.
authors.forename: Takuichi
authors.initials: T
authors.lastname: Tsubata
authors.affiliation: From the Department of Cell and Developmental Biology, Graduate School of Biostudies, Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan and.
authors.forename: Eisuke
authors.initials: E
authors.lastname: Nishida
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000502:physiology; D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D019556:COS Cells; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism*; D056404:Chaperonin Containing TCP-1 / Q000235:genetics / Q000378:metabolism*; D002522:Chlorocebus aethiops; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D048728:MAP Kinase Kinase Kinase 1 / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D000097603:Dyrk Kinases
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D056404:Chaperonin Containing TCP-1; D004268:DNA-Binding Proteins; D048728:MAP Kinase Kinase Kinase 1; D017346:Protein Serine-Threonine Kinases; D011505:Protein-Tyrosine Kinases; D000097603:Dyrk Kinases; C515143:DCAF7 protein, human; C091435:DDB1 protein, human; C482576:MAP3K1 protein, human
keywords: DYRK1A; Heat Shock Protein (HSP); Molecular Chaperone; Nuclear Translocation; Phosphoproteomics; Protein Phosphorylation; TCP1; TRiC/CCT; WD40 Repeat; WDR68