The Molecular Chaperone TRiC/CCT Binds to the Trp-Asp (WD) 40-repeat Protein WDR68 and Promotes Its Folding, Protein Kinase DYRK1A-binding, and Nuclear Accumulation [Protein Structure and Folding]

October 22nd, 2014 by Miyata, Y., Shibata, T., Aoshima, M., Tsubata, T., Nishida, E.

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 phospho-proteomic 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 7-bladed β-propeller ring. Experiments with a series of deletion mutants in combination with the structural modelling showed that 3 out of 7 of the 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.
  • Posted in Journal of Biological Chemistry, Publications
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