Biochemical and Biophysical Research Communications
Molecular characterization of human ninein protein: two distinct subdomains required for centrosomal targeting and regulating signals in cell cycle☆
Section snippets
Materials and methods
Plasmid construction. A full-length ninein was constructed to pEGFP C2 vector (Clontech) and fused at the restriction sites BamHI and EcoRI. The N-terminal (1–471 a.a), CCI (461–1193 a.a ), CCII (1303–1930 a.a.), and truncated versions (as described in legend) of CCII domains of ninein were amplified by PCR with the Taq polymerase (TaKaRa). These amplified fragments were digested by restriction enzyme and constructed into pEGFP C2 vector. A full-length centrin was amplified from human testis
The identification of ninein self-binding domains using the yeast two-hybrid system
Previously, we identified a human ninein protein whose C-terminal domain (1303–1930) interacts with GSK3β. Later on, the ninein protein was designated as GSK3β interacting protein (NCBI database, AF212162). To further study the physiological role of ninein, we performed yeast two-hybrid to screen the interaction proteins for binding this coiled-coil II (CCII) region. Here, we report the results of a yeast two-hybrid screen which show that CCII domain could not only interact with CCII domain
Ninein protein complex formation at centrosome may be through its intensive coiled-coil domains
The features of ninein protein include a potential GTP-binding site, a large coiled-coil domain together with four leucine-zipper domains, and a GSK3β-binding site [13]. In general, leucine-zippers have been shown to mediate homo- or heterodimmerization of proteins and to play a role in protein–DNA binding [31]. This motif has also been found to mediate protein–protein interactions in a wide array of proteins. In this study, we report the results of a yeast two-hybrid screen which show that
Acknowledgements
This work was supported by NSC 90-2745-P-037-001 (Taiwan, ROC); NSC 91-3112-B-037-003 to SLH and NSC 91-2320-B-037-040 to YRH.
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hNinein is required for targeting spindle-associated protein Astrin to the centrosome during the S and G2 phases
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