Molecular characterization of human ninein protein: two distinct subdomains required for centrosomal targeting and regulating signals in cell cycle

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Abstract

The centrosomal protein ninein has been identified as a microtubules minus end capping, centriole position, and anchoring protein, but the true physiological function remains to be determined. In this report, using immunofluorescence analysis and GFP-fusions we show that coiled-coil II domain (CCII domain, 1303–2096) co-localized with γ-tubulin and centrin at the centrosome. We further narrow down within 83 amino acids and classify a new centrosomal targeting signal. Interestingly, antibodies raised against CCII domain reveal that ninein protein declines from spindle poles during mitosis, but reaccumulates at centrosomes at the end of cell division. Moreover, the data also suggest that fragment 1783–1866 may be attributed to declined signal of ninein. In kinase assay, we show that CCII domain could readily be phosphorylated by AIK and PKA. Taken together, our results suggest that ninein protein contains two distinct subdomains which are required for targeting and regulating asymmetry centrosomes. Importantly, the decline of ninein during mitosis implies that this centrosomal protein may play a role to regulate the process of chromosome segregation without discrimination. The model we propose here will foster a clearer picture of how two asymmetric centrosomes could direct and ensure the correct segregation of chromosomes during the mitotic stage.

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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