Centrosomal protein required in the positioning and anchorage of the microtubule minus-end in epithelial cells. May also act as a centrosome maturation factor. May play a role in microtubule nucleation. Overexpression does not perturb nucleation or elongation of microtubules but suppresses release of microtubules.
Centrosome is the major microtubule organizing center in mammalian cells that plays a critical role in a variety of cellular events by the microtubule arrays emanating from it. Despite its significance, the molecular mechanisms underlying the structure and function of the centrosome are still not clear. Herein we describe the identification of three isotypes of human ninein by expression library screening with autoimmune sera from CREST patients. All three ninein isotypes exhibit centrosomal localization throughout the cell cycle when GFP-tagged fusion proteins are expressed transiently in mammalian cells. Construction of serial deletions of GFP-tagged ninein reveals that a stretch of three leucine zippers with a flanking sequence is required and sufficient for centrosomal targeting. Overexpression of ninein results in mislocalization of gamma-tubulin, recruiting it to ectopic (noncentrosomal) ninein-containing sites which are not active in nucleating microtubules. In these cells, nucleation of microtubules from the centrosome is also inhibited. These results thus suggest a regulatory role for ninein in microtubule nucleation.
Interacting selectively and non-covalently with any protein or protein complex (a complex of two or more proteins that may include other nonprotein molecules).
Evidence
1:
Inferred from Physical InteractionHGNC
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 gamma-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.
Evidence
2:
Inferred from Physical InteractionUniProtKB
Int. J. Oncol. 37, 429-436 (2010)[PubMed:20596670]
Aurora-A is involved in chromosome alignment, centrosome maturation, mitotic spindle assembly and regards to an oncogene. Aurora-A is also known to bind to several other proteins affecting its up-regulation or down-regulation and localization. However, how these different binding signals work together to regulate Aurora-A is not properly known. To explore more Aurora-A interacting proteins, the low-copy yeast two-hybrid screening using Aurora-A as bait protein was performed. One novel gene, AIBp, was demonstrated to associate with Aurora-A by the yeast two-hybrid method and in vitro GST pull-down assay. Molecular characterization showed that AIBp possessed a binding site at the C-terminal with Aurora-A (kinase domain). Interestingly, AIBp also interacts with hNinein at the N-terminal, which overlaps with a previously reported hNinein and GSK3beta binding site. Using a kinase assay, AIBp interacts with the Aurora-A kinase domain functions as a positive regulator, whereas AIBp binding to hNinein appears to block the phosphorylation of hNinein by both Aurora-A and GSK3beta. siRNA-mediated elimination of AIBp from HeLa cells, results in a doughnut-like shape, asymmetrical spindle pole and multiple spindle pole formation. We also demonstrated that both AIBp and Aurora-A are co-overexpressed in various brain tumors. These studies demonstrate that AIBp may not only be required for the dynamic movement of Aurora-A at the centrosomes and spindle apparatus during the cell cycle, but may also be important during brain tumorigenesis.
Evidence
3:
Inferred from Physical InteractionHGNC
A centrosomal-associated protein, ninein is a microtubules minus end capping, centrosome position, and anchoring protein, but the underlying structure and physiological functions are still unknown. To identify the molecules that regulate the function of human ninein in centrosome, we performed yeast two-hybrid screen and isolated the SUMO-conjugating E2 enzyme, Ubc9, and SUMOylation enhancing enzymes, including PIAS1 and PIASxalpha, as binding partners of hNinein. These interactions as well as the interaction between hNinein and SUMO-1 are also confirmed by a glutathione S-transferase (GST) pull-down experiment. Furthermore, the C-terminal region of hNinein can be SUMOylated in vitro and in HeLa cells transfected with a plasmid expressing GFP-hNinein. Our findings firstly place SUMOylation target on the centrosome structure protein, hNinein, which results in the switch localization from centrosome to nucleus, suggesting the importance of the SUMOylation of hNinein and probably other centrosomal proteins may also be involved in the centrosome activity.
Evidence
4:
Inferred from Physical InteractionIntAct
Using human glycogen synthase kinase 3beta (GSK-3beta) as bait in the yeast two-hybrid system, we identified a novel human centrosome associated protein, hNinein. When the full length cDNA of hNinein was sequenced, it showed that an open reading frame encoded a protein consisting of 2047 amino acids with a predicted molecular mass of 239 kDa. The features of this protein include a potential GTP binding site, a large coiled-coil domain together with four leucine zipper domains and a GSK-3beta binding site. Fluorescence microscopy experiment showed that hNinein is localized in the pericentriolar matrix of the centrosome. In addition, hNinein also showed to react with centrosomal autoantibody sera. Our findings suggest that hNinein may be involved in the formation of centrosome matrix and interacts with the GSK-3beta, implying that it may also be regulated by GSK-3beta phosphorylation signaling.
Evidence
5:
Inferred from Physical InteractionIntAct
To explore more hNinein interacting proteins, the yeast two-hybrid screening using ninein C-terminal domain as bait protein was performed. One novel gene, CGI-99, was demonstrated to associate with hNinein in the yeast two-hybrid method and in vitro GST pull-down assay. Molecular characterization also showed that CGI-99 possessed a transcriptional activity at the N-terminal. In addition, CGI-99 formed a dimer with the C-terminal, which overlapped with hNinein binding site. In kinase assay, CGI-99 binds to hNinein and completely blocks the phosphorylation of hNinein by GSK3beta. Moreover, CGI-99 was highly expressed in all brain tumors which is in agreement with the Northern blot analysis. Taken together, we have isolated a novel protein CGI-99, which may be involved in the functional regulation of human ninein in the centrosome structure and may also be important in brain development and tumorigenesis.
A centrosomal-associated protein, ninein is a microtubules minus end capping, centrosome position, and anchoring protein, but the underlying structure and physiological functions are still unknown. To identify the molecules that regulate the function of human ninein in centrosome, we performed yeast two-hybrid screen and isolated the SUMO-conjugating E2 enzyme, Ubc9, and SUMOylation enhancing enzymes, including PIAS1 and PIASxalpha, as binding partners of hNinein. These interactions as well as the interaction between hNinein and SUMO-1 are also confirmed by a glutathione S-transferase (GST) pull-down experiment. Furthermore, the C-terminal region of hNinein can be SUMOylated in vitro and in HeLa cells transfected with a plasmid expressing GFP-hNinein. Our findings firstly place SUMOylation target on the centrosome structure protein, hNinein, which results in the switch localization from centrosome to nucleus, suggesting the importance of the SUMOylation of hNinein and probably other centrosomal proteins may also be involved in the centrosome activity.
A reference proteome is a set of protein sequences derived from a complete proteome which constitutes a defined standard for a particular user community. Reference proteomes are manually defined according to a number of criteria. They cover the proteomes of well- studied model organisms and other proteomes of interest for biomedical and biotechnological research. Reference proteomes have been selected to provide broad coverage of the tree of life, and constitute a representative cross-section of the taxonomic diversity to be found within UniProtKB.
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