Molecular characterization of human ninein protein: two distinct subdomains required for centrosomal targeting and regulating signals in cell cycle.
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.
Chen
Chang-Han
CH
Graduate Institute of Biochemistry, Kaohsiung Medical University, Kaohsiung 807, Taiwan, ROC.
Howng
Shen-Long
SL
Cheng
Tai-Shan
TS
Chou
Meng-Hui
MH
Huang
Chi-Ying
CY
Hong
Yi-Ren
YR
D000596:Amino Acids / Q000737:chemistry; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002453:Cell Cycle; D002455:Cell Division; D018385:Centrosome / Q000378:metabolism / Q000648:ultrastructure*; D002868:Chromosomal Proteins, Non-Histone; D003598:Cytoskeletal Proteins; D019084:Fluorescent Antibody Technique, Indirect; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000235:genetics*; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008938:Mitosis; D008954:Models, Biological; D009687:Nuclear Proteins; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D014404:Tubulin / Q000737:chemistry; D020798:Two-Hybrid System Techniques; D001616:beta-Galactosidase / Q000378:metabolism
D000596:Amino Acids; D002135:Calcium-Binding Proteins; D002868:Chromosomal Proteins, Non-Histone; D003598:Cytoskeletal Proteins; D008164:Luminescent Proteins; C512806:NIN protein, human; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D014404:Tubulin; C056608:caltractin; D049452:Green Fluorescent Proteins; D001616:beta-Galactosidase; D019204:GTP-Binding Proteins