Characterization and functional aspects of human ninein isoforms that regulated by centrosomal targeting signals and evidence for docking sites to direct gamma-tubulin.
The functions of centrosomal protein ninein may be involved in microtubule minus end capping, centriole positioning, protein anchoring and microtubule nucleation, but the true physiological function of various human hNinein isoforms remains to be determined. Here we describe the identification of four diverse CCII-termini of human hNinein isoforms, including a novel isoform 6, by differential expression in a tissue-specific manner. These hNinein isoforms exhibit centrosomal (concentrated) and noncentrosomal (aggregated) localization when GFP-tagged fusion proteins are expressed transiently in mammalian cells. In a kinase assay, we show that the CCII region of hNinein provides a differential phosphorylation site by GSK3beta. In addition, our data indicate that either N-terminal or CCIIZ domain disruption may cause hNinein conformational change which recruits gamma-tubulin to centrosomal or noncentrosomal hNinein-containing sites, implying that the gamma-tubulin localization may be hNinein-dependent. Further, our RNA interference experiment against all hNinein isoforms caused a significant decrease in the gamma-tubulin signal in the centrosome. In domain swapping, we clearly show that the CCIIX-CCIIY region provides docking sites for gamma-tubulin. Moreover, our data also show that nucleation of microtubules from the centrosome is significantly affected by the presence of either the full -length hNinein or CCIIX-CCIIY region overexpression. Taken together, these results show that the centrosomal targeting signals of hNinein have a role not only in regulating hNinein conformation, resulting in localization change, but also provide docking sites to recruit gamma-tubulin at centrosomal and noncentrosomal sites.
Lin
Ching-Chih
CC
Graduate Institute of Biochemistry, Kaosiung Medical University, Kaohsiung, Taiwan.
Cheng
Tai-Shan
TS
Hsu
Ching-Mei
CM
Wu
Che-Hsiang
CH
Chang
Long-Sen
LS
Shen
Zhi-Shiang
ZS
Yeh
Hung-Ming
HM
Chang
Li-Kwan
LK
Howng
Shen-Long
SL
Hong
Yi-Ren
YR
D000595:Amino Acid Sequence; D018385:Centrosome / Q000737:chemistry / Q000648:ultrastructure*; D003598:Cytoskeletal Proteins / Q000737:chemistry* / Q000502:physiology*; D005786:Gene Expression Regulation; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry* / Q000502:physiology*; D011485:Protein Binding; D011487:Protein Conformation; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution; D014404:Tubulin / Q000737:chemistry*
D003598:Cytoskeletal Proteins; C512806:NIN protein, human; D009687:Nuclear Proteins; D020033:Protein Isoforms; D014404:Tubulin; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; D038362:Glycogen Synthase Kinase 3