[Nuclear localization of insulin-like growth factor binding protein-6].
OBJECTIVE
To study the nuclear localization of insulin-like growth factor binding protein-6(IGFBP-6) in PC-3M cells.


METHODS
The two fragments of the nuclear localization sequence (NLS)-deleted IGFBP-6 and the NLS-mutated IGFBP-6 were obtained by overlapping PCR, and then the fragment was inserted into a pEGFP-C1 vector. PC-3M cells were transfected with the expression constructs containing wild-type IGFBP-6 or the two mutants (pEGFP-C1-BP6 Delta NLS and pEGFP-C1-BP6-Mut), and the different distribution of the three EGFP-fusion proteins was observed by confocal laser microscope. The statistical analysis of the ratio of the nuclear fluorescence to the cytoplasmic fluorescence (Fn/c) was performed. Results Confocal microscopic images of transfected cells showed that the green fluorescence of EGFP-IGFBP-6 was concentrated mostly in the nuclei, whereas the control cells expressing EGFP showed green fluorescence distributed uniformly. The results of Fn/c from EGFP and EGFP-IGFBP-6 were significant different (P<0.05). The NLS-deleted IGFBP-6 completely eliminated nuclear accumulation of the green fluorescent signal; in contrast, nuclear accumulation was only slightly reduced for the NLS-mutated IGFBP-6; compared with wild-type IGFBP-6, both mutants were significantly different (P<0.05). Conclusions IGFBP-6 can be translocated to the nucleus in PC-3M cell that is mediated by a putative NLS sequence. Our study provides new evidence for further studies on the insulin-like growth factor-independent activity of IGFBP-6.
Han
Jian-jun
JJ
National Laboratory of Medical Molecular Biology, Institute of National Laboratory of Medical Molecular Biology, Beijing 100005, China.
Huang
Bing-ren
BR
Wang
Xin
X
Ma
Xiao-li
XL
Chen
Hong
H
D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism*; D005822:Genetic Vectors; D006801:Humans; D018976:Insulin-Like Growth Factor Binding Protein 6 / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D021381:Protein Transport / Q000235:genetics; D014162:Transfection
D018976:Insulin-Like Growth Factor Binding Protein 6