Screening and identification of proteins interacting with nucleostemin.
OBJECTIVE
To identify the proteins interacting with nucleostemin (NS), thereby gaining an insight into the function of NS.


METHODS
Yeast two-hybrid assay was performed to screen a human placenta cDNA library with the full length of NS as a bait. X-Gal assay and beta-galactosidase filter assay were subsequently conducted to check the positive clones and the gene was identified by DNA sequencing. To further confirm the interaction of two proteins, the DNA fragment coding NS and the DNA fragment isolated from the positive clone were inserted into the mammalian expression vector pcDNA3 and pcDNA3-myc, respectively. Then, two plasmids were cotransfected into the COS-7 cells by DEAE-dextron. The total protein from the cotransfected cells was extracted and coimmunoprecipitation and Western blot were performed with suitable antibodies sequentially.


RESULTS
Two positive clones that interacted with NS were obtained from human placenta cDNA library. One was an alpha isoform of human protein phosphatase 2 regulatory subunit B (B56) (PPP2R5A) and the other was a novel gene being highly homologous to the gene associated with spondylo paralysis. The co-immunoprecipitation also showed that NS specifically interacted with PPP2R5A.


CONCLUSIONS
NS and PPP2R5A interact in yeast and mammalian cells, respectively, which is helpful for addressing the function of NS in cancer development and progression.
Yang
Hai-Xia
HX
Department of Biochemistry and Molecular Biology, Peking University School of Oncology and Beijing Institute for Cancer Research, Beijing 100034, China.
Jin
Geng-Lin
GL
Meng
Ling
L
Zhang
Jian-Zhi
JZ
Liu
Wen-Bin
WB
Shou
Cheng-Chao
CC
D000818:Animals; D019556:COS Cells; D002352:Carrier Proteins / Q000378:metabolism*; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D005260:Female; D019204:GTP-Binding Proteins; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism*; D010920:Placenta / Q000378:metabolism; D010957:Plasmids; D011247:Pregnancy; D011994:Recombinant Proteins / Q000378:metabolism
D002352:Carrier Proteins; C469949:GNL3 protein, human; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D019204:GTP-Binding Proteins