Screening and identification of proteins interacting with nucleostemin

Abstract

Aim: 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.

Conclusion: NS and PPP2R5A interact in yeast and mammalian cells, respectively, which is helpful for addressing the function of NS in cancer development and progression.

Figure 1

Identification of the recombinant clone of NS and its expression in yeast cell. A: Analysis of pGBKT7-NS with restriction enzyme digestion. M: DNA markers; lane 1: undigested pGBKT7-NS; lane 2: digestion of pGBKT7-NS with BamHI/XhoI and 1 680-bp fragment was released; B: Western blotting analysis of NS expression by pGBKT7-NS in yeast. Total protein from yeast AH109 transferred with pGBKT7-NS was subjected to Western blot and the NS mAb was used to detect the NS protein. Lane 1: total proteins from pGBKT7-NS transferred AH109; lane 2: total proteins from pGBKT7 transferred AH109.

Figure 2

Restriction enzyme analysis of recombinant pcDNA3-NS and pcDNA3-myc-PPP2R5A. M: DNA markers; lane 1: undigested pCDNA3-NS; lane 2: digestion of pCDNA3-NS by BamHI/XhoI and 1 680-bp fragment was released; lane 3: undigested pcDNA3-myc-PPP2R5A; lane 4: digestion of pcDNA3-myc-PPP2R5A with BamHI/XhoI and 2 300 bp fragment was released.

Figure 3

Co-immunoprecipitation of protein NS and PPP2R5A. Total proteins from cells co-transferred with plasmids pCDNA3-NS and pcDNA3-myc-PPP2R5A were used for immunoprecipitation with antibody against NS or c-myc and antibody against c-myc or NS was used in Western blot. Mouse IgG was used in immunoprecipitation as negative control.