Identification Of NDRG2 Interaction Proteins And Correlation Of Its Expression With The Differentiation Of NB4 Cells

NDRG2 ,which was first identified and cloned from normal human whole brain cDNA library in our lab in 1999, belongs to a new family of differentiation-related genes, the NDRG family. This family includes four related members: NDRG1-4 . Database search and phylogenetic analysis revealed that NDRG2 genes are highly conserved in mammals. Moreover, mammalian NDRG family members show high homology to each other, except for their C-and N-terminal regions, pointing out the important functions of this gene family. Bioinformation analysis does not indicate any known motif or domain in NDRG2 and other members of NDRG family. The precise molecular and cellular functions of NDRG2 are still unknow so far. Some preliminary researches suggest that NDRG2 may play a role in growth arrest and cell differentiation, and it may be regulated by cellular stress. So, identification of Ndrg2 interaction proteins may provide important informations for its function research. For this purpose, we performed yeast two-hybrid screening of a adult brain cDNA library using Ndrg2 as a bait protein. 96 clones were obtained after 1.65 x10~6 clones were screened by reporter genes. 12 clones were got after interactions were retested in yeast, which represents 5 different gene fragments.Of these 5 final isolates, one fragment encoded FHOS (forming homologue overexpression spleen), adifferentiation-related protein; The other fragment encoded common C-terminal part of MSP58(58-KDa microspherule protein), MCRSl (Microspherule protein 1), MCRS2(Microspherule protein 2). They are isforms of each other; we named it MCRS2-C. The remained other three fragments encoded a hypothetical protein, a one of the randomly sampled human cDNA clone and a fragment of 3 ' terminal untranslated region of Syntaxin 1A mRNA.Among these positive clones, MCRS2-C was choosed for the further investigation. Sequence analysis revealed that fragment contains a conserved FHA domain and a coiled-coil domain. GST-pull down and co-immunoprecipitation northerly showed that Ndrg2 and MCRS2-C possessed physical interactions both in vitro and in vivo. To test the subcellular localization of Ndrg2 and MCRS2, pDsRed2-Nl-M)ï¿¡G2 and pEGFP-c3-MCRS2 recombinant plasmid was constructed and transfected into HHCC cells. The fusion proteins were expressed and observed by fluorescence microscopy, and confocal microscopy. We found that Red-Ndrg2 localized in cytoplasm while MCRS2-GFP localized in nucleus. Ndrg2 was reported to shuttle from cytoplasm to nucleus after the cell was treated by some stress stimulus. So the cells were treated by mitomycin C stress stimulus for 10 hours, and the localization of Ndrg2 and MCRS2-C were observed again. This time Ndrg2 was found to translocalize and colocalize with MCRS2 in nucleus. These results suggested that the interaction between Ndrg2 and MCRS2 (or MSP58, MCRSl) might regulated by some kind of stress stimulus. Because stress stimulus resulted in translocation of Ndrg2 from cytoplasm to nucleus. Colocalization of Ndrg2 and MCRS2 provide spatial possibility for the physical interactions of these two proteins. Todelineate the interaction region(s) of Ndrg2 and MCRS2 (or MSP58, MCRSl), various deletions of two proteins were engineered and subjected to analyze in yeast two hybrid assay. We found that the FHA domain of MCRS2-C is responsible for the interaction with Ndrg2, while amino acid residues between 100 to 257aa are responsible for its interaction with MCRS2(or MSP58, MCRSl).The two-hybrid analysis is of exceptional value for the detection of pairwise and transient associations. However, yeast two-hybrid approaches do not seem to be particularly suited for characterization of protein complexes.This supports the view that complex formation is more than the sum of binary interactions. Success of the TAP/MS approach for the characterization of protein complexes relies on its maintaining protein concentration, localization and post-translational modifications in a manner that closely approximates normal physiology. Yeast two-hybrid analysis and TAP/MS method are ideally complementary. In this research, we successfully constructed a TAP tag eukaryotic expression vector, and fused this tag to the C-terminus of Ndrg2. This recombinant was stably transfected to HHCC cells to develop an Ndrg2 sepcific mammalian TAP expression system. This work built up basis of identifying Ndrg2 interaction protein complex with TAP method.Some previous researches have demonstrated that NDRG2 is involved in the differentiation of some kinds of myelomonocytic leukemia cells. To investigate the relationship between NDRG2 and NB4 cells, a kind of myelomonocytic leukemia cells, a classic differentiation cell model was made with NB4 cell (a human APL cell line) treated by ATRA and AS2O3 at low concerntration. Expression of Ndrg2 is preliminary analized during differentiation of NB4 cells. Expression of Ndrg2 was up-regulated in the...