Identification Of Key Element Of Spindlin1 Localization And Function

The development and mature of oocyte is a complex and continuum process,in which some genes and proteins play important roles.The reasaerch of these genes and proteins is important and pop in life science.From 1970s,there were more and more studies on the proteins which involved in the development of sperm cells,oocyte cells and zygote.Among these proteins,a 30KDA protein was concerned by several reaearch group.In 1997, Bermseok cloned and named spindlin in his research of mice oocyte development.The Spin/Ssty gene family exists in oocyte development, and have a high protein expression in this period.To identify the function of protein correlated to the development and mature of oocyte is important for realizing the origination of lifes. The phosphorilated spindlin take a effect in MOS/MAPK signal pathway,and have correlation to spindle in meiosis process.It is reported that spindlin express in bird and fish germ cell,indicating that spindlin and Ssty form a family,Spin/Ssty family,and the function of this family needs to be deeply researched.Spindlin1, a novel human gene recently isolated by our laboratory, It was found to localize in cell nuclei.The transfected cells, which are prone to grow as cancer cells in nude mice, showed a complete morphological change. Overexpression of spindlin1 might lead to variations in cell cycle distribution during mitosis. Spindlin1 may regulate the cell cycle through theβ-catenin/ T cell factor (TCF-4) pathway. The previous studies indicate that the structure of Spindlin1 is similar as Spin/Ssty's structure which has 4-5β-fold in every domain.So it is important to identify the key element for protein localization and function.1.Construction of Spindlin 1mutant vectorBased on bioinformatics analysis, a series of mutant fused with enhanced green fluorescent protein (EGFP) expression vector were constructed successfully.We mutant the 14th,84th,99th Ser to Ala ,and mutant Ser14M,Ser84M,Ser99M, Ser14+84M,Ser14+99M,Ser14+84+99M were constructed for further research.2.Effect of spindlin 1 mutant to localization and function To determine the function domain of spindlin1 protein for subcellular location and the correlation between the structure and function of new gene spindlin1.To find the key amino acid point of spindlin protein function. We transiently transfected HeLa cells with wild type and mutant spindlin1 construct. The subcellular location was observed under fluorescence microscope. Infect Hela cells with pEGFP-C1,pEGFP-spindlin1,TCF-4 luciferase reporter pTOPFlash andβ-galactosidase constructs.Identify the interaction between spindlin1 and TCF-4 with GST pull-down assay.Results:Wild type spindlin 1 protein locate in the nuclei of Hela cell. The EGFP fusion proteins with mutations Ser14M,Ser84M,Ser99M and Ser14+99M gave the similar subcellular location as wild type. But different from the wild type, the EGFP fluorescence signal of mutations Ser14+84M,Ser84+99M,Ser14+84+99M were distributed uniformly in cytoplasm. Conclusion: Ser84 play a key role in nuclear targeting, which need the cooperation of Ser14 and Ser99,indicate that mutation of Ser84 with either Ser14 or Ser99 could abolish the promotion of spindlin1 on TCF-4 reporter activity. These results lay foundation for further study on the structure and function of spindlin1.3.The research of Spindlin1 interact with kinase Aurora AHuman Aurora A localizes at chromosome 20q13.2, 403aa.Recently more and more evidence indicate that Aurora A is a oncogene.Aurora Ainteract with many subtrate,involved in many different signal pathways which form a signal network.Spindlin1 and Aurora A have many common in feature and function.We construct vectr to express GST- spindlin1 fusion protein,validate different IPTG inducement way. Identify the interaction between spindlin1 and Ser/Thr kinase Aurora A with GST-pulldown assay.We add restriction enzyme site EcoR I and BamH I,and construct prokaryotic expression vector pGEX-2T-spindlin1.Express GST-spindlin1 fusion protein in E.coli BL21,and collect the protein,then GST-pulldown .Conclusion:spindlin1 is the substrate of Ser kinase Aurora A.