关键词: SH2D4A, 酵母双杂交技术, GST pull-down, 蛋白质相互作用, SH2结构域

Abstract: SH2D4A is a member of SH2 signaling protein family, which is involved in the signal transduction mediated by protein tyrosine kinase-related receptor, cell growth, proliferation, differentiation, and thereby affects the development of human disorders. To determine the role of SH2D4A in the cell signal transduction pathway, SH2D4A interactive proteins were screened using yeast two-hybrid system, and yeast mating and GST pull-down assays were carried out to further confirm the interaction. We successfully generated a bait protein expression construct-pGBKT7-SH2D4A, screened the human kidney cDNA library, and obtained 46 positive yeast clones. After isolation of positive colonies, DNA sequencing, and sequence alignment analysis with BLAST software, we obtained 5 potential SH2D4A interactive proteins, AZGP1, DAD1, HSD17B10, KAT5, and PKM2, which were predicted by NetPhos 2.0 Server software and were all shown to be phosphory-lated tyrosine (pY)-containing proteins except for HSD17B10. KAT5 and HSD17B10 were selected to perform yeast mating and GST pull-down experiments, indicating their direct binding to SH2D4A.

Key words: SH2D4A, yeast two-hybrid system, GST pull-down, protein interaction, SH2 domain