Yeast Two-hybrid Method To Find Erbin Of Interacting Proteins And Their Interactions, The Physiological Function Of A Preliminary Study

A protein named ERBIN that belongs to the LAP (for Leucine rich and PDZ domain containing proteins) protein family. ERBIN specifically interacts with Erb-B2 and is direct downstream target molecule, however,its exact molecular mechanism and signaling pathway remains unclear.In this study, we investigated Erbin associated proteins in human leucocyte cDNA library by yeast 2 hybrid system with Leucine rich and PDZ domain of erbin as baits The results from following sequencing and bioinfomational analysis shows type I human T lymphoid cell leukemia interact with erbin protein,Pull-down and immunoprecipatation experiment were utilized to confirm the specificity of the interaction identified. Erbin bound to taxi in specific manner, In addition, the mutation experiment suggests tax1bound selectively t PDZ domain of Erbin,not PDZ domain of Zo-1,Htr or LNX protein.Furthermore, the interaction is mainly mediated by the PDZ domain of erbin and several important amino acids in the c-terminal of tax1.To further understand the physiological significance of the interaction between tax1 and erbin,we established stable cell line overexpressing Tax1 gene and found the gene products promote the MCF-7 cell proliferation.Importantly the effect may be mediated by ras and NF-κB pathway. Meanwhile, mutation experiment showed that ras signaling pathway play key role in the effect of the interaction between erbin and tax1.Taken together,tax1 is specific binding protein of erbin and functions in signaling pathway to promote cell proliferation,in which the interaction between them play a critical role in cell growth. In addition our result indicates that taxi could inhibit obviously apoptosis signaling.