GIT2 Involved In Negative Regulation Of NF-κB Signal Pathway

Nuclear factorκB (NF-κB) plays a pivotal role in inflammation,immunity, stress responses, and apoptosis. Canonical activation of NF-κB is dependent on the phosphorylation of the inhibitory subunit IκBαthat is mediated by a multimeric, high molecular weight complex, called IκB kinase (IKK) complex. This is composed of two catalytic subunits IKKαand IKKβ, and a regulatory subunit NEMO/IKKг. The latter protein is essential for the activation of IKKs and NF-κB, but its mechanism of action is not well understood. It is of great significance to discover and characterize the proteins that interact with NEMO in the development of IKK activated and NF-κB transcriptional activity. Therefore, we identified GIT2 as NEMO-interacting partner by yeast two-hybrid screen of fetal liver cDNA library using NEMO as a bait ,in other way, we identified NEMO,TRAF1 ,TNIP1 and A20 which participate in the NF-κB as GIT2-interacting partner by yeast two-hybrid screen of fetal liver cDNA library using GIT2 as a bait .The GIT proteins had a complex domain structure and were now known to bind many proteins. They appeared to have important functions in the control of cytoskeletal dynamics and membrane trafficking between the plasma membrane and recycling endosomes. In previous research, GIT2 had interacted with some important proteins in NF-κB, we presumed GIT2 may participate in NF-κB.So we explored the interacting proteins of GIT2 and researched GIT2 how to influence NF-κB.We constructed of the A20,TRAF1,TNIP1 and the GIT2 deletion mutants eukaryotic expression vectors and GTI2,NEMO prokaryotic expression vectors by PCR. Then we detected their expression in the eukaryotic or prokaryotic cells. In coimmunprecipitation assay, GIT2 could interacted with NEMO, TRAF1 and TNIP1;the ARFGAP and PBS domains of GIT2 and the CC domain of NEMO were the binding domains of each other, GIT2(1-347aa)interacted with TRAF1. In GST pull-down assay, GIT2 interacted with NEMO. GIT2,A20,TRAF1 and NEMO were inhibitors of NF-κB which GIT2 could influence with A20 and TRAF1;but TNIP1 got a reverse activity.GIT2 could inhibited TRAF2-mediated NF-κB activitation, but could not inhibited IKKβ-mediated NF-κB activitation. We used GIT2 ,A20and NEMO antibody and found them can be express in HEK293 cell.We also found A20siRNA and GIT2siRNA could knocked down A20 and GIT2 in HEK293 cell by Western blot and RT-PCR. GIT2 promoted association of A20 with NEMO.Ferthermore, We overexpressed and knocked down GIT2 in HEK293 cells and found that GIT2 mediated the De-ubiquitinating activity of A20 on NEMO and inhibited NF-κB.But GIT2 how to influence NF-κB need to investigate in the future.In conclusion, GIT2 plays an important role in NF-κB signaling .GIT2 ,a new gene that influences NF-κB, provides some important clues for investigating NF-κB.