Two Pairs Of Interaction Study Of The Human Protein (arrb1/gnmt, And Nlk/smad4)

This study is focused on two protein-protein interaction (ARRB1/GNMT and NLK/SMAD4). We carried out yeast two-hybrid screening in a human liver cDNA library and identified an ARRB1 (β-arrestin 1) binding protein GNMT (glycine N-methyltransferase), as well as a SMAD4 binding protein NLK (nemo-like kinase).In part 1, we identified the interaction between GNMT and ARRB1, and found GNMT can promote the cytoplasm to nucleus translocalization of ARRB1 and ARRB1 can attenuate the inhibition of cell proliferation of SK-HEP-1 via GNMT. ARRB1 play an important role in GCPR signaling, and its novel function as a cytoplasm-nucleus messenger in the pathway has been identified recently. Also, recent studies showed that GNMT is a potential tumor suppressor gene. However, the mechanism is unclear yet. We discovered and identified the interaction between GNMT and ARBB1. Their special interaction was confirmed by GST pull-down and co-immunoprecipitation assay. The interaction was not dependent on the tetrameric structure and enzyme activity of GNMT. Further, we identified GNMT can promote nuclear accumulation of ARRB1 in HeLa. Also, we found increased expression of GNMT can inhibit the cell proliferation of hepatoma cell line SK-HEP-1 and the inhibition via GNMT is attenuated when co-transfected with ARRB1. The results in this study provided a clue to explore the role of GNMT in tumorigenesis and the mechanism of the cytoplasm to nucleus translocalization of ARRB1 in GPCR signaling.In part 2, we found the phosphorylation of SMAD4 via NLK and its negative effect on the expression level of SMAD4. In the former work of our laboratory, we had discovered and identified the special interaction between NLK and SMAD4. SMAD4 is the core member in TGF-βsignaling while NLK is one of the key kinases in p38 MAPK pathway and suppresses the activity of a wide range of transcription factors. Here, we identified the phosphorylation of SMAD4 via NLK in vitro, and found the phosphorylation sites were within the MH1 domain and the linker sequence. Truncated mutants assay displayed that the linker sequence between the MH1 domain and MH2 domain of SMAD4 are sufficient and necessary for this specific interaction. And we found their interaction is kianse-activity-independent. Further, we found that over-expression of NLK in HEK293T cells can result in the reduced expression level of SMAD4. The results in this study indicated that the interaction between SMAD4 and NLK and their phosphorylation may play an important role in regulation of the function of SMAD4 and TGF-βsignaling.