Location Of Kinase Nuak1 And Research Into Nuak1 In Nf-kb Pathway

Objective: As one of AMPK family, NUAK1 is a tumor related kinase, involves apoptosis, invation and metastasis in the formation of cancer. Here we make sure the subcellular location of NUAK1 and whether NUAK1 is involved in NF-kB pathway, and proceed detailed research will follow with the ultimate aim of validating if NUAK1 is a key therapy target of cancer.Methods: First prepared plasmids NUAK1-pEGFP, NUAK1-pc DNA3.1 and dominant negative mutant NUAK1( D178N ) -pcDNA 3.1 following the classical instruction of plasmid construction, and the dominant negative mutant was cloned by the way of Two-step PCR. To make sure the location of NUAK1, after NUAK1-pEGFP transiently expressed in transfected 293, cells nuclear DNA was counterstained with DAPY and the cells were observed under fluorescence microscope system. Before the detection of NF-kB luciferase activation, to verify the expression of NUAK1-pcDNA3.1 and NUAK1( D178N )- pcDNA 3.1 in the 293 cell line, western blotting performed by anti-myc antibody which is a tag of the vector pcDNA3.1. Then, To test the NF-kB luciferase activation under or without the stimulation of TNFα, with pcDNA3.1 as the control, NUAK1-pcDNA3.1 and the dominant negative NUAK1 ( D178N )-pcDNA 3.1 were overexpressed in a 293 cell line which was stably expressing NF-kB luciferase promoter reporter, and a plasmid dose response experiment was performed for further study.Results: The results of the sequence detection show that NUAK1- pEGFP, NUAK1-pcDNA3.1 and dominant negative mutant NUAK1 ( D178N )-pcDNA 3.1 were correctly constructed. And under fluorescence microscope system, in 293 cells, NUAK1 was constitutively distributed in the nucleus. The figure of western blotting show that both of NUAK1-pcDNA3.1 and dominant negative mutant NUAK1 ( D178N )-pcDNA 3.1 could expressed stably in 293 cell line. And under the stimulation of TNFα, NUAK1 inhibited the NF-kB luciferase activation and had a significantly positive correlation with the expression of NUAK1.Conclusion: Due to both of nuclear localization and the remarkable inhibition in the test of the NF-kB luciferase activation under the stimulation of TNFα, NUAK1 might be involved in the transcriptional regulation of NF-kB pathway .