The Functional Analysis Of The Gene Relative To Blood Glucose Regulation

A series of genes relative to blood-glucose regulation had been cloned in our lab by messenger RNA differential display. One of the genes had been named Fangl (GenBank Accession NO.AF399873). By the NCBI BLAST analysis, Fangl shared high similarity (86%) with its human homologue (GenBank Accession N0.AK001644), which was identified as the pantothenate kinase gene (PANK4). Then the fang-1 was suggested as the SD rat PANK4. From the NCBI analysis, Rat PANK4 was highly conserved among its homologues from mouse to Homo sapiens. It has two conserved domains, KOG2201 and KOG4584. KOG2201 homologue is fumble, which is relative to the cell division. KOG4584 homologue is DUF89, which exists in a prokaryotic protein pfam01937 and the function is unknown.When transfected Rat PANK4 fused with green fluorescent protein (GFP) into L-6TG, HEK293T and Hela cells, PANK4 protein was found to localize in cytoplasm. The PANK4 antibody was obtained by immunizing rabbit and purified, then immunohistochemicalized to SD rat muscle, heart muscle, lung, liver, kidney, pancreas, hypothyroid, brain and small intestine tissues, the PANK4 protein was found to mainly express in some kinds of cells which have high energetic and biosynthetic requirements, such as cell division, secretory duct. By yeast two-hybrid using the PANK4 as bait protein, PANK4 protein was found to interact with Rattus norvegicus pyruvate kinase (Pkm2), as novel binding partners of PANK4. The interaction of PANK4 with pyruvate kinase was further confirmed by glutathione-S-transferase pull-down assay and co-immunoprepcipitation experiment. Fluorescence microscopic analysis using anti-FLAG MoAb revealed that FLAG-Pkm2 transiently expressed in HEK293T mainly exhibited distribution in the cytoplasm with co-expressed PANK4. After the co-immunoprecipitation experiment between the two domains of PANK4 respectively with Pkm2, we found that both domains can interact with Pkm2 respectively. It will provide some ideas to identify KOG4584 domain's function. PANK4 can interact with Pkm2 directly, which proved that PANK4 might involve in regulating the glucose metabolism by regulating the activity of Pkm2.Pkm2 is a key enzyme of glycolysis. Pkm2 can be found in cells in two major forms,...