分子選殖與特性化二個新的肝醣合成激酶3b相關蛋白-Human Dynamin-like和Human Ninein

Over the past decade, the study of glycogen metabolism was an important subject in human. In 1990, Woodgett et, al. found that a serine-threonine kinase first characterized for its role in glycogen metabolism has shown itself to be a key player in numerous processes, in organisms ranging from yeast to mammals. The protein was Glycogen synthase kinase-3??(GSK-3b). GSK-3b was first discovered by virtue of its ability to phosphorylate glycogen synthase and regulatory enzyme of glycogen synthesis in mammals. Recently, GSK-3b have been identified that can modulate transcriptional factors and effect their functions. It has also been implicated in the regulation of cell fate in animals. On the other words, GSK3b is very important factor in mammalian cells. Using human GSK-3b as a bait and human fetal liver cDNA library as a prey in the yeast two-hybrid system, we identified two novel human GSK-3b interaction proteins. One is human dynamin-like protein (HdynIV) and the other is human ninein protein. (hNinein). Human dynamin-like protein is a large GTP-binding protein. It is a member of dynamin family. Dynamin was initially identified as a microtubule-binding protein. When cells process endocytosis, dynamin can pitch the vesicle from cell membrane to cytosol. Dynamin also can help transportation of protein and take microbiology into lysosome. Perviously studies, dynamin could be classified three forms. There were Dynamin I、Dynamin II and Dynamin III. The human dynamin-like protein that we found is designated HdynIV. The full length cDNA of HdynIV has 699 amino acids. Its C-terminal of HdynIV can interact with GSK-3b using yeast two-hybrid system. By Northern blot and isoform specific PCR, we found that HdynIV is ubiquitously expressed in all human tissues. In pathology, we extracted total RNA of human brain tumor tissues, and we found at least four human dynamin-like isoforms by RT-PCR. There were human dynamin-like-wild type (HdynIV-WT), human dynamin-like-11(HdynIV-11), human dynamin-like-26 (HdynIV-26) and human dynamin-like-37 (HdynIV-37). Our data show that HdynIV-26 overexpressed in human brain tumor tissues. We suggested that HdynIV-26 may play a role in brain tumorigenesis. On the other way, we identified that three forms of rat dynamin-like protein (DLP1) in rat brain cDNA library. One of these forms is a novel gene that is not be reported yet. We examined the interaction of these three rat brain DLP1 with GSK3b using the yeast two-hybrid. By Northern blot, we found that rat DLP1is expressed ubiquitously in all tissues. In vitro kinase assay data showed that DLP1 acts as a substrate of GSK3b. Another candidate of GSK3b is human Ninein (hNinein). The full length cDNA of hNinein encoded a protein consisting 2096 amino acids. The features of this protein include a potential GTP binding site,in its N-terminal, a large coiled-coil domain together with four leucine zipper domains. Using the yeast two-hybrid, we identify the C terminal of hNinein that can interact with GSK3b. Our results also demonstrate that GSK3b can co-localize with human ninein in HeLa cells. By Northern blot and isoform specific PCR, hNinein is ubiquitously expressed in all human tissues. When we transfected GFP-hNinein into HeLa cells, we found that hNinein is localized in the centrosome. In cell cycle experiments Ninein was diminish in metaphase and anaphase, but reaccumulated in telophase.. In pathology, hNinein also was showed to react with autoantibody sera. The Ninein genome was found to correspond to 29 exons and 28 introns of genomic sequence on human chromosome 14q22. In addition, an alternatively splicing form can be found from genome sequence. Promoter analysis predicts that hNinein contains a TATA, two CCAAT, and three GC box. The promoter also exhibits the following potential transcription factor binding sites including Sp1, p300 and AP-1. In conclusion, we suggesting that HdynIV and hNinein proteins may play an important role in anchoring of GSK3b to ER and mitochoria or centrosome for signal transduction and metabolic regulation.