Preliminary Study Of The Function Of M-type Pyruvate Kinase In Zebrafish

Pyruvate kinase regulates the final rate-limiting step of glycolysis, which catalyzes phosphoenolpyruvate (PEP) to yields one molecule of pyruvate and one molecule of adenosine triphosphate (ATP).Two pyruvate kinase forms exist in mammals,PKLR and PKM.The pyruvate kinase M1 (PKM1) and M2 (PKM2) isoforms result from mutually exclusive alternative splicing of the PKM, reflecting the inclusion of either exon 9 (PKM1) or exon10 (PKM2), respectively.PKM1 expression is found predominantly in differentiated adult tissues with high ATP requirements, such as the heart,brain,and muscle. PKM2 is expressed during development and all cancers.Recent studies show that PKM2 not only as pyruvate kinase processes in glycolysis, but also has the protein kinase activity. PKM2 has become a hot area in research, scientists have focused on the role of PKM2 in tumor cells. They Found that, PKM2 regulates the Warburg effect in tumor cells, results that the tumor cells metabolize by the way of aerobic glycolysis in the presence of oxygen although. In normal cells, PKM2 exists mainly in the form of tetramer, as pyruvate kinase; and the dimeric form of PKM2 mainly in the tumor cells,plays a role as protein kinase activity.Recent studies have confirmed that PKM2 plays an important function in the development and progression of tumors, and its basic mechanism has been elucidated, has become a hot research in cancer in recent years.Previous studies demonstrated that hypoxia induces PKM2 mRNA expression in tumor cells, and also confirmed that PKM2 as a target genes regulate by HIF-1α directly.Bioinformatic analysis of the human PKM2 gene sequence reveals a HRE within the first intron,which can be distinguished by HIF-1α,activating PKM2 transcription.By the amino acid sequence analysis found that PKM2 has a carboxyl-terminal nuclear localization signal (Arg399/400), which plays a key role in inducting PKM2 into the nucleus under the conditions of hypoxia. In the nucleus, PKM2 interacts with HIF-1α and activates the transcriptional activity of HIF-la, thereby promotes the transcription of target genes, including glycolysis-related glucose transporter and lactate dehydrogenase A.PKM2 as HIF-la’s target gene, its transcriptional activity can be regulated also, so that a feedback regulation forms between PKM2 and HIF-la.However, although there is no doubt that PKM2 can interact with HIF-1α,all these studies were conducted in vitro.So far there has no studies prove the interaction between PKM2 and HIF-la and the biological function of PKM2 in vivo, especially whether PKM2 plays a role in the early embryonic development and whether it involves in the HIF role in embryonic development is still blank. Now the idea is that cancer is a problem in developmental biology. Since PKM2 is essential for tumors, suggesting that it is likely to play an important role in embryonic development. The zebrafish as a new animal model, used in a wide range of developmental biology.By the analysis of zebrafish gene database shows that there are two pkm gene in zebrafish, named pkma and pkmb, is located at 18th and 25th chromosome, respectively. Structural analysis shows that it is the highly conserved with mammalian. The pkma were spliced into two transcripts,result from mutually exclusive alternative splicing, according to its cDNA nucleotides number named pkma-2404 and pkma-2412, respectively, pkma-2412 contains exon 10 and pkma-2404 excludes exon 10 but specific intron-containing part of the original 9. Through bioinformatics analysis, we found that the pkma and pkmb exists nuclear localization signal, and exists the HRE in the upstream of sequence also.By RT-PCR we found that pkma-2404 and pkmb are expressed during embryonic development and adult fish, but pkma-2412 only express on early embryonic development and adult fish. In addtion pkma and pkmb expression levels were upregulated by hypoxia on zebrafish embryos and adult fish, then we transfected transiently the expression plasmids tagged with GFP into HEK293T cells, p2.1 as the reported plasmid, the dual luciferase activity illustrate that Pkma has a significant increase in the activity of dual luciferase, indicating that Pkma can interact with HIF-la. Hela cells were transfected transiently the expression plasmids also, found that Pkma and Pkmb can enter into the nucleus under hypoxic conditions. After that, we overexpressed HIF-1α in zebrafish embryos, found that overexpression of HIF-1α can increase pkma mRNA expression levels, suggesting that pkma is the target gene of HIF-1α.In summary, the pkm genes of zebrafish are regulated by hypoxia,through experiments in vivo demonstrate that pkma may be a target gene of HIF-1α, And we also have a certain understanding the function of pkm in early embryonic development.