Rgulation Pathway Of Polo-like Kinase 1(Plk1) In Murine Embryonic Stem Cells

Polo-like kinase 1 play key roles during multiple stages of mitosis including prophase, metaphase, anaphase, and cytokinesis. Plk1 are defined by two conserved features- an N-terminal Ser/Thr kinase domain and a C-terminal duplicated Polo-box region. Numerous studies have demonstrated that Plk1 gene expression is frequently upregulated in tumor tissues, a statistically significant correlation was found between high Plk1 expression and low patient survival, suggesting that Plk1 can be used as a negative prognostic indicator for some tumor types.Though extensive research in the past several years has defined the molecular pathway of Plk1,the exact molecular mechanism by which mammalian Plk1 regulate cell cycle checkpoint remains to be elucidated.For further investigating the function of Plk1 in cell cycle checkpoint. Protein moleculars interacted with Plk1 were screened with yeast two hybridation system, Plk1 cDNA was amplified from total RNA of murine embryonic stem cells by RT-PCR. The mouse embryonic stem cells cDNA library was screened by yeast two-hybrid assay,some candidate molecules were get,including DDX39 (DEAD (Asp-Glu-Ala-Asp) box polypeptide 39), BAT1A(HLA-B-associated transcript 1A), NAC1 ( nucleus accumbens-1 ) , BicD2 (bicaudal D homolog 2),MLL3 (myeloid/lymphoid or mixed-lineage leukemia 3). NAC1 and MLL3 were associated with regulation of gene transcription. DDX39 andBAT1A were associated with pre-mRNA processing and mRNA export from nucleus to cytoplasm. BicD2 was associated with the regulation of Golgi body.The subcellular location of DDX39,BAT1A,NAC1 and Plk1 were determined by fluorescent microscope.The interaction of Plk1 and candidate molecules,including DDX39,BAT1A,and NAC1 were confirmed by co-immunoprecipitation in vivo and GST-pulldown in vitro. The data indicated that Plk1 coud interact directly with DDX3,BAT1A and NAC1 both in vitro and in vivo. The specific region of DDX39,BAT1A and NAC1 binding with Plk1 were confirmed.The molecules including DDX39,BAT1A and NAC1 could be phosphorylated by Plk1 in vitro. After Plk1 being knockdown,the protein synthesis slowed down obviously,this may be caused by the decreased activation of DDX39 because of Plk1 level downregulation.All these data indicated that Plk1 was associated with the regulation of gene transcription ,pre-mRNA processing and mRNA export.