Porcine MELK Inhibiting BCL-G Mediated Apoptosis By STS

The maternal embryonic leucine zipper kinase(MELK) participates in a wide range of cellular processes. such as cell apoptosis and cell proliferation and so on. The N-terminal catalytic domain is the key domain in MELK. And its activity was affected by UBA domain and TP-rich domain. In breast carcinogenesis:the kinase phosphorylates Bcl-GL, inhibiting the latter’s proapoptotic function. Our lab had cloned the porcine MELK(pMELK) gene and porcine BCL-G(pBCL-G) gene. And had certified that pMELK protein can interact with pBCL-G protein. So we focus on the investigate whether pMELK can affected pBCL-G cell apoptosis. Our results are as followings:1. We constructed pMELK and catalytic domain(cd) of pMELK expression vectors: pEGFP-MELK and pEGFP-cd. Fluorescence microscope and western blot showed that fusion protein GFP-pMELK and GFP-cd was expressed.2. Transiently over-expressing GFP-pMELK or GFP-cd protein cells induced by staurosporine(STS) was obseved by light microscopy, cells appeared apoptotic phenomenon, such as shrinkage and fragmentation of cells. After staining with Hoechst33342, transiently over-expressing GFP-pMELK or GFP-cd protein cells induced by STS showed chromatin condensation and nuclear fragmentation, compared with control group. And Annexin V-PE/PI staining was used. Analysis of flow cytometry showed that over-expressing GFP-pMELK or GFP-cd protein not significantly affected apoptosis induced by STS. It suggested that pMELK had the same result to the catalytic domain. We transiently transfected recombinant plasmid pCMV-HA-pMELK into cells expressing GFP-pBCL-G protein stably for further study, and then induced by STS(500nM). Flow cytometry analysis showed that pMELK can significantly inhibit pBCL-G and reduce cell apoptosis.