The Functions Of Golgi-resident Protein STK16 And G Protein GNG7 In Mitosis And Membrane Trafficking

The disorder of mitosis and membrane trafficking will both lead to diseases such as tumors. For many years, the two key processes were recognized as separate processes, whereas recent studies have suggested a close relationship between them.So far, however, there is a relatively lack of research on the proteins and their regulatory mechanisms involving both in mitosis and membrane trafficking. So the discovery of more proteins that play dual roles in the two steps will shed light on elucidating the pathogenesis of tumor and other diseases. Here we employed the Golgi-resident protein STK16 and Gy subunit GNG7 as the targets,to gain more knowledge on the mechanisms of the two proteins in regulating mitosis and membrane trafficking through combination with cell biology, biochemistry and molecular biology. The main results are listed below:1. The function of STK16 in mitosis and membrane trafficking.STK16 kinase was mainly located at the Golgi apparatus and the cell membrane.STK16 in the Golgi was co-localized with the antibody marked medial cisterna of Golgi in interphase cells. In comparison, it was dissociated from its original localization in mitosis. STK16 knockdown or kinase inhibition caused not only the delayed mitotic entry, prolonged mitosis, but also the arrest of cells at G2 phase,prometaphase, and cytokinesis. Moreover, the disassembly of Golgi from G2 stage to metaphase,and the reassembly of Golgi ribbon from anaphase to cytokinesis were affected by STK16 knockdown or kinase inhibition. Further study showed that STK16 directly binds to actin and regulated actin dynamics in a concentration- and kinase activity-dependent manner. Therefore, we concluded that STK16 regulates actin dynamics directly to control Golgi structure and participate in mitotic progression.The transfection of cells with VSVG-GFP plasmids showed that STK16 did not affect the anterograde trafficking from endoplasmic reticulum to Golgi and then to plasma membrane. However, STK16 was involved in the classical endocytosis pathway, the recycling of transferrin and the degradation of EGFR, the trafficking pathways between cell membrane and early endosomes, and between Golgi and endosomes, and the autophagy pathway.The results of site directed mutagenesis showed that the myristoylated and palmitoylated modifications were the premise of STK16 binding to the plasma membrane and Golgi. The acylation modifications regulated the phenotype of its effect on kinetics of actin polymeriation, Golgi integrity and nucleocytoplasmic shuttling. We speculated that STK16 may act as a transcription factor to regulate the expression and function of other proteins.2. The function of GNG7 in mitosis and autophagy.It was reported that Gy subunit GNG7 was low expressed in several tumor cells.Through semi-quantitative RT-PCR, we also found that the transcriptional levels of GNG7 in tumor cells were significantly lower than that in normal cells, suggesting that GNG7 might be a tumor suppressor protein. Therefore, we established GNG7 overexpressed HeLa and U20S cells. The comparison of cell growth curve showed that GNG7 overexpression induced cell growth inhibition and cell death. In combination with flow cytometry, cell synchronization and RNA interference techniques, the mechanism of GNG7 inhibiting cell growth was investigated. The results showed that both GNG7 overexpression and knockdown caused mitotic arrest,resulting in a large number of bi/multinucleated cells. In addition, GNG7 was up-regulated in mitosis, especially in cytokinesis. All these suggested that GNG7 may play a critical role in cytokinesis. On this basis, we analyzed the effect of GNG7 on F-actin. GNG7 led to abnormal cytokinesis through disruption of the dynamic balance of actin in vivo.The pathways GNG7 inducing cell death were detected by using of flow cytometry, cell staining and western blot. The results gave out that GNG7 overexpression promoted apoptosis and autophagy. The proteins interacting with GNG7 during autophagy were screened by immunoprecipitation, and the mammalian target of rapamycin, mTOR, was obtained. Further experiments showed that GNG7 could also act on mTOR signaling pathways and induce autophagy, its knockdown inhibited the autophagic flux caused by mTOR inhibitors and EBSS starvation. Thus,the good inhibitory effect on cancer of GNG7 was caused by the combination of its inhibition on cell growth and the induction of autophagy death.In this study, we first identified Serine/Threonine kinases located in the Golgi complex could bind to actin and affect the whole mitosis through regulating Golgi integrity. This will provide basis for studies on the regulation model of Serine/Threonine kinases in mitosis. And STK16 was involved in several membrane trafficking pathways. For another thing, GNG7 was the first found Gy subunit taking part in autophagy. It interacted with mTOR and positively regulated autophagy, and acted on mitosis by affecting actin. Therefore,STK16 and GNG7 were the key proteins linking mitosis and membrane trafficking. The mechanism study of them will not only provide strong evidence for the close connection between the two processes,but also lay the foundation for further researches.