Mageh1 Domain Preliminary Study Of The Cell Cycle

A widely accepted concept in neural biology field is that mature neurons are terminally differentiated cells and are therefore no longer able to re-enter cell cycles or to proliferate. However, previous studies in our lab have demonstrated that some end-differentiated neurons still possess the ability to proliferate under certain conditions.Investigation on the molecular mechanisms governing neurons withdrawn from the cell cycle will provide theoretical and practical significance. Two MAGEⅡfamily members, MageD1 and MageH1, share a similar sequence of protein segment yet distinct or even opposite distribution and function. The investigation of their roles in the regulation of cell proliferation and differentiation may provide a breakpoint to better understand the mechanisms explaining why the mature neurons lose the ability to divide. Here we mainly focus on the function of MageH1 in the regulation of cell cycle. A fusion protein between His-tag and N terminal region(amino acids 1-89) of MageH1, predicted as the major B cell epitopes, was expressed in bacterium and specific antibody was prepared.It has been showed that PC12 cells transiently overexpressing MageH1 can be significantly arrested at the G0-G1 phase in our previous work. This raises a question that which part of MageH1 plays a key role in such a regulation. Constructing a variety of MageH1 fusion proteins with the GFP expressing plasmid disclosed that N90-194aa of MageH1 contributes to the arrest of cell cycle.Yeast two-hybridization screening was employed to identify the interacting proteins of MageH1. Among the selected proteins, MAPKK1 has a close relationship with the modulation of cell cycle, and the interaction between MAPKK1 and MageH1 was further proved by Co-IP. Owing to MageH1 has the ability to arrest cell at G0-G1 phase and MAPKK1 is the upstream kinase of ERK1/2, it is presumed that MageH1 may have an impact on the phosphorylation of ERK1/2 through the interaction with MAPKK1. However, our following experiment demonstrated that there is no obvious difference in the phosphorylation of ERK1/2 between native PC12 cells and PC12 cells stably overexpressing MageH1 under NGF treatment, indicating that MageH1 has no effect on the activation of ERK1/2.Moreover, an effective lentivirus interference plasmid targeting at MageH1 was constructed, which may help us better understand the function of MageH1 in maintenance of the resting state of postmiotic neurons in the following study.