P52SHC3 And Its Domains Influence The Cell Cycle Of PC12 Cell

Objective:SHC3 protein belongs to Shc family which contains SHC1, SHC2, HC3 and SHC4. SHC1(also named ShcA or Shc) which is found earlier than other members, is expressed universally in mammalian tissues, but not in the mature neurons; SHC2(also named ShcB) widely exits in the neural system, but not in other tissues; SHC3(also named N-Shc, ShcC or Rai) can be not found in any other tissues except mature neurons; SHC4(also named ShcD) is a new member of Shc family, while it is reported that it exits in skeletal muscle, but whether other tissues express SHC4 is still not clear. All the members in Shc family have two functional domains: PTB domain and SH2 domain which can bind to phosphotyrosine and a central region (denoted CH1) that contains critical tyrosine phosphorylation sites. Both PTB domain and SH2 domain can bind to activated tyrosine kinase receptors, while CH1 domain is supposed to bind to Grb2 and trigger the downstream cell signaling, but some people insist that CH1 domain can be involved in the downstream signaling only by locating Shc to the cell membrane, without binding to Grb2. SHC3 protein has two isoforms which are p66SHC3 and p52SHC3 with the difference of their N-terminals Compared to p52SHC3, the p66SHC3 contains a central-region-like CH2 domain. It is said that SHC3 can promote the differentiation of neural precursor cells and maintain the survival of neural precursor cells and neurons. But whether SHC3 can influence the cell cycle of PC12 cell is still not studied by other researchers. Our study tries to investigate the influence that p52SHC3 and its domains bring to the cell cycle of PC12 cell and further understand how p52SHC3 and its domains affect the cell cycle of other dividing cells and get the message of the function of p52SHC3 and its domains.Methods: First, Western Blot was used in our study to conform the distribution of SHC3 in rat's brain, lung, liver and heart. Second, p52SHC3, its PTB domain, SH2 domain, PTB+CH1 domain and CH1+SH2 domain were inserted into pEGFP-N1 vector to construct their eukaryotic expression vectors. Third, the eukaryotic vectors and the control vector (without insertion) were transfected into PC12 cells. When all vectors were transfected in PC12 cells in 36hours and 60 hours, the percentage of cells in G0/G1 phase of GFP positive cells that accounted to all GFP positive cells was determined by flow cytometry.Results: Our research showed that there was expression of p52SHC3 in rat's brain, but there was none in rat's lung, liver and heart(consistent with the reports of the reference); the flow cytometry demonstrated that compared with the PC12cells that was transfected with controlled vector, in the p52SHC3 transfected PC12 cells, the percentage of cells in G0/G1 period of GFP positive cells was higher in contrast with all GFP positive cells, and the percentage of G0/G1 period cells was also obviously higher both in PTB domain and PTB+CH1 domain transfected PC12 cells, but in CH1+SH2 domain transfected PC12 cells, the percentage of G0/G1 period cells was lower, SH2 domain has no significant effect on cell cycle of PC12 cells. Conclusion: (1)Full length of p52SHC3 could prohibit the proliferation of PC12 cells and the PTB domain and PTB+CH1 domain of p52SHC3 also could prohibit PC12 cells to G0/G1 period of cell cycle, but the CH1+SH2 domain of p52SHC3 could promote the proliferation of PC12 cells .SH2 domain has no significant effect on cell cycle of PC12 cells; (2)p52SHC3 might influence the cell cycle of PC12 cells through its PTB domain; (3)PTB domain of p52SHC3 might affect the cell cycle of PC12 cells without the signaling of CH1 domain involved, but SH2 domain could promote the proliferation of PC12 cells with the participation of CH1 domain.Two domains that might have different function both exiting in p52SHC3 suggested that the influence of p52SHC3 to cell cycle may be more complicated than we have expected before. This needs to be revealed in the next study.