p52SHC3及其结构域对PC12细胞周期的影响
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摘要
目的:SHC3蛋白属于Shc家族,Shc家族包括SHC1、SHC2、SHC3和SHC4四种蛋白。SHC1(又称ShcA或Shc)发现最早,在哺乳动物的组织中广泛表达,但不存在于成熟的神经元中;SHC2(又称ShcB)在神经系统中广泛存在,但在其他组织中不表达;SHC3(又称N-Shc、ShcC和Rai)只在成熟的神经元中表达,在其它组织中不表达;SHC4(又称ShcD)是新近发现的成员,在骨骼肌中表达,其它组织中的表达情况还不清楚。Shc家族都含有两种功能性结构域:能和磷酸化酪氨酸结合的PTB结构域和SH2结构域以及一个含有磷酸化酪氨酸的中央区域结构域CH1。PTB结构域和SH2结构域都可以和活化的酪氨酸激酶受体结合,CH1结构域则被认为可以和Grb2结合,参与下游的信号通路,也有人认为CH1结构域可以将Shc蛋白定位于细胞膜上,不经Grb2即可传递上游信号。SHC3蛋白有两种异构体,p66SHC3和p52SHC3,两者只是在N-末端不同。和p52SHC3相比,p66SHC3多了一个类似于中央区结构域的CH2结构域。有研究认为SHC3可以促进神经前体细胞的分化,维持神经前体细胞和神经元的存活,但是SHC3对可分裂细胞周期的影响还没有人研究过。本项研究旨在通过对p52SHC3及其结构域对PC12细胞周期的影响,探讨p52SHC3及其结构域对其它可分裂细胞周期的影响,进一步了解p52SHC3及其不同结构域的功能。
方法:应用Western Blot技术验证SHC3在大鼠大脑、肺、肝和心脏等不同组织中的分布,将p52SHC3全长及其PTB结构域、SH2结构域、PTB+CH1结构域和CH1+SH2结构域插入载体pEGFP-N1中,构建其真核表达载体,使用脂质体转染的方法,将构建好的含有p52SHC3全长及其PTB结构域、SH2结构域、PTB+CH1结构域和CH1+SH2结构域的表达载体以及空载体转染进PC12细胞中,在转染后36h和60h,使用流式细胞术检测GFP阳性的细胞中G0/G1期的细胞所占的百分比。
结果:p52SHC3在大鼠的大脑中表达量比较高,在大鼠的肺、肝和心脏中则没有表达;流式细胞术检测的结果显示,和转染空载的PC12细胞相比,转染p52SHC3全长的PC12细胞中,G0/G1期的细胞比例上升,转染PTB结构域和PTB+CH1结构域的PC12细胞中,G0/G1期的细胞所占的比例明显上升,而CH1+SH2结构域的PC12细胞中,G0/G1期的细胞所占的比例下降,转染SH2结构域的PC12细胞G0/G1期的细胞所占的比例无明显变化。
结论:(1) p52SHC3全长可以抑制PC12细胞的增殖,PTB结构域和PTB+CH1结构域也可以将PC12细胞抑制在G0/G1期,CH1+SH2结构域则可以促进PC12细胞的增殖,SH2结构域对PC12细胞周期无明显作用;(2) p52SHC3有可能是通过其PTB结构域影响可分裂细胞的增殖;(3)PTB结构域对可分裂细胞周期的影响可能不经过CH1介导的信号通路,但CH1结构域的存在可以明显提高SH2结构域对可分裂细胞的增殖的促进作用。在p52SHC3中存在对细胞周期起截然相反的作用的两个结构域,这说明SHC3对细胞周期的影响是比较复杂的,进一步研究这两个结构域的功能将是非常有意义的。
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.
方法:应用Western Blot技术验证SHC3在大鼠大脑、肺、肝和心脏等不同组织中的分布,将p52SHC3全长及其PTB结构域、SH2结构域、PTB+CH1结构域和CH1+SH2结构域插入载体pEGFP-N1中,构建其真核表达载体,使用脂质体转染的方法,将构建好的含有p52SHC3全长及其PTB结构域、SH2结构域、PTB+CH1结构域和CH1+SH2结构域的表达载体以及空载体转染进PC12细胞中,在转染后36h和60h,使用流式细胞术检测GFP阳性的细胞中G0/G1期的细胞所占的百分比。
结果:p52SHC3在大鼠的大脑中表达量比较高,在大鼠的肺、肝和心脏中则没有表达;流式细胞术检测的结果显示,和转染空载的PC12细胞相比,转染p52SHC3全长的PC12细胞中,G0/G1期的细胞比例上升,转染PTB结构域和PTB+CH1结构域的PC12细胞中,G0/G1期的细胞所占的比例明显上升,而CH1+SH2结构域的PC12细胞中,G0/G1期的细胞所占的比例下降,转染SH2结构域的PC12细胞G0/G1期的细胞所占的比例无明显变化。
结论:(1) p52SHC3全长可以抑制PC12细胞的增殖,PTB结构域和PTB+CH1结构域也可以将PC12细胞抑制在G0/G1期,CH1+SH2结构域则可以促进PC12细胞的增殖,SH2结构域对PC12细胞周期无明显作用;(2) p52SHC3有可能是通过其PTB结构域影响可分裂细胞的增殖;(3)PTB结构域对可分裂细胞周期的影响可能不经过CH1介导的信号通路,但CH1结构域的存在可以明显提高SH2结构域对可分裂细胞的增殖的促进作用。在p52SHC3中存在对细胞周期起截然相反的作用的两个结构域,这说明SHC3对细胞周期的影响是比较复杂的,进一步研究这两个结构域的功能将是非常有意义的。
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.
引文
1 Luzi, L., S. Confalonieri, P. P. Di Fiore, et al Evolution of shc functions from nematode to human. Curr. Opin. Genet, 2000, 10:668~674
2 O’Bryan, J. P, Z. Songyang, L. Cantley, et al. A mammalian adaptor protein with conserved Src homology 2 and phosphotyrosine-binding domains is related to Shc and is specifically expressed in thebrain. Proc. Natl. Acad. Sci, 1996, 93:2729~2734
3 Pelicci, G.L. Dente, A. De Giuseppe, et al A family of Shc related proteins with conserved PTB, CH1 and SH2 regions. Oncogene, 1996, 13:633~641
4 Pelicci, G. L. Lanfrancone, F. Grignani, et al A novel transforming protein(SHC) with an SH2 domain is implicated in mitogenic signal transduction. Cell, 1992, 70:93~104
5 Jones N, Hardy WR, Friese MB, et al Analysis of a Shc family adaptor protein, ShcD/Shc4, that associates with muscle-specific kinase. Mol Cell Biol, 2007, 27(13):4759~4773
6 Sakai, R., J. T. Henderson, J. P. O’Bryan, et al The mammalian ShcB and ShcC phosphotyrosine dockingproteins function in the maturation of sensory and sympathetic neurons. Neuron, 2000, 28:819~833
7 Conti, L., S. Sipione, L. Magrassi, et al Shc signaling in differentiating neural progenitor cells. Nat.Neurosci, 2001, 4:579~586
8 Ravichandran KS, Lorenz U, Shoelson SE, et al Interaction of Shc with Grb2 regulates association of Grb2 with mSOS. Mol Cell Biol, 1995, 15(2):593~600
9 Kojima T, Yoshikawa Y, Takada S, et al Genomic organization of the Shc-related phosphotyrosine adapters and characterization of the full-length Sck/ShcB: specific association of p68-Sck/ShcB with pp135.Biochem Biophys Res Commun, 2001, 284(4):1039~1047
10 Ganju, P., J. P. O’Bryan, C. Der, et al Differential regulation of SHC proteins by nerve growth factor in sensory neurons and PC12 cells. Eur. J. Neurosci, 1998, 10:1995~2008
11 Nakamura, T., M. Komiya, N. Gotoh, et al Discrimination between phosphotyrosine-mediated signaling properties of conventional and neuronal Shc adapter molecules. Oncogene, 2002, 21:22~31
12 Nakamura, T.S. Muraoka, R. Sanokawa, et al Their differential regional expression in the brain and roles in neurotrophin and Srcsignaling. J.Biol.Chem, 1998, 273: 6960~6967
13 L.A. Greene, D.R. Kaplan, Early events in neurotrophin signaling via Trk and p75 receptors. Curr. Opin. Neurobiol,1995, 5: 579~587
14 Y. Hashimoto, K. Matuoka, T. Takenawa, et al Different interactions of Grb2rAsh molecule with the NGF and EGF receptors in rat pheochromocytoma PC12 cells. Oncogene, 1994, 9:869~875
15 A. Obermeier, R.A. Bradshaw, K. Seedorf, et al Neuronal differentiation signals are controlled by nerve growth factor receptorrTrk binding sites for SHC and PLC gamma. EMBO J, 1994, 13: 1585~1590
16 A.M. Rozakis, J. McGlade, G. Mbamalu, et al Association of the Shc and Grb2rSem5 SH2-containing proteins is implicated in activation of the Ras pathway by tyrosine kinases. Nature, 1992, 360: 689~692
17 John P. O’Bryan, Que T. Lambert, et al The Src Homology 2 and Phosphotyrosine Binding Domains of the ShcC Adaptor Protein Function as Inhibitors of Mitogenic Signaling by the Epidermal Growth Factor Receptor. J. Biol. Chem, 1998, 273: 20431~20437
18 Nakamura T, Muraoka S, Sanokawa R, et al N-Shc and Sck, two neuronally expressed Shc adapter homologs. Their differential regional expression in the brain and roles in neurotrophin and Src signaling. J Biol Chem. 1998, 273(12):6960~6967
19 O'Bryan JP, Songyang Z, Cantley L, et al A mammalian adaptor protein with conserved Src homology 2 and phosphotyrosine-binding domains is related to Shc and isspecifically expressed in the brain. Proc Natl Acad Sci U S A,1996, 93(7):2729~2734
20 Nakamura T, Sanokawa R, Sasaki Y, et al N-Shc: a neural -specific adapter molecule that mediates signaling from neurotrophin/Trk to Ras/MAPK pathway. Oncogene, 1996 , 13(6):1111~1121
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16 Migliaccio E, Mele S, Salcini AE, et al Opposite effects of the p52shc/p46shc and p66shc splicing isoforms on the EGF receptor-MAP kinase-fos signalling pathway. MBO J, 1997, 16:706~716
17 Sakai R, Henderson JT, O'Bryan JP, et al The mammalian ShcB and ShcC phosphotyrosine docking proteins function in the maturation of sensory and sympathetic neurons. Neuron, 2000, 28: 819~833
18 Kousteni S, Bellido T, Plotkin LI, et al Nongenotropic, sex-nonspecific signaling through the estrogen or androgen receptors: dissociation from transcriptional activity. Cell, 2001, 104(5):719~730
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24 Bonfni L, Migliaccio E, Pelicci G, et al Not all Shc's roads lead to Ras. Trends Biochem Sci, 1996, 21(7):257~261
25 Lai KM, Olivier JP, Gish GD, et al A Drosophila shc gene product is implicated in signaling by the DER receptor tyrosine kinase.Mol. Cell.Biol, 1995, 15,4810~4818
2 O’Bryan, J. P, Z. Songyang, L. Cantley, et al. A mammalian adaptor protein with conserved Src homology 2 and phosphotyrosine-binding domains is related to Shc and is specifically expressed in thebrain. Proc. Natl. Acad. Sci, 1996, 93:2729~2734
3 Pelicci, G.L. Dente, A. De Giuseppe, et al A family of Shc related proteins with conserved PTB, CH1 and SH2 regions. Oncogene, 1996, 13:633~641
4 Pelicci, G. L. Lanfrancone, F. Grignani, et al A novel transforming protein(SHC) with an SH2 domain is implicated in mitogenic signal transduction. Cell, 1992, 70:93~104
5 Jones N, Hardy WR, Friese MB, et al Analysis of a Shc family adaptor protein, ShcD/Shc4, that associates with muscle-specific kinase. Mol Cell Biol, 2007, 27(13):4759~4773
6 Sakai, R., J. T. Henderson, J. P. O’Bryan, et al The mammalian ShcB and ShcC phosphotyrosine dockingproteins function in the maturation of sensory and sympathetic neurons. Neuron, 2000, 28:819~833
7 Conti, L., S. Sipione, L. Magrassi, et al Shc signaling in differentiating neural progenitor cells. Nat.Neurosci, 2001, 4:579~586
8 Ravichandran KS, Lorenz U, Shoelson SE, et al Interaction of Shc with Grb2 regulates association of Grb2 with mSOS. Mol Cell Biol, 1995, 15(2):593~600
9 Kojima T, Yoshikawa Y, Takada S, et al Genomic organization of the Shc-related phosphotyrosine adapters and characterization of the full-length Sck/ShcB: specific association of p68-Sck/ShcB with pp135.Biochem Biophys Res Commun, 2001, 284(4):1039~1047
10 Ganju, P., J. P. O’Bryan, C. Der, et al Differential regulation of SHC proteins by nerve growth factor in sensory neurons and PC12 cells. Eur. J. Neurosci, 1998, 10:1995~2008
11 Nakamura, T., M. Komiya, N. Gotoh, et al Discrimination between phosphotyrosine-mediated signaling properties of conventional and neuronal Shc adapter molecules. Oncogene, 2002, 21:22~31
12 Nakamura, T.S. Muraoka, R. Sanokawa, et al Their differential regional expression in the brain and roles in neurotrophin and Srcsignaling. J.Biol.Chem, 1998, 273: 6960~6967
13 L.A. Greene, D.R. Kaplan, Early events in neurotrophin signaling via Trk and p75 receptors. Curr. Opin. Neurobiol,1995, 5: 579~587
14 Y. Hashimoto, K. Matuoka, T. Takenawa, et al Different interactions of Grb2rAsh molecule with the NGF and EGF receptors in rat pheochromocytoma PC12 cells. Oncogene, 1994, 9:869~875
15 A. Obermeier, R.A. Bradshaw, K. Seedorf, et al Neuronal differentiation signals are controlled by nerve growth factor receptorrTrk binding sites for SHC and PLC gamma. EMBO J, 1994, 13: 1585~1590
16 A.M. Rozakis, J. McGlade, G. Mbamalu, et al Association of the Shc and Grb2rSem5 SH2-containing proteins is implicated in activation of the Ras pathway by tyrosine kinases. Nature, 1992, 360: 689~692
17 John P. O’Bryan, Que T. Lambert, et al The Src Homology 2 and Phosphotyrosine Binding Domains of the ShcC Adaptor Protein Function as Inhibitors of Mitogenic Signaling by the Epidermal Growth Factor Receptor. J. Biol. Chem, 1998, 273: 20431~20437
18 Nakamura T, Muraoka S, Sanokawa R, et al N-Shc and Sck, two neuronally expressed Shc adapter homologs. Their differential regional expression in the brain and roles in neurotrophin and Src signaling. J Biol Chem. 1998, 273(12):6960~6967
19 O'Bryan JP, Songyang Z, Cantley L, et al A mammalian adaptor protein with conserved Src homology 2 and phosphotyrosine-binding domains is related to Shc and isspecifically expressed in the brain. Proc Natl Acad Sci U S A,1996, 93(7):2729~2734
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