Effect of Phorbol 12-Myristate 13-Acetate Induced Protein Kinase C Activation on the TorsinA Subcellular Distribution_West China Medical Journal

Authors：

HU Xiaohui , ZHENG Zhenzhen , SHENG Liqin , PAN Pinglei , SONG Wei , HUANG Rui ,  SHANG Huifang

Department of Neurology, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, P. R. China;

Corresponding author：

SHANG Huifang, Email: smilewheeler@163.com

Keywords：

佛波酯; 蛋白激酶C; 扭转蛋白A; 肌张力障碍

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【摘要】　目的　探讨佛波酯激活的蛋白激酶C与扭转蛋白A在亚细胞成分中的表达之间的关系。　方法　采用免疫荧光法观察扭转蛋白A在原代培养的神经元和小鼠胚胎成纤维细胞（NIH 3T3细胞）中的分布。运用蛋白质印迹法分析蛋白激酶C和扭转蛋白A在细亚细胞成分中的表达。　结果　扭转蛋白A在NIH 3T3细胞中的表达类似于神经元。扭转蛋白A在细胞质溶质、膜成分中均有分布。佛波酯活化蛋白激酶C后并不引起扭转蛋白A在细胞质成分和膜成分中表达含量的变化。　结论　扭转蛋白A可能是膜相关蛋白，细胞氧化应激中扭转蛋白A表达上调和重分布变化不是由佛波酯诱导的蛋白激酶C活化途径来实现的。鉴于扭转蛋白A表达上调具有潜在的治疗原发性早发扭转性肌张力障碍的前景，影响其分布和表达的分子机制需要进一步研究。
【Abstract】　Objective　 To investigate the relationship between the phorbol 12-myristate 13-acetate (PMA) activated protein kinase C (PKC) and the subcellular expression of TorsinA protein.　Methods　 The expression of TorsinA in the primary cultured neurons and the NIH 3T3 cells was detected by immunofluorescence. The expression of PKC and TorsinA in subcellular fraction was analyzed by the western blotting.　Results　 The expression pattern of TorsinA in NIH 3T3 cells was similar to neuron. PMA, an activator of PKC, did not promote the up-expression of TorsinA or redistribution in the subcellular fraction of NIH 3T3 cells.　Conclusions　 TorsinA may be a membrane-associated protein. The up-regulation and redistribution of TorsinA is not caused by the pathway of the PMA activating PKC after cells insulted by oxidative stress. We should pay more attention on the mechanisms of the expression of TorsinA protein for the potential therapies to early-onset primary torsion dystonia (DYT1).

Citation： HU Xiaohui,ZHENG Zhenzhen,SHENG Liqin,PAN Pinglei,SONG Wei,HUANG Rui,SHANG Huifang. Effect of Phorbol 12-Myristate 13-Acetate Induced Protein Kinase C Activation on the TorsinA Subcellular Distribution. West China Medical Journal, 2011, 26(3): 321-324. doi: Copy

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2.	Zorzi G, Zibordi F, Garavaglia B, et al. Early onset primary dystonia[J]. Eur J Paediatr Neuro, 2009, 13(6): 488-492.
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7.	Ozelius LJ, Hewett JW, Page CE, et al. The early-onset torsion dystonia gene (DYT1) encodes an ATP-binding protein[J]. Nat Genet, 1997, 17(1): 40-48.
8.	Nery FC, Zeng J, Niland BP, et al. TorsinA binds the KASH domain of nesprins and participates in linkage between nuclear envelope and cytoskeleton[J]. J Cell Sci, 2008, 121(Pt 20): 3476-3486.
9.	Kazi JU, Soh JW. Isoform-specific translocation of PKC isoforms in NIH3T3 cells by TPA[J]. Biochem Bioph Res Co, 2007, 364(2): 231-237.
10.	Quest AF, Bell RM. The regulatory region of protein kinase C gamma. Studies of phorbol ester binding to individual and combined functional segments expressed as glutathione S-transferase fusion proteins indicate a complex mechanism of regulation by phospholipids, phorbol esters, and divalent cations[J]. J Biol Chem, 1994, 269(31): 20000-20012.〖ZK)〗.
11.	Gerace L. TorsinA and torsion dystonia: unraveling the architecture of the nuclear envelope[J]. Proc Natl Acad Sci USA, 2004, 101(24): 8839-8840.
12.	Gopalakrishna R, Jaken S. Protein kinase C signaling and oxidative stress[J]. Free Radical Bio Med, 2000, 28(9): 1349-1361.
13.	Hewett J, Gonzalez-Agosti C, Slater D, et al. Mutant torsinA, responsible for early-onset torsion dystonia, forms membrane inclusions in cultured neural cells[J]. Hum Mol Genet, 2000, 9(9): 1403-1413.
14.	de Carvalho Aguiar PM, Ozelius LJ. Classification and genetics of dystonia[J]. Lancet Neurol, 2002, 1(5): 316-325.
15.	Lee SH, Dominguez R. Regulation of actin cytoskeleton dynamics in cells[J]. Mol Cells, 2010, 29(4): 311-325.

1. [10　Hsieh CH, Rau CS, Jeng SF, et al. Identification of the potential target genes of microRNA-146a induced by PMA treatment in human microvascular endothelial cells[J]. Exp Cell Res, 2010, 316(7): 1119-1126.
2. 　Zorzi G, Zibordi F, Garavaglia B, et al. Early onset primary dystonia[J]. Eur J Paediatr Neuro, 2009, 13(6): 488-492.
3. 　Hu XH, Chen XP, Huang R, et al. Role of DYT1 gene in early-onset primary torsion dystonia[J]. NRR, 2010, 5(18): 1429-1434.
4. 　Chen XP, Hu XH, Wu SH, et al. RNA interference-mediated inhibition of wild-type Torsin A expression increases apoptosis caused by oxidative stress in cultured cells[J]. Neurochem Res, 2010, 35(8): 1214-1223.
5. 　Hewett J, Ziefer P, Bergeron D, et al. TorsinA in PC12 cells: localization in the endoplasmic reticulum and response to stress[J]. J Neurosci Res, 2003, 72(2): 158-168.
6. 　Zhao Y, Xiao J, Ueda M, et al. Glial elements contribute to stress-induced torsinA expression in the CNS and peripheral nervous system[J]. Neuroscience, 2008, 155(2): 439-453.
7. 　Ozelius LJ, Hewett JW, Page CE, et al. The early-onset torsion dystonia gene (DYT1) encodes an ATP-binding protein[J]. Nat Genet, 1997, 17(1): 40-48.
8. 　Nery FC, Zeng J, Niland BP, et al. TorsinA binds the KASH domain of nesprins and participates in linkage between nuclear envelope and cytoskeleton[J]. J Cell Sci, 2008, 121(Pt 20): 3476-3486.
9. 　Kazi JU, Soh JW. Isoform-specific translocation of PKC isoforms in NIH3T3 cells by TPA[J]. Biochem Bioph Res Co, 2007, 364(2): 231-237.
10. 　Quest AF, Bell RM. The regulatory region of protein kinase C gamma. Studies of phorbol ester binding to individual and combined functional segments expressed as glutathione S-transferase fusion proteins indicate a complex mechanism of regulation by phospholipids, phorbol esters, and divalent cations[J]. J Biol Chem, 1994, 269(31): 20000-20012.〖ZK)〗.
11. 　Gerace L. TorsinA and torsion dystonia: unraveling the architecture of the nuclear envelope[J]. Proc Natl Acad Sci USA, 2004, 101(24): 8839-8840.
12. 　Gopalakrishna R, Jaken S. Protein kinase C signaling and oxidative stress[J]. Free Radical Bio Med, 2000, 28(9): 1349-1361.
13. 　Hewett J, Gonzalez-Agosti C, Slater D, et al. Mutant torsinA, responsible for early-onset torsion dystonia, forms membrane inclusions in cultured neural cells[J]. Hum Mol Genet, 2000, 9(9): 1403-1413.
14. 　de Carvalho Aguiar PM, Ozelius LJ. Classification and genetics of dystonia[J]. Lancet Neurol, 2002, 1(5): 316-325.
15. 　Lee SH, Dominguez R. Regulation of actin cytoskeleton dynamics in cells[J]. Mol Cells, 2010, 29(4): 311-325.

West China Medical Journal

Effect of Phorbol 12-Myristate 13-Acetate Induced Protein Kinase C Activation on the TorsinA Subcellular Distribution

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