mP19对细胞骨架和增殖的影响及其相互作用分子筛选
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摘要
本课题组前期实验证实MARVELD1是一个在多种肿瘤细胞中呈现低表达的候选抑癌基因,其功能研究有待进一步深入。同源比对分析发现来自小鼠的mP19与人源MARVELD1氨基酸序列同源性达88%,且结构域高度保守。因此,本课题通过生化和细胞生物学技术探讨mP19的基本功能,旨在阐明mP19和MARVELD1的功能异同,为后续建立小鼠模型提供一定参考。本课题中通过生物信息学方法,我们推断mP19及其种属同源分子仅在脊椎动物中表达且在高等哺乳类中呈现高度保守性。mP19具有四个潜在的蛋白激酶C(PKC)磷酸化位点(Thr15, Ser19, Thr51, Thr120),而人源MARVELD1仅存在一个(Thr51),我们采用免疫沉淀技术及Western Blot方法检测证实mP19存在可被磷酸化的苏氨酸,而Thr51因其在人鼠序列中的高度保守性被认为是mP19的PKC磷酸化关键苏氨酸。我们采用免疫荧光技术,在共聚焦显微镜下观察到mP19呈现周期依赖性细胞定位:间期时定位于细胞核及细胞核周,分裂期时与纺锤体微管及胞质分裂器有明显共定位。佛波酯醇(PMA)激活PKC能促进mP19的核转位。通过GST-pull down和免疫共沉淀技术,我们证实mP19与α-tubulin存在相互作用,且秋水仙素能促进mP19向细胞膜转位。我们利用相差显微镜、共聚焦显微镜及扫描电镜从不同角度观察到过表达mP19抑制细胞微丝骨架组装、促进细胞表面超微结构和丝状伪足的形成。此外,我们证实在NIH3T3细胞中过表达mP19能明显抑制细胞增殖、造成细胞G1期阻滞、抑制细胞迁移运动。
Our previous study shows that candidate tumor suppressor MARVELD1 was downregulated in multiple primary tumors. Phylogenetic analysis demonstrates that mouse mP19 is 88% conserved to its human counterpart MARVELD1 with highly consensus domains. Therefore, in this study, we prefer to elucidate the cellular function of mP19 by means of the biochemical and cytology methods to assess the similarities and differences between mP19 and MARVELD1, which may provide important insights into the foundation of mP19-transgenic mouse.
Bioinformatics analysis shows that mp19 and its homologs exist in the genomes of vertebrates and highly conserved in various mammalian species. By BLAST searching, four candidate PKC phosphorylation sites (Thr15, Ser19, Thr51, Thr120) were predicted to locate in mP19, while only one (Thr51) in human MARVELD1. According to the clues, we identified the Thr phosphorylation site of mP19 using immunoprecipitation and western blot analysis and considered that Thr51 could be the direct phosphorylation site by PKC according to its high identity between mP19 and human MARVELD1. Further immunofluorescence analysis shows that mP19 exhibits a cell cycle-dependent cellular localization. It was observed to be in the nucleus and at the perinuclear region at interphase, while with the clear mitotic spindle and the midbody during mitosis.
In addition, our study suggests that mP19 translocates into the nucleus induced by PMA, a PKC activator. By co-immunoprecipitation assay,α-tubulin was identified to be the interacted protein of mP19. Moreover, treatment of cells with colchicine, a microtubule-depolymerizing agent, translocation of the whole fraction of mMARVELD1 was observed. We also found that overexpressed mP19 affected the assembly of microfilament, cellular surface micro-structure and stress fiber. Finally, overexpression of mMARVELD1 in NIH3T3 cells resulted in a remarkable inhibition of cell proliferation, G1-phase arrest, and reduced cell migration.
Our previous study shows that candidate tumor suppressor MARVELD1 was downregulated in multiple primary tumors. Phylogenetic analysis demonstrates that mouse mP19 is 88% conserved to its human counterpart MARVELD1 with highly consensus domains. Therefore, in this study, we prefer to elucidate the cellular function of mP19 by means of the biochemical and cytology methods to assess the similarities and differences between mP19 and MARVELD1, which may provide important insights into the foundation of mP19-transgenic mouse.
Bioinformatics analysis shows that mp19 and its homologs exist in the genomes of vertebrates and highly conserved in various mammalian species. By BLAST searching, four candidate PKC phosphorylation sites (Thr15, Ser19, Thr51, Thr120) were predicted to locate in mP19, while only one (Thr51) in human MARVELD1. According to the clues, we identified the Thr phosphorylation site of mP19 using immunoprecipitation and western blot analysis and considered that Thr51 could be the direct phosphorylation site by PKC according to its high identity between mP19 and human MARVELD1. Further immunofluorescence analysis shows that mP19 exhibits a cell cycle-dependent cellular localization. It was observed to be in the nucleus and at the perinuclear region at interphase, while with the clear mitotic spindle and the midbody during mitosis.
In addition, our study suggests that mP19 translocates into the nucleus induced by PMA, a PKC activator. By co-immunoprecipitation assay,α-tubulin was identified to be the interacted protein of mP19. Moreover, treatment of cells with colchicine, a microtubule-depolymerizing agent, translocation of the whole fraction of mMARVELD1 was observed. We also found that overexpressed mP19 affected the assembly of microfilament, cellular surface micro-structure and stress fiber. Finally, overexpression of mMARVELD1 in NIH3T3 cells resulted in a remarkable inhibition of cell proliferation, G1-phase arrest, and reduced cell migration.
引文
1 R. Puertollano, M. A. Alonso. MAL, an Integral Element of the Apical Sorting Machinery, Is an Itinerant Protein that Cycles between the Trans-Golgi Network and the Plasma Membrane. Mol. Biol. Cell. 1999, 10:3435~3447
2 M. Furuse, T. Hirase, M. Itoh, et al. Occludin: a Novel Integral Membrane Proteins Localizing at Tight Junctions. J. Cell Biol. 1993, 123:1777~1788
3 L. Sánchez-Pulido, F. Martín-Belmonte, A. Valencia, et al. MARVEL: a Conserved Domain Involved in Membrane Apposition Events. Trends Biochem Sci. 2002, 27:599~601
4 K. Hübner, R. Windoffer, H. Hutter, et al. Tetraspan Vesicle Membrane Proteins: Synthesis, Subcellular Localization, and Functional Properties. Int Rev Cytol. 2002, 214:103~159
5 C. P. Arthur, M. H. Stowell. Structure of Synaptophysin: a Hexameric MARVEL-Domain Channel Protein. Structure, 2007, 15:707~714
6 http://www.ncbi.nlm.nih.gov
7 J. Ikenouchi, M. Furuse, K. Furuse, et al. Tricellulin Constitutes a Novel Barrier at Tricellular Contacts of Epithelial Cells. J Cell Biol. 2005, 171:939~945
8 S. Riazuddin, Z. M. Ahmed, A. S. Fanning, et al. Tricellulin Is a Tight-Junction Protein Necessary for Hearing. Am J Hum Genet. 2006, 79:1040~1051
9 D. R. Raleigh, A. M. Marchiando, Y. Zhang, et al. Tight Junction-associated MARVEL Proteins Marveld3, Tricellulin, and Occludin Have Distinct but Overlapping Functions. Mol Biol Cell. 2010, 21:1200~1213
10 E. Steed, N. T. Rodrigues, M. S. Balda, et al. Identification of MarvelD3 as a Tight Junction-associated Transmembrane Protein of the Occludin Family. BMC Cell Biol. 2009, 10:95
11 S. Wang, Y. Li, F. Han, et al. Identification and Characterization of MARVELD1, a Novel Nuclear Protein that Is Down-regulated in Multiple Cancers and Silenced by DNA Methylation. Cancer Lett. 2009, 282:77~86
12 W. Han, P. Ding, M. Xu, et al. Identification of Eight Genes Encoding Chemokine-like Factor Superfamily Members 1-8 (CKLFSF1-8) by in Silico Cloning and Experimental Validation. Genomics. 2003, 81:609~617
13 Y. Wang, J. Li, Y. Cui, et al. CMTM3, Located at the Critical Tumor Suppressor Locus 16q22.1, Is Silenced by CpG Methylation in Carcinomas and Inhibits Tumor Cell Growth Through Inducing Apoptosis. Cancer Res. 2009, 69:5194~5201
14 L. Shao, Y. Cui, H. Li, et al. CMTM5 Exhibits Tumor Suppressor Activities and IsFrequently Silenced by Methylation in Carcinoma Cell Lines. Clin Cancer Res. 2007, 13:6543~6562
15 C. Jin, P. Ding, Y. Wang, et al. Regulation of EGF Receptor Signaling by the MARVEL Domain-containing Protein CKLFSF8. FEBS Letter. 2005, 579:6375~6382
16 L. Shao, X. Guo, M. Plate, et al. CMTM5-v1 Induces Apoptosis in Cervical Carcinoma Cells. Biochem Biophys Res Commun. 2009, 379:866~871
17 D. Li, C. Jin, C. Yin, et al. An Alternative Splice Form of CMTM8 Induces Apoptosis. Int J Biochem Cell Biol. 2007, 39:2107~2119
18 B. K. Lydersen, D. E. Pettijohn. Human-specific Nuclear Protein that Associates with the Polar Region of the Mitotic Apparatus: Distribution in a Human/Hamster Hybrid Cell. Cell. 1980, 22:489~499
19 C. H. Yang, E. J. Lambie, et al. NuMA: an Unusually Long Coiled-coil Related Protein in the Mammalian Nucleus. J Cell Biol. 1992, 116:1303~1317.
20 T. Wittmann, M. Wilm, E. Karsenti, et al. TPX2, A Novel Xenopus MAP Involved in Spindle Pole Organization. J Cell Biol. 2000, 149:1405~1418
21 T. Raemaekers, K. Ribbeck, J. Beaudouin, et al. NuSAP, a Novel Microtubule-Associated Protein Involved in Mitotic Spindle Organization. J Cell Biol. 2003, 162:1017~1029
22 E. Shen, Y. Lei, Q. Liu, et al. Identification and Characterization of INMAP, a Novel Interphase Nucleus and Mitotic Apparatus Protein that Is Involved in Spindle Formation and Cell Cycle Progression. Exp Cell Res. 2009, 315:1100~1116
23 K. Shinmura, R. A. Bennett, P. Tarapore, et al. Direct Evidence for the Role of Centrosomally Localized p53 in The Regulation of Centrosome Duplication. Oncogene. 2007, 26:2939~2944
24 L. C. Hsu, R. L. White. BRCA1 Is Associated with the Centrosome during Mitosis. Proc Natl Acad Sci USA. 1998, 95:12983~12988
25 A. C. Newton. Protein Kinase C: Structure, Function and Regulation. J Biol Chem. 1995, 270:28495~28498
26 A. Besson, T. L. Wilson, V. W. Yong. The Anchoring Protein RACK1 Links Protein Kinase C-εto IntegrinβChains Requirement for Adhesion and Motility. J Biol Chem. 2002, 277:22073~22084
27 X. A. Zhang, A. L. Bontrager, M. E. Hemler. Transmembrane-4 Superfamily Proteins Associate with Activated Protein Kinase C (PKC) and Link PKC to Specificβ1 integrins. J Biol Chem. 2001, 276:25005~25013
28 A. Pascale, D. L. Alkon, M. Grimaldi. Translocation of Protein kKinase C-betaII in Astrocytes Requires Organized Actin Cytoskeleton and Is Not Accompanied bySynchronous RACK1 Relocation. Glia. 2004, 46:169~182
29 E. O. Harrington, J. L?ffler, P. R. Nelson, et al. Enhancement of Migration by Protein Kinase C-αand Inhibition of Proliferation and Cell Cycle Progression by Protein Kinase C-δin Capillary Endothelial Cells. J Biol Chem. 1997, 272:7390~7397
30 J. Liliental, D. D. Chang. Rack1, a Receptor for Activated Protein Kinase C, Interacts with IntegrinβSubunit. J Biol Chem. 1998, 273:2379~2383
31 A. M. Martelli, C. Evangelisti, M. Nyakern, et al. Nuclear Protein Kinase C. Biochimica et Biophysica Acta. 2006, 1761:542~551
32 L. M. Neri, A. M. Billi, A. M. Martelli. Selective Nuclear Translocation of Protein Kinase Cαin Swiss 3T3 Cells Treated with IGF-I, PDGF and EGF. FEBS Lett. 1994, 347:63~68
33 S. Wagner, C. Harteneck, F. Hucho, et al. Analysis of the Subcellular Distribution of Protein Kinase CαUsing PKC-GFP Fusion Proteins. Exp. Cell Res. 2000, 258:204~214
34 A. Alison. How to Spot a Protein in a Crowd. Nature. 1999, 402:715~720
35 L. Lu, G. Tai, W. Hong. Interaction of Arl1 GTPase with the GRIP Domain of Golgin-245 as Assessed by GST (Glutathione-S-transferase) Pull-down Experiments. Methods Enzymol. 2005. 404:432~441
36 X. Huang, Z. Shi, W. Wang, et al. Identification and Characterization of a Novel Protein ISOC2 that Interacts with p16INK4a. Biochem Biophys Res Commun. 2007, 361:287~293
37王山.候选肿瘤抑制基因MARVELD1的表达及功能研究.哈尔滨工业大学博士学位论文. 2009:44~63
38高大. mP19假定蛋白的亚细胞定位及其磷酸化状态的分析.哈尔滨工业大学硕士学位论文. 2008:30~40
39王山,肖晟等.核蛋白MARVELD1与核质输入蛋白importinβ1相互作用的鉴定.中国生物化学与分子生物学报. 2009, 25:735~739
40 D. Du, F. Xu, L. Yu, et al. The Tight Junction Protein, Occludin, Regulates the Directional Migration of Epithelial Cells. Dev Cell. 2010, 18:52~63
41 M. D. Landis, D. D. Seachrist, M. E. Monta?ez-Wiscovich, et al. Gene Expression Profiling of Cancer Progression Reveals Intrinsic Regulation of Transforming Growth Factor-beta Signaling in ErbB2/Neu-induced Tumors from Transgenic Mice. Oncogene. 2005, 24:5173~5190
42 P. Kirstetter, M. B. Schuster, O. Bereshchenko, et al. Modeling of C/EBP Alpha Mutant Acute Myeloid Leukemia Reveals a Common Expression Signature ofCommitted Myeloid Leukemia-initiating Cells. Cancer Cell. 2008, 13:299~310
43吕冰洁.肿瘤相关新基因结构及功能研究的方法和策略.哈尔滨医科大学博士学位论文. 2004:74~79
44 C. C. Mello, D. J. Conte, Revealing the World of RNA Interference. Nature. 2004, 431:338~342
2 M. Furuse, T. Hirase, M. Itoh, et al. Occludin: a Novel Integral Membrane Proteins Localizing at Tight Junctions. J. Cell Biol. 1993, 123:1777~1788
3 L. Sánchez-Pulido, F. Martín-Belmonte, A. Valencia, et al. MARVEL: a Conserved Domain Involved in Membrane Apposition Events. Trends Biochem Sci. 2002, 27:599~601
4 K. Hübner, R. Windoffer, H. Hutter, et al. Tetraspan Vesicle Membrane Proteins: Synthesis, Subcellular Localization, and Functional Properties. Int Rev Cytol. 2002, 214:103~159
5 C. P. Arthur, M. H. Stowell. Structure of Synaptophysin: a Hexameric MARVEL-Domain Channel Protein. Structure, 2007, 15:707~714
6 http://www.ncbi.nlm.nih.gov
7 J. Ikenouchi, M. Furuse, K. Furuse, et al. Tricellulin Constitutes a Novel Barrier at Tricellular Contacts of Epithelial Cells. J Cell Biol. 2005, 171:939~945
8 S. Riazuddin, Z. M. Ahmed, A. S. Fanning, et al. Tricellulin Is a Tight-Junction Protein Necessary for Hearing. Am J Hum Genet. 2006, 79:1040~1051
9 D. R. Raleigh, A. M. Marchiando, Y. Zhang, et al. Tight Junction-associated MARVEL Proteins Marveld3, Tricellulin, and Occludin Have Distinct but Overlapping Functions. Mol Biol Cell. 2010, 21:1200~1213
10 E. Steed, N. T. Rodrigues, M. S. Balda, et al. Identification of MarvelD3 as a Tight Junction-associated Transmembrane Protein of the Occludin Family. BMC Cell Biol. 2009, 10:95
11 S. Wang, Y. Li, F. Han, et al. Identification and Characterization of MARVELD1, a Novel Nuclear Protein that Is Down-regulated in Multiple Cancers and Silenced by DNA Methylation. Cancer Lett. 2009, 282:77~86
12 W. Han, P. Ding, M. Xu, et al. Identification of Eight Genes Encoding Chemokine-like Factor Superfamily Members 1-8 (CKLFSF1-8) by in Silico Cloning and Experimental Validation. Genomics. 2003, 81:609~617
13 Y. Wang, J. Li, Y. Cui, et al. CMTM3, Located at the Critical Tumor Suppressor Locus 16q22.1, Is Silenced by CpG Methylation in Carcinomas and Inhibits Tumor Cell Growth Through Inducing Apoptosis. Cancer Res. 2009, 69:5194~5201
14 L. Shao, Y. Cui, H. Li, et al. CMTM5 Exhibits Tumor Suppressor Activities and IsFrequently Silenced by Methylation in Carcinoma Cell Lines. Clin Cancer Res. 2007, 13:6543~6562
15 C. Jin, P. Ding, Y. Wang, et al. Regulation of EGF Receptor Signaling by the MARVEL Domain-containing Protein CKLFSF8. FEBS Letter. 2005, 579:6375~6382
16 L. Shao, X. Guo, M. Plate, et al. CMTM5-v1 Induces Apoptosis in Cervical Carcinoma Cells. Biochem Biophys Res Commun. 2009, 379:866~871
17 D. Li, C. Jin, C. Yin, et al. An Alternative Splice Form of CMTM8 Induces Apoptosis. Int J Biochem Cell Biol. 2007, 39:2107~2119
18 B. K. Lydersen, D. E. Pettijohn. Human-specific Nuclear Protein that Associates with the Polar Region of the Mitotic Apparatus: Distribution in a Human/Hamster Hybrid Cell. Cell. 1980, 22:489~499
19 C. H. Yang, E. J. Lambie, et al. NuMA: an Unusually Long Coiled-coil Related Protein in the Mammalian Nucleus. J Cell Biol. 1992, 116:1303~1317.
20 T. Wittmann, M. Wilm, E. Karsenti, et al. TPX2, A Novel Xenopus MAP Involved in Spindle Pole Organization. J Cell Biol. 2000, 149:1405~1418
21 T. Raemaekers, K. Ribbeck, J. Beaudouin, et al. NuSAP, a Novel Microtubule-Associated Protein Involved in Mitotic Spindle Organization. J Cell Biol. 2003, 162:1017~1029
22 E. Shen, Y. Lei, Q. Liu, et al. Identification and Characterization of INMAP, a Novel Interphase Nucleus and Mitotic Apparatus Protein that Is Involved in Spindle Formation and Cell Cycle Progression. Exp Cell Res. 2009, 315:1100~1116
23 K. Shinmura, R. A. Bennett, P. Tarapore, et al. Direct Evidence for the Role of Centrosomally Localized p53 in The Regulation of Centrosome Duplication. Oncogene. 2007, 26:2939~2944
24 L. C. Hsu, R. L. White. BRCA1 Is Associated with the Centrosome during Mitosis. Proc Natl Acad Sci USA. 1998, 95:12983~12988
25 A. C. Newton. Protein Kinase C: Structure, Function and Regulation. J Biol Chem. 1995, 270:28495~28498
26 A. Besson, T. L. Wilson, V. W. Yong. The Anchoring Protein RACK1 Links Protein Kinase C-εto IntegrinβChains Requirement for Adhesion and Motility. J Biol Chem. 2002, 277:22073~22084
27 X. A. Zhang, A. L. Bontrager, M. E. Hemler. Transmembrane-4 Superfamily Proteins Associate with Activated Protein Kinase C (PKC) and Link PKC to Specificβ1 integrins. J Biol Chem. 2001, 276:25005~25013
28 A. Pascale, D. L. Alkon, M. Grimaldi. Translocation of Protein kKinase C-betaII in Astrocytes Requires Organized Actin Cytoskeleton and Is Not Accompanied bySynchronous RACK1 Relocation. Glia. 2004, 46:169~182
29 E. O. Harrington, J. L?ffler, P. R. Nelson, et al. Enhancement of Migration by Protein Kinase C-αand Inhibition of Proliferation and Cell Cycle Progression by Protein Kinase C-δin Capillary Endothelial Cells. J Biol Chem. 1997, 272:7390~7397
30 J. Liliental, D. D. Chang. Rack1, a Receptor for Activated Protein Kinase C, Interacts with IntegrinβSubunit. J Biol Chem. 1998, 273:2379~2383
31 A. M. Martelli, C. Evangelisti, M. Nyakern, et al. Nuclear Protein Kinase C. Biochimica et Biophysica Acta. 2006, 1761:542~551
32 L. M. Neri, A. M. Billi, A. M. Martelli. Selective Nuclear Translocation of Protein Kinase Cαin Swiss 3T3 Cells Treated with IGF-I, PDGF and EGF. FEBS Lett. 1994, 347:63~68
33 S. Wagner, C. Harteneck, F. Hucho, et al. Analysis of the Subcellular Distribution of Protein Kinase CαUsing PKC-GFP Fusion Proteins. Exp. Cell Res. 2000, 258:204~214
34 A. Alison. How to Spot a Protein in a Crowd. Nature. 1999, 402:715~720
35 L. Lu, G. Tai, W. Hong. Interaction of Arl1 GTPase with the GRIP Domain of Golgin-245 as Assessed by GST (Glutathione-S-transferase) Pull-down Experiments. Methods Enzymol. 2005. 404:432~441
36 X. Huang, Z. Shi, W. Wang, et al. Identification and Characterization of a Novel Protein ISOC2 that Interacts with p16INK4a. Biochem Biophys Res Commun. 2007, 361:287~293
37王山.候选肿瘤抑制基因MARVELD1的表达及功能研究.哈尔滨工业大学博士学位论文. 2009:44~63
38高大. mP19假定蛋白的亚细胞定位及其磷酸化状态的分析.哈尔滨工业大学硕士学位论文. 2008:30~40
39王山,肖晟等.核蛋白MARVELD1与核质输入蛋白importinβ1相互作用的鉴定.中国生物化学与分子生物学报. 2009, 25:735~739
40 D. Du, F. Xu, L. Yu, et al. The Tight Junction Protein, Occludin, Regulates the Directional Migration of Epithelial Cells. Dev Cell. 2010, 18:52~63
41 M. D. Landis, D. D. Seachrist, M. E. Monta?ez-Wiscovich, et al. Gene Expression Profiling of Cancer Progression Reveals Intrinsic Regulation of Transforming Growth Factor-beta Signaling in ErbB2/Neu-induced Tumors from Transgenic Mice. Oncogene. 2005, 24:5173~5190
42 P. Kirstetter, M. B. Schuster, O. Bereshchenko, et al. Modeling of C/EBP Alpha Mutant Acute Myeloid Leukemia Reveals a Common Expression Signature ofCommitted Myeloid Leukemia-initiating Cells. Cancer Cell. 2008, 13:299~310
43吕冰洁.肿瘤相关新基因结构及功能研究的方法和策略.哈尔滨医科大学博士学位论文. 2004:74~79
44 C. C. Mello, D. J. Conte, Revealing the World of RNA Interference. Nature. 2004, 431:338~342