Analysis of Intermediate Filament Binding Proteins

中间丝结合蛋白的分析

• 批准号: 13480241
• 负责人: INAGAKI Masaki
• 金额: $ 9.66万
• 依托单位: Aichi Cancer Center
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13480241/
• 关键词: Intermediate filament; Protein phosphorylation; site-and phosphorylation state-specific antibody; Aurora-B kinase; IF bunding protein; 中間径フィラメント; ケラチン; TNF; TRADD; Densin-180

1. We identified Aurora-B as a cleavage furrow (CF) kinase. Aurora-B phosphorylates type III intermediate filaments (Ifs), vimentin, GFAP, and desmin, and the phosphorylation causes reduction of their filament forming ability. We next determined the phosphorylation sites of these Ifs by Aurora-B. IF mutants, in which phosphorylation sites by Aurora-B and/or Rho-kinase are changed to Ala or Gly, cause dramatic defect of filament separation between daughter cells in cytokinesis. These results indicate that Aurora-B coordinately regulates cytokinesis with Rho-kinase through the cleavage furrow-specific phosphorylation of type III Ifs.2. We identified human TNF receptor 1 (TNFR1)-associated death domain protein (TRADD) to be the keratin 18 (K18)-interacting protein. Endogenous TRADD coimmunoprecipitated with K18, and colocalized with K8/18 filaments in human mammary epithelial cells. Overexpression of the NH_2- terminus (aa 1-270) of K18 containing the TRADD-binding domain as well as overexpression of K8/18 in SW13 cells, which are devoid of keratins, rendered the cells more resistant to killing by TNF. These results suggest that K18 may sequester TRADD to attenuate interactions of TRADD with activated TNFR1 and moderate TNF-induced apoptosis in simple epithelial cells.3. We isolated δ-catenin/neural plakophilin-related armadillo-repeat protein (NPRAP) as a Densin- 180-interacting protein. Densin-180 is associated in vivo with δ-catenin/NPRAP and may be involved in organization of the synaptic cell-cell junction through interaction with theδ-catenin/NPRAP-N- cadherin complex.

1. 我们确定 Aurora-B 是一种裂解沟 (CF) 激酶。 Aurora-B 磷酸化 III 型中间丝 (Ifs)、波形蛋白、GFAP 和结蛋白，磷酸化导致其丝形成能力降低。接下来我们通过 Aurora-B 确定了这些 If 的磷酸化位点。 IF 突变体，其中 Aurora-B 和/或 Rho-激酶的磷酸化位点变为 Ala 或 Gly，导致胞质分裂中子细胞之间的细丝分离出现严重缺陷。这些结果表明，Aurora-B 通过 III 型 Ifs.2 的裂解沟特异性磷酸化与 Rho 激酶协调调节胞质分裂。我们确定人 TNF 受体 1 (TNFR1) 相关死亡域蛋白 (TRADD) 是角蛋白 18 (K18) 相互作用蛋白。内源性 TRADD 与 K18 共免疫沉淀，并与人乳腺上皮细胞中的 K8/18 丝共定位。含有TRADD结合结构域的K18的NH_2-末端（氨基酸1-270）的过表达以及K8/18在缺乏角蛋白的SW13细胞中的过表达，使得细胞对TNF的杀伤具有更强的抵抗力。这些结果表明，K18可能会隔离TRADD，从而减弱TRADD与激活的TNFR1的相互作用，并减轻TNF诱导的简单上皮细胞的凋亡。3．我们分离了 δ-连环蛋白/神经斑亲蛋白相关的犰狳重复蛋白 (NPRAP) 作为 Densin-180 相互作用蛋白。 Densin-180 在体内与 δ-连环蛋白/NPRAP 相关，并且可能通过与 δ-连环蛋白/NPRAP-N-钙粘蛋白复合物相互作用参与突触细胞-细胞连接的组织。

项目成果

Minoshima, Y., et al.: "Aurora B Phosphorylates MgcRacGAP and Induces PhoGAP Activity during M Phase : Identification of a RhoGAP Indispensable for Cytokinesis"Dev.Cell. 4. 549-560 (2003)

Minoshima, Y. 等人：“Aurora B 磷酸化 MgcRacGAP 并在 M 期诱导 PhoGAP 活性：鉴定细胞分裂所必需的 RhoGAP”Dev.Cell。

Goto, H., et al.: "Aurora-B phosphorylates histone H3 at serine28 with regard to the mitosic chromosome condensation"Genes to Cells. (in press).

Goto, H. 等人：“Aurora-B 在丝氨酸 28 处磷酸化组蛋白 H3，与有丝分裂染色体浓缩有关”Genes to Cells。

Goto, H., et al.: "Aurora-B phosphorylates histone H3 at serine28 with regard to the mitosic chromosome condensation"Genes to Cells. 7. 11-17 (2002)

Goto, H. 等人：“Aurora-B 在丝氨酸 28 处磷酸化组蛋白 H3，与有丝分裂染色体浓缩有关”Genes to Cells。

Goto, H., et al.: "Aurora-B phosphorylates histone H3 at serine28 wish regard to the mitosis chromosome condensation"Genes to Cells. 7. 11-17 (2002)

Goto, H. 等人：“Aurora-B 在丝氨酸 28 处磷酸化组蛋白 H3，希望考虑到有丝分裂染色体浓缩”基因到细胞。

Izawa, I., et al.: "Densin-180 interacts with δ-catenin/neural plakophilin-related armadillo-repeat protein at synapses"J.Biol.Chem. 277. 5345-5350 (2002)

Izawa, I., et al.：“Densin-180 在突触处与 δ-连环蛋白/神经斑亲核蛋白相关的犰狳重复蛋白相互作用”J.Biol.Chem. 277. 5345-5350 (2002)