Molecular mechanism of endosomal sorting of growth factors and receptors

生长因子和受体内体分选的分子机制

• 批准号: 15370053
• 负责人: KITAMURA Naomi
• 金额: $ 9.79万
• 依托单位: Tokyo Institute of Technology
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15370053/
• 关键词: growth factor; growth factor receptor; early endosome; sorting; Hrs; STAM; deubiquitinating enzyme UBPY; ALG-2; ユビキチン化; STAM; Alix

Upon stimulation with growth factors, their receptors are rapidly internalized as a complex with bound ligands, and are transported to the endosome. From the endosome, ligand-bound receptors are transported to the lysosome for degradation. In this study, we examined the molecular mechanisms how Hrs and STAM regulate the endosomal sorting of growth factor/receptor complexes, and obtained the following results.1.STAM mutants lacking the Hrs-binding activity were defective in causing the enlargement of early endosomes, accumulating ubiquitinated proteins on this aberrant organelle, and inhibiting the degradation of ligand-activated epidermal growth factor receptors(EGFR). These results indicate that association with Hrs on the early endosome is a prerequisite for STAM function.2.Overexpression of a deubiquitinating enzyme UBPY that is a STAM binding protein, reduced the ubiquitination level of EGFR and delayed its degradation in EGF-stimulated cells. In contrast overexpression of a catalytically-inactive UBPY mutant did not show such effects. These results indicate that UBPY plays an important role through reduction of the ubiquitination level of EGFR in regulation of EGFR sorting.3.ALG-2 was identified as a novel Hrs-binding protein by a yeast two-hybrid screening. Hrs, ALG-2 and an ALG-2 binding protein Alix were bound in a Ca^<2+>-dependent manner. Treatment of cells with a Ca^<2+> ionophore induced colocalization of these proteins to the early endosome. These results suggest that Hrs regulates endosomal sorting by recruiting Alix through ALG-2 to the early endosome in a Ca^<2+>-dependent manner.

在生长因子刺激后，它们的受体迅速内化为与结合配体的复合物，并被转运至内体。配体结合受体从内体转运至溶酶体进行降解。本研究探讨了Hrs和STAM调节生长因子/受体复合物内体分选的分子机制，得到了以下结果：1.缺乏Hrs结合活性的STAM突变体无法引起早期内体的增大，在异常细胞器上积累泛素化蛋白，并抑制配体激活的降解。 表皮生长因子受体（EGFR）。这些结果表明，早期内体上Hrs的结合是STAM功能的先决条件。2.STAM结合蛋白去泛素化酶UBPY的过度表达，降低了EGFR的泛素化水平，延缓了EGF刺激细胞的降解。相反，催化失活的 UBPY 突变体的过度表达没有显示出这样的效果。这些结果表明UBPY通过降低EGFR的泛素化水平在EGFR分选的调控中发挥重要作用。3.酵母双杂交筛选将ALG-2鉴定为一种新型的Hrs结合蛋白。小时，ALG-2和ALG-2结合蛋白Alix以Ca 2+ 依赖性方式结合。用Ca 2+ 离子载体处理细胞诱导这些蛋白质共定位至早期内体。这些结果表明，Hrs通过ALG-2以Ca 2+ 依赖性方式将Alix募集到早期内体来调节内体分选。

项目成果

K.Nakano: "Cofilin phosphorylation and actin polymerization by NRK/NESK, a member of the germinal center kinase family"Exp.Cell Res.. 287. 219-227 (2003)

K.Nakano：“生发中心激酶家族成员 NRK/NESK 的肌动蛋白丝切蛋白磷酸化和肌动蛋白聚合”Exp.Cell Res.. 287. 219-227 (2003)

Activation of c-Met (hepatocyte growth factor receptor) in human gastric cancer tissue

• DOI: 10.1111/j.1349-7006.2004.tb02185.x
• 发表时间: 2004-10-01
• 期刊: CANCER SCIENCE
• 影响因子: 5.7
• 作者: Inoue, T;Kataoka, H;Miyazawa, K
• 通讯作者: Miyazawa, K

A scaffold protein JIP-1b enhances amyloid precursor protein phosphorylation by JNK and its association with kinesin light chain 1

• DOI: 10.1074/jbc.m212160200
• 发表时间: 2003-06-20
• 期刊: JOURNAL OF BIOLOGICAL CHEMISTRY
• 影响因子: 4.8
• 作者: Inomata, H;Nakamura, Y;Kitamura, N
• 通讯作者: Kitamura, N

STAM protein bind ubiquitinated proteins on the early endosome via the VHS domain and ubiquitin-interacting motif

STAM 蛋白通过 VHS 结构域和泛素相互作用基序结合早期内涵体上的泛素化蛋白

• 发表时间: 2003
• 期刊: Mol.Biol.Cell 14
• 作者: Matsumoto;M.;Yada;M.;Hatakeyama;S.;Ishimoto;H.;Tanimura;T.;Tsuji;S.;Kakizuka;A.;Kitagawa;M.;Nakayama;K.I.;E.Mizuno
• 通讯作者: E.Mizuno

Involvement of down regulation of Cdk2 activity in hepatocyte growth factor-induced cell cycle arrest at G1 in the human hepatocellular carcinoma cell line HepG2

Cdk2活性下调参与肝细胞生长因子诱导的人肝癌细胞系HepG2细胞周期阻滞在G1期

• 发表时间: 2004
• 期刊: J.Biochem. 136
• 作者: Kano;F.;山内忍;村田昌之;Fumi Kano;Fumi Kano;Satomi Nadanaka;J.Ichinose;加納ふみ;加納ふみ;田中亜路;M.Nakamura;M.Komada;H.Kakinuma;H.Tanaka;J.Han;E.Mizuno;T.Hori;E.Mizuno;C.Morino;H.Itoh;T.Inoue;Y.Tsukada
• 通讯作者: Y.Tsukada