Role of Bcl-2 phosphorylation after microtubule disruption

Bcl-2 磷酸化在微管破坏后的作用

• 批准号: 13680726
• 负责人: SIMIZU Siro
• 金额: $ 2.3万
• 依托单位: RIKEN
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 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/grant/KAKENHI-PROJECT-13680726/
• 关键词: apoptosis; ERKs; phoslactomycin; PP2A; Bcl-2; Erk; Bc1-2; ミトコンドリア; リン酸化; 細胞周期; ERK2

To clarify the role of Bcl-2 phosphorylation, we established the wild-type and several mutant-form of Bcl-2-expressing cell lines. We found that transmembrane region ?-deleted Bcl-2 (Bcl-2ΔTM), which was not localized at mitochondria, was constitutively phosphorylated in the cells. We identified Ser-87 residue within Bcl-2ΔTM protein as the phosphorylation site. To identify the responsible kinase for phosphorylation of Bcl-2ΔTM, we used several kinase inhibitors, and found that PD98059 and U0126, MEK1 inhibitors, suppressed phosphorylation of Bcl-2ΔTM. Moreover, in vitro kinase assay showed that ERK, a downstream kinase of MEK1, phosphorylated at Ser-87 residue of Bcl-2ΔTM. Both wild-type Bcl-2 and Bcl-2ΔTM could bind to ERK. Wild-type Bcl-2 could associate with PP2A, thus, wild-type Bcl-2 was not phosphorylated constitutively. On the other hand, Bcl-2ΔTM could interact with ERK, but not with PP2A. Moreover, Bcl-2 protein was constitutively phosphorylated in normal human blood cells, whereas Bcl-2 was not phosphorylated in human tumor cell lines.

为了阐明Bcl-2磷酸化的作用，我们建立了野生型和几种突变型Bcl-2表达细胞系。我们发现了跨膜区？-缺失的Bcl-2（Bcl-2ΔTM）在细胞中呈组成性磷酸化。我们确定Bcl-2ΔTM蛋白中的Ser-87残基为磷酸化位点。为了确定Bcl-2ΔTM磷酸化的负责激酶，我们使用了几种激酶抑制剂，发现PD 98059和U 0126，MEK 1抑制剂，抑制Bcl-2ΔTM的磷酸化。体外激酶活性测定结果表明，MEK 1的下游激酶ERK在Bcl-2ΔTM的Ser-87残基上发生磷酸化。野生型Bcl-2和Bcl-2ΔTM均能与ERK结合。野生型Bcl-2可以与PP 2A结合，因此，野生型Bcl-2不是组成性磷酸化的。Bcl-2ΔTM与ERK相互作用，而与PP 2A不相互作用。此外，Bcl-2蛋白在正常人血细胞中组成性磷酸化，而Bcl-2在人肿瘤细胞系中不磷酸化。

项目成果

Kawatani, M., Uchi, M., Simizu, S., Osada, H., Imoto, M.: "Trnasmembrane domain of Bcl-2 is required for inhibition of ceramide synthesis, but not cytochrome c release in the pathway of inostamycin-induced apoptosis"Experimental Cell Research. (in press).

Kawatani, M.、Uchi, M.、Simizu, S.、Osada, H.、Imoto, M.：“Bcl-2 的 Trnasmembrane 结构域对于抑制神经酰胺合成是必需的，但在肌霉素途径中不需要抑制细胞色素 c 的释放

Simizu, S., Ishida, K., Wierzba, M. K., Sato, T. & Osada, H.: "Expression of heparanase in human tumor cell lines and human head and neck tumors"Cancer Letters. (in press).

西米祖，S.，石田，K.，维尔兹巴，M.K.，佐藤，T.

Kawatani, M., Uchi, M., Simizu, S., Osada, H. & Imoto, M.: "Transmembrane domain of Bcl-2 is required for inhibition of ceramide synthesis, but not cytochrome c release in the pathway of inostamycin-induced apoptosis"Experimental Cell Research. (in press)

川谷，M.，内，M.，西水，S.，长田，H.

Nagashima, R, Kondoh, M., Kawase, M., Simizu, S., Osada, H., Fujii, M., Watanabe, Y., Sato, M. & Asakawa, Y.: "Apoptosis-inducing properties of ent-kaurene-type diterpenoids from the liverwort Jungermannia truncata"Planta Medica. (in press).

长岛，R，近藤，M.，河濑，M.，西米祖，S.，大田，H.，藤井，M.，渡边，Y.，佐藤，M.

清水史郎, 長田裕之: "微小管作用薬の抗がん作用"分子細胞治療. 2. 366-369 (2001)

Shiro Shimizu、Hiroyuki Nagata：“微管作用药物的抗癌作用”《分子细胞疗法》2. 366-369 (2001)。