BCL-2 Is Phosphorylated and Inactivated by an ASK1/Jun N-Terminal Protein Kinase Pathway Normally Activated at G2/M
BCL-2 Is Phosphorylated and Inactivated by an ASK1/Jun N-Terminal Protein Kinase Pathway Normally Activated at G2/M
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DOI：
10.1128/mcb.19.12.8469
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发表时间：
1999-12
影响因子：
5.3
通讯作者：
Kazuhito Yamamoto;H. Ichijo;S. Korsmeyer
中科院分区：
文献类型：
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作者：
Kazuhito Yamamoto;H. Ichijo;S. Korsmeyer
摘要 多种信号转导途径能够修饰 BCL-2 家族成员以重置细胞凋亡的易感性。我们使用二维肽图谱和测序来鉴定 BCL-2 非结构化环内的三个残基（Ser70、Ser87 和 Thr69），这些残基响应微管损伤剂而被磷酸化，微管损伤剂也会将细胞阻滞在 G2/M 期。将这些位点改为丙氨酸可在生理死亡信号和紫杉醇发出后赋予 BCL-2 更强的抗凋亡活性，表明磷酸化正在失活。对通过淘洗富集的细胞周期不同阶段的循环细胞进行的检查表明，BCL-2 在细胞周期的 G2/M 期被磷酸化。事实证明，G2/M 期细胞更容易受到死亡信号的影响，BCL-2 的磷酸化似乎是其中的原因，因为 Ser70Ala 取代恢复了对细胞凋亡的抵抗力。我们注意到 ASK1 和 JNK1 通常在 G2/M 期激活，并且 JNK 能够磷酸化 BCL-2。一系列野生型和显性失活激酶的表达表明 ASK1/Jun N 末端蛋白激酶 1 (JNK1) 途径在体内磷酸化 BCL-2。此外，显性失活 ASK1、(dnASK1)、dnMKK7 和 dnJNK1 的组合可抑制紫杉醇诱导的 BCL-2 磷酸化。因此，应激反应激酶在细胞周期进展过程中磷酸化 BCL-2，作为正常生理过程，在 G2/M 时使 BCL-2 失活。
ABSTRACT Multiple signal transduction pathways are capable of modifying BCL-2 family members to reset susceptibility to apoptosis. We used two-dimensional peptide mapping and sequencing to identify three residues (Ser70, Ser87, and Thr69) within the unstructured loop of BCL-2 that were phosphorylated in response to microtubule-damaging agents, which also arrest cells at G2/M. Changing these sites to alanine conferred more antiapoptotic activity on BCL-2 following physiologic death signals as well as paclitaxel, indicating that phosphorylation is inactivating. An examination of cycling cells enriched by elutriation for distinct phases of the cell cycle revealed that BCL-2 was phosphorylated at the G2/M phase of the cell cycle. G2/M-phase cells proved more susceptible to death signals, and phosphorylation of BCL-2 appeared to be responsible, as a Ser70Ala substitution restored resistance to apoptosis. We noted that ASK1 and JNK1 were normally activated at G2/M phase, and JNK was capable of phosphorylating BCL-2. Expression of a series of wild-type and dominant-negative kinases indicated an ASK1/Jun N-terminal protein kinase 1 (JNK1) pathway phosphorylated BCL-2 in vivo. Moreover, the combination of dominant negative ASK1, (dnASK1), dnMKK7, and dnJNK1 inhibited paclitaxel-induced BCL-2 phosphorylation. Thus, stress response kinases phosphorylate BCL-2 during cell cycle progression as a normal physiologic process to inactivate BCL-2 at G2/M.