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视网膜母细胞瘤蛋白在细胞凋亡中的内部裂解特性

Characterization of interior cleavage of retinoblastoma protein in apoptosis.

作者信息

Fattman C L, An B, Dou Q P

机构信息

Department of Pharmacology, University of Pittsburgh, School of Medicine, Pennsylvania, USA.

出版信息

J Cell Biochem. 1997 Dec 1;67(3):399-408.

PMID:9361194
Abstract

Previously we reported that at the onset of apoptotic execution, retinoblastoma protein (RB) was cleaved in its interior region, resulting in production of two major fragments, p48 and p68, and that the RB interior cleavage was mediated by a caspase-like activity. Here, we further characterized the RB interior cleavage process in human leukemia cells treated with the anticancer agent etoposide. We found that the RB interior cleavage activity was much more sensitive to two specific tetrapeptide caspase inhibitors, YVAD-CMK and DEVD-FMK, than the poly(ADP-ribose) polymerase cleavage activity, suggesting that two distinct caspases are involved in these processes. Several Asp residues are located in amino acids 341-421 of RB protein, and cleavage of any one of these sites by a caspase would generate a p48, which contains the amino terminus, and a p68 fragment, which contains the A/B pocket and the carboxyl terminus. This hypothesis was supported by the fact that the p48 and p68 fragments had selective binding affinity to different RB antibodies and that the p48 was found only in the low-salt-extracted cytoplasmic fraction, while the p68 was only in the nuclear fraction, of the apoptotic cells. However, the nuclear binding partner of the p68 RB fragment is not the transcription factor E2F-1 since a specific E2F-1 antibody coimmunoprecipitated only the unphosphorylated form of RB, but not the p68 fragment. Lastly, we confirmed that RB also underwent dephosphorylation and carboxyl terminal cleavage during apoptosis, as we and others reported previously.

摘要

此前我们报道过,在凋亡执行开始时,视网膜母细胞瘤蛋白(RB)在其内部区域被切割,产生两个主要片段,p48和p68,并且RB内部切割是由一种类似半胱天冬酶的活性介导的。在此,我们进一步对用抗癌药物依托泊苷处理的人白血病细胞中的RB内部切割过程进行了表征。我们发现,RB内部切割活性比聚(ADP - 核糖)聚合酶切割活性对两种特异性四肽半胱天冬酶抑制剂YVAD - CMK和DEVD - FMK更为敏感,这表明两个不同的半胱天冬酶参与了这些过程。几个天冬氨酸残基位于RB蛋白的氨基酸341 - 421中,半胱天冬酶对这些位点中任何一个的切割都会产生一个包含氨基末端的p48和一个包含A/B口袋及羧基末端的p68片段。这一假设得到以下事实的支持:p48和p68片段对不同的RB抗体具有选择性结合亲和力,并且在凋亡细胞中,p48仅存在于低盐提取的细胞质部分,而p68仅存在于核部分。然而,p68 RB片段的核结合伴侣不是转录因子E2F - 1,因为一种特异性E2F - 1抗体共免疫沉淀的仅是未磷酸化形式的RB,而非p68片段。最后,我们证实,正如我们和其他人之前报道的那样,RB在凋亡过程中也经历了去磷酸化和羧基末端切割。

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