Cisplatin is a widely used chemotherapeutic agent, yet its efficacy is limited by nephrotoxicity. The severity of nephrotoxicity is associated with the extent of kidney cell death. Previously, we found that cisplatin-induced kidney cell death was dependent on Cdk2 activation, and inhibition of Cdk2 protected cells from cisplatin-induced apoptosis. Using an in vitro kination assay, we showed that Cdk2 phosphorylated Bcl-xL, an anti-apoptotic member of Bcl-2 family proteins, at serine 73. We also found that this phosphorylated Bcl-xL participated in cell death, as a phosphomimetic mutant of Bcl-xL at the serine 73 site (S73D-Bcl-xL) activated caspases. We now find that S73D-Bcl-xL was cleaved at D61 and D76, which are putative caspase cleavage sites, to generate 15-kDa and 12-kDa fragments. Unlike full-length Bcl-xL, these cleavage products of Bcl-xL were previously reported to be pro-apoptotic. We sought to determine whether these Bcl-xL fragments were necessary for the induction of cell death by S73D-Bcl-xL. Mutation of these caspase cleavage sites prevented the formation of the 15-kDa and 12-kDa Bcl-xL cleavage products, but apoptosis still persisted in a S73D modified Bcl-xL. Our findings show that Cdk2 phosphorylation of Bcl-xL at Ser73, but not the Bcl-xL cleavage products, is necessary and sufficient to induce cell death.

中文翻译：

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在Ser73上模仿Bcl-xL的Cdk2磷酸化会导致胱天蛋白酶激活和Bcl-xL裂解。

顺铂是一种广泛使用的化学治疗剂，但其功效受到肾毒性的限制。肾毒性的严重程度与肾细胞死亡的程度有关。以前，我们发现顺铂诱导的肾细胞死亡取决于Cdk2活化，而抑制Cdk2保护细胞免受顺铂诱导的细胞凋亡。使用体外激酶测定，我们显示Cdk2磷酸化Bcl-xL，Bcl-2家族蛋白的抗凋亡成员，在丝氨酸73处。我们还发现，该磷酸化Bcl-xL作为拟磷酸酶突变体参与细胞死亡。丝氨酸73位点的Bcl-xL（S73D-Bcl-xL）激活的胱天蛋白酶。现在我们发现，S73D-Bcl-xL在假定的半胱天冬酶裂解位点D61和D76处裂解，产生15-kDa和12-kDa片段。与全长Bcl-xL不同，Bcl-xL的这些裂解产物以前被报道是促凋亡的。我们试图确定这些Bcl-xL片段对于S73D-Bcl-xL诱导细胞死亡是否必需。这些半胱天冬酶切割位点的突变阻止了15 kDa和12 kDa Bcl-xL切割产物的形成，但是凋亡仍然在S73D修饰的Bcl-xL中持续存在。我们的发现表明，在Ser73上Bcl-xL的Cdk2磷酸化，而不是Bcl-xL裂解产物，是诱导细胞死亡的必要和充分条件。