Mimicking Cdk2 phosphorylation of Bcl-xL at Ser73 results in caspase activation and Bcl-xL cleavage.

Ns Seng,J Megyesi,Pm Price,A Tarcsafalvi

doi:10.1038/cddiscovery.2016.1

Abstract

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.

Highlights

Cis-diamminedichloroplatinum (II) or cisplatin has been widely used to treat various solid tumors, such as ovarian, testicular, head and neck, lung, and bladder cancers.[1]
We report that Cdk[2] phosphorylation of Bcl-xL at Ser[73] is an upstream event resulting in perinuclear mitochondrial clustering, caspase activation, and subsequent Bcl-xL cleavage after D61 and D76
As S73D-Bcl-xL mimics the phosphorylation of Bcl-xL by Cdk[2] after cisplatin treatment, we investigated whether endogenous Bcl-xL would be cleaved after cisplatin treatment

Summary

Introduction

Cis-diamminedichloroplatinum (II) or cisplatin has been widely used to treat various solid tumors, such as ovarian, testicular, head and neck, lung, and bladder cancers.[1]. Apart from its role in cell-cycle regulation, its role in cell death pathways is poorly understood but has become increasingly recognized.[3,4,5,6,7]. Our laboratory found that cisplatin-induced kidney cell death was dependent on Cdk[2] activation, and inhibition of Cdk[2] by p21, dominant-negative (DN)-Cdk[2], or chemical inhibitors such as roscovitine or purvalanol protected kidney cells from cisplatininduced apoptosis both in vitro and in vivo.[8,9,10] the mechanisms of how Cdk[2] mediates apoptosis are still unknown

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Results

Conclusion