N-Benzyladriamycin-14-Valerate (AD198) Induces Apoptosis through Protein Kinase C-δ–Induced Phosphorylation of Phospholipid Scramblase 3

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Abstract
Phospholipid scramblase 3 (PLS3) is an enzyme that plays a critical role in mitochondrial morphology, functions, and apoptotic response. During apoptosis, activated protein kinase C-δ (PKC-δ) translocates to mitochondria and phosphorylates PLS3. Here, we utilize an extranuclear-targeted anthracycline N-benzyladriamycin-14-valerate (AD198), a PKC-δ activator, to investigate the mechanism of PLS3 phosphorylation by PKC-δ. Overexpression of PLS3 enhanced, whereas down-regulation of PLS3 by small interfering RNA decreased, the sensitivity of AD198-induced apoptosis. Overexpression of PKC-δ, but not the kinase-defective PKC-δ, and AD198 treatment enhanced threonine phosphorylation of PLS3. The phosphorylated threonine was mapped to Thr21 of PLS3. Mutation of Thr21 to alanine did not affect mitochondrial localization of PLS3 but abolished threonine phosphorylation by PKC-δ in vitro and AD198-induced PLS3 phosphorylation in vivo. Expression of PLS3(T21A) in cells could not enhance AD198-induced apoptosis compared with expression of the wild-type PLS3. Using benzyloxycarbonyl-Val-Ala-Asp-(OMe) fluoromethyl ketone and cyclosporine A, we also showed that AD198-induced PLS3 phosphorylation occurs upstream of caspase activation and independent of mitochondrial permeability transition. These studies establish that AD198-activated PKC-δ induces phosphorylation of mitochondrial PLS3 at Thr21 and that PLS3 is a critical downstream effector of PKC-δ in AD198-induced apoptosis.