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

¹ Huntsman Cancer Institute and Department of Internal Medicine, University of Utah, Salt Lake City, Utah and ² Department of Pharmacology and University of Tennessee Cancer Institute, University of Tennessee Health Science Center, Memphis, Tennessee

Requests for reprints: Ray M. Lee, Huntsman Cancer Institute at University of Utah, 2000 Circle of Hope, Suite 5244, Salt Lake City, UT 84112. Phone: 801-585-0611; Fax: 801-585-0900; E-mail: ray.lee{at}hci.utah.edu.

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- (PKC-) translocates to mitochondria and phosphorylates PLS3. Here, we utilize an extranuclear-targeted anthracycline N-benzyladriamycin-14-valerate (AD198), a PKC- activator, to investigate the mechanism of PLS3 phosphorylation by PKC-. Overexpression of PLS3 enhanced, whereas down-regulation of PLS3 by small interfering RNA decreased, the sensitivity of AD198-induced apoptosis. Overexpression of PKC-, but not the kinase-defective PKC-, 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- 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- induces phosphorylation of mitochondrial PLS3 at Thr²¹ and that PLS3 is a critical downstream effector of PKC- in AD198-induced apoptosis.

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