title: Phosphorylation of mitochondrial phospholipid scramblase 3 by protein kinase C-delta induces its activation and facilitates mitochondrial targeting of tBid.
abstract: Phospholipid scramblase 3 (PLS3) is a member of the phospholipid scramblase family present in mitochondria. PLS3 plays an important role in regulation of mitochondrial morphology, respiratory function, and apoptotic responses. PLS3 is phosphorylated by PKC-delta at Thr21 and is the mitochondrial target of PKC-delta-induced apoptosis. Cells with overexpression of PLS3, but not the phosphoinhibitory mutant PLS3(T21A), are more susceptible to apoptosis induced by AD198, an extranuclear targeted anthracycline that activates PKC-delta. Here we report that the phosphomimetic mutant of PLS3(T21D) by itself can induce apoptosis in HeLa cells. Using proteoliposomes with addition of pyrene-labeled phosphatidylcholine (PC) at the outer leaflet, we measured the lipid flip-flop activity of PLS3 and its phosphorylation mutant. PLS3(T21D) is more potent than wild-type PLS3 or PLS3(T21A) to transfer pyrene-PC from the outer leaflet to the inner leaflet of liposomes. Based on our previous finding that PLS3 enhances tBid-induced mitochondrial damages, we tested the hypothesis that PLS3 enhances cardiolipin translocation to mitochondrial surface and facilitates tBid targeting. Fluorescein-labeled tBid(G94E) was used as a probe to quantify cardiolipin on the surface of mitochondria. Mitochondria from cells treated with AD198 or cells expressing PLS3(T21D) had a higher level of tBid-binding capacity than control cells or cells expressing wild-type PLS3. These findings indicate that phosphorylation of PLS3 by PKC-delta induces PLS3 activation to facilitate mitochondrial targeting of tBid and apoptosis.
authors.affiliation: Kunming Medical College, Kunming, Yunnan, P.R. China.
authors.forename: Yongwen
authors.initials: Y
authors.lastname: He
authors.forename: Jihua
authors.initials: J
authors.lastname: Liu
authors.forename: Douglas
authors.initials: D
authors.lastname: Grossman
authors.forename: David
authors.initials: D
authors.lastname: Durrant
authors.forename: Trevor
authors.initials: T
authors.lastname: Sweatman
authors.forename: Leonard
authors.initials: L
authors.lastname: Lothstein
authors.forename: Raquel F
authors.initials: RF
authors.lastname: Epand
authors.forename: Richard M
authors.initials: RM
authors.lastname: Epand
authors.forename: Ray M
authors.initials: RM
authors.lastname: Lee
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D051023:BH3 Interacting Domain Death Agonist Protein / Q000378:metabolism*; D015536:Down-Regulation / Q000187:drug effects; D004317:Doxorubicin / Q000031:analogs & derivatives / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D008928:Mitochondria / Q000187:drug effects / Q000201:enzymology*; D050505:Mutant Proteins / Q000378:metabolism; D049294:Phospholipid Transfer Proteins / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D051745:Protein Kinase C-delta / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects; D019076:Transgenes
chemical_list: D051023:BH3 Interacting Domain Death Agonist Protein; D050505:Mutant Proteins; C493424:PLSCR3 protein, human; D049294:Phospholipid Transfer Proteins; D004317:Doxorubicin; C046808:N-benzyladriamycin-14-valerate; D051745:Protein Kinase C-delta