title: Selective inhibition of juxtanuclear translocation of protein kinase C betaII by a negative feedback mechanism involving ceramide formed from the salvage pathway.
abstract: In a previous study, we showed that protein kinase C betaII (PKC betaII) translocated to a novel juxtanuclear compartment as observed in several cell types (Becker, K. P., and Hannun, Y. A. (2003) J. Biol. Chem. 278, 52747-52754). In this study, we noted the absence of this translocation in MCF-7 breast cancer cells, and we examined the mechanisms underlying this selectivity of response. We show that sustained stimulation of PKC betaII with 4beta-phorbol 12-myristate 13-acetate (PMA) resulted in accumulation of ceramide in MCF-7 cells but not in those cells that showed juxtanuclear translocation of PKC betaII. Addition of exogenous ceramides or formation of endogenous ceramide by the action of bacterial sphingomyelinase prevented PMA-induced translocation of PKC betaII in HEK 293 cells. On the other hand, inhibition of ceramide accumulation with fumonisin B1 restored the ability of PMA to induce translocation of PKC betaII in MCF-7 cells. Taken together, the results showed that endogenous ceramide is both necessary and sufficient for preventing juxtanuclear translocation of PKC betaII in response to PMA. Investigation of the mechanisms of ceramide generation in response to PMA revealed that PMA activated the salvage pathway of ceramide formation and not the de novo pathway. This conclusion was based on the following: 1) the ability of fumonisin B1 but not myriocin to inhibit ceramide formation, 2) the ability of PMA to induce increases in palmitate-labeled ceramide only under chase labeling but not acute pulse labeling, 3) the induction of the levels of sphingosine but not dihydrosphingosine in response to PMA, and 4) induction of sphingomyelin hydrolysis in response to PMA. Together, these results define a novel pathway of regulated formation of ceramide, the salvage pathway, and they define a role for this pathway in regulating juxtanuclear translocation of PKC betaII.
authors.affiliation: Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
authors.forename: Kevin P
authors.initials: KP
authors.lastname: Becker
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Kitatani
authors.forename: Jolanta
authors.initials: J
authors.lastname: Idkowiak-Baldys
authors.forename: Jacek
authors.initials: J
authors.lastname: Bielawski
authors.forename: Yusuf A
authors.initials: YA
authors.lastname: Hannun
mesh_terms: D001669:Biochemical Phenomena; D001671:Biochemistry; D002460:Cell Line; D045744:Cell Line, Tumor; D002518:Ceramides / Q000378:metabolism*; D002794:Choline / Q000378:metabolism; D002853:Chromatography, Liquid; D011992:Endosomes / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D037341:Fumonisins / Q000494:pharmacology; D006801:Humans; D050356:Lipid Metabolism; D013058:Mass Spectrometry; D018613:Microscopy, Confocal; D008954:Models, Biological; D019308:Palmitic Acid / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors* / Q000378:metabolism; D064546:Protein Kinase C beta; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection
chemical_list: D002518:Ceramides; D004791:Enzyme Inhibitors; D037341:Fumonisins; D034741:RNA, Small Interfering; D019308:Palmitic Acid; C056933:fumonisin B1; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D002794:Choline; D013755:Tetradecanoylphorbol Acetate