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.
Highlights
The isoenzymes of protein kinase C (PKC),1 designated as the classical PKCs, comprise a subfamily of closely related isoenzymes that share similar structural features and mechanisms of action and regulation
We show that sustained activation of PKC over 30 – 60 min activates the salvage pathway of sphingolipid metabolism (Fig. 1) by which sphingoid bases generated from hydrolysis of complex sphingolipids are recycled into ceramide synthesis
Translocation of GFP-PKCII to a Subset of Recycling Endosomes in HEK 293 but Not in MCF-7 Cells—The novel translocation of classical PKC isoenzymes, ␣ and II, to a subset of recycling endosomes concentrated around the microtubule-organizing center/centrosome was observed in several cell types including HEK 293, HeLa, HT-1080, DLD-1, A549, and COS-1 cells (1)
Summary
Materials—Eagle’s minimal essential media, RPMI, and HEPES were from Invitrogen. The MCF-7 and HEK 293 cell lines were purchased from American Tissue Culture Collection (Manassas, VA). [3H]Palmitic acid was from PerkinElmer Life Sciences. 4-Phorbol 12myristate 13-acetate (PMA) was purchased from Calbiochem. Transfected cells were grown for 12 h 10% fetal bovine serum media. Palmitate Labeling—For chase experiments, MCF-7 cells were labeled (16 –24 h) in 35-mm dishes with 3.0 Ci/ml [3H]palmitic acid in RPMI 1640 supplemented with 10% fetal bovine serum and 0.1% delipidated bovine serum albumin. Cells were washed three times with PBS, and serum-free RPMI, 0.1% bovine serum albumin media was added. Cells were placed in RPMI 1640 containing 0.1% bovine serum albumin for 24 h and treated with PMA in concurrence with labeling with 3 Ci/ml [3H]palmitic acid for 1 h. Lipid Extraction and Analysis—Following stimulation, the culture media were removed, and the cells were washed rapidly three times with 1 ml of ice-cold PBS.
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