Selective Inhibition of Juxtanuclear Translocation of Protein Kinase C βII by a Negative Feedback Mechanism Involving Ceramide Formed from the Salvage Pathway

Abstract

In a previous study, we showed that protein kinase C βII (PKC βII) 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 βII with 4β-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 βII. Addition of exogenous ceramides or formation of endogenous ceramide by the action of bacterial sphingomyelinase prevented PMA-induced translocation of PKC βII 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 βII in MCF-7 cells. Taken together, the results showed that endogenous ceramide is both necessary and sufficient for preventing juxtanuclear translocation of PKC βII 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 βII. In a previous study, we showed that protein kinase C βII (PKC βII) 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 βII with 4β-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 βII. Addition of exogenous ceramides or formation of endogenous ceramide by the action of bacterial sphingomyelinase prevented PMA-induced translocation of PKC βII 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 βII in MCF-7 cells. Taken together, the results showed that endogenous ceramide is both necessary and sufficient for preventing juxtanuclear translocation of PKC βII 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 βII. The isoenzymes of protein kinase C (PKC), 1The abbreviations used are: PKC, protein kinase C; DAG, diacylglycerol; GFP, green fluorescent protein; GCS, glucosylceramide synthase; PMA, 4β-phorbol 12-myristate 13-acetate; FB1, fumonisin B1; small interfering ribonucleic acid, siRNA; SMase, sphingomyelinase; SPT, serine palmitoyltransferase; LC/MS, liquid chromatography/mass spectrometry; PBS, phosphate-buffered saline; ISs, internal standards.1The abbreviations used are: PKC, protein kinase C; DAG, diacylglycerol; GFP, green fluorescent protein; GCS, glucosylceramide synthase; PMA, 4β-phorbol 12-myristate 13-acetate; FB1, fumonisin B1; small interfering ribonucleic acid, siRNA; SMase, sphingomyelinase; SPT, serine palmitoyltransferase; LC/MS, liquid chromatography/mass spectrometry; PBS, phosphate-buffered saline; ISs, internal standards. designated as the classical PKCs, comprise a subfamily of closely related isoenzymes that share similar structural features and mechanisms of action and regulation. This group consists of PKC α, PKC βI, PKC βII, and PKC γ that share highly homologous regulatory domains and are activated by diacylglycerol (DAG) and calcium. Thus, they serve as signal transducers for the action of extracellular agonists that activate phospholipases C, which results in the generation of DAG and calcium (2Mellor H. Parker P.J. Biochem. J. 1998; 332: 281-292Crossref PubMed Scopus (1352) Google Scholar, 3Ohno S. Nishizuka Y. J. Biochem. (Tokyo). 2002; 132: 509-511Crossref PubMed Scopus (154) Google Scholar). Activation of PKC requires the translocation of PKC to the plasma membrane in response to acutely generated DAG (3Ohno S. Nishizuka Y. J. Biochem. (Tokyo). 2002; 132: 509-511Crossref PubMed Scopus (154) Google Scholar, 4Kraft A.S. Anderson W.B. Nature. 1983; 301: 621-623Crossref PubMed Scopus (1000) Google Scholar). This translocation also places PKC in proximity to specific membrane substrates. Thus, the dynamic interaction of PKC with membranes has emerged as a key mechanism in regulating its function. Previous studies from our laboratory (1Becker K.P. Hannun Y.A. J. Biol. Chem. 2003; 278: 52747-52754Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), supported by the recent results from Hu and Exton (5Hu T. Exton J.H. J. Biol. Chem. 2004; 279: 35702-35708Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), have demonstrated a novel translocation of PKC α and βII to a juxtanuclear region. This novel translocation is seen with prolonged stimulation of these two isoenzymes of PKC by PMA (for at least 30 min) and follows the initial translocation of PKC to the plasma membrane (seen as early as 2–5 min following stimulation with PMA). Most interestingly, this novel translocation operates selectively on PKC α and βII in that it is not observed with PKC βI or any of the nonclassical PKCs (1Becker K.P. Hannun Y.A. J. Biol. Chem. 2003; 278: 52747-52754Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 6Becker K.P. Hannun Y.A. J. Biol. Chem. 2004; 279: 28251-28256Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Mechanistically, this translocation involves activation of PLD (6Becker K.P. Hannun Y.A. J. Biol. Chem. 2004; 279: 28251-28256Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar) and the interaction of PKC with PLD (5Hu T. Exton J.H. J. Biol. Chem. 2004; 279: 35702-35708Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). This novel juxtanuclear compartment is centered around and beyond the centrosome and the microtubule-organizing center, and therefore we have described it as the “pericentrion.” In the course of studying this novel translocation of PKC, we noted that although it is seen in most cell types, we could not observe it in a few cell lines such as MCF-7 breast carcinoma cells and LnCAP prostate cancer cells. Because PKC activation in LnCAP was previously associated with formation of ceramide (7Garzotto M. White-Jones M. Jiang Y. Ehleiter D. Liao W.C. Haimovitz-Friedman A. Fuks Z. Kolesnick R. Cancer Res. 1998; 58: 2260-2264PubMed Google Scholar), we wondered if ceramide could regulate this translocation of PKC. Ceramide is a neutral lipid that serves as the hub of sphingolipid synthesis, analogous to the role of DAG in glycerolipid metabolism. However, unlike DAG, which appears to be involved in the regulation of mitogenesis and tumor promotion, ceramide has been implicated in regulating cell cycle arrest, apoptosis, and cell senescence (8Hannun Y.A. Obeid L.M. J. Biol. Chem. 2002; 277: 25847-25850Abstract Full Text Full Text PDF PubMed Scopus (740) Google Scholar, 9Merrill Jr., A.H. J. Biol. Chem. 2002; 277: 25843-25846Abstract Full Text Full Text PDF PubMed Scopus (486) Google Scholar, 10Olivera A. Spiegel S. Prostaglandins Other Lipid Mediat. 2001; 64: 123-134Crossref PubMed Scopus (146) Google Scholar). Indeed, the regulation of ceramide metabolism has received increasing attention over the past 15 years with increasing appreciation that many of the enzymes of ceramide metabolism are regulated and that this regulation may subserve specific cellular functions. Thus far, studies have primarily focused on two major pathways of ceramide formation (Fig. 1) as follows: the first through activation of sphingomyelinases, which hydrolyze sphingomyelin and liberate ceramide (11Levade T. Jaffrezou J.P. Biochim. Biophys. Acta. 1999; 1438: 1-17Crossref PubMed Scopus (283) Google Scholar, 12Marchesini N. Hannun Y.A. Biochem. Cell Biol. 2004; 82: 27-44Crossref PubMed Scopus (268) Google Scholar); and the second involves activation of the de novo pathway, which commences with the condensation of serine and palmitate through the action of serine palmitoyltransferase (SPT) (13Perry D.K. Ann. N. Y. Acad. Sci. 2000; 905: 91-96Crossref PubMed Scopus (37) Google Scholar). The list of activators of these pathways continues to grow and includes cytokines, chemotherapeutic agents, cytotoxic peptides (e.g. amyloid β peptide), and other environmental stresses. Other studies are also beginning to show regulation of enzymes of ceramide clearance such as ceramidases, glucosylceramide synthase, and sphingomyelin synthase (14Hannun Y.A. Luberto C. Argraves K.M. Biochemistry. 2001; 40: 4893-4903Crossref PubMed Scopus (440) Google Scholar). In this study, we provide evidence for a specific role for endogenous ceramide in inhibiting the novel juxtanuclear translocation of PKC to the pericentrion. Most importantly, 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. This is the first description of regulation of this pathway in response to an agonist. Thus, the results define a negative feedback mechanism in the activation of PKC involving PKC-induced formation of endogenous ceramide through the salvage pathway. 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. 4β-Phorbol 12-myristate 13-acetate (PMA) was purchased from Calbiochem. C6-ceramide was from Avanti Polar Lipids (Alabaster, AL). Phospholipid standards were from Avanti Polar Lipids. Fumonisin B1 was from Alexis (San Diego, CA). Whatman Silica Gel 60 TLC plates were from Fisher. Bacterial SMase and all other chemicals were from Sigma. Cell Culture—MCF-7 cells were maintained in RPMI, and HEK 293 cells were maintained in Eagle's minimal essential media supplemented with 10% (v/v) fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin in a 5% CO2 incubator at 37 °C. Cells were passaged every 3–4 days to maintain cells in logarithmic growth. Plasmid were following The was described previously J. T. Hannun Y.A. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). of was with to the cells were for 10% fetal bovine of was in these Cells green fluorescent were in media or following a with 10% to 37 °C. were with an with an and is of over a of and all were at the of the were at fluorescent were and the Life were in for chase MCF-7 cells were in with acid in supplemented with 10% fetal bovine and bovine Cells were with PBS, and RPMI, bovine media was Cells were to in media at 37 for 30 min to Cells were with PMA for the and at the noted were at 37 °C. pulse labeling cells were in bovine for and with PMA in with labeling with acid for for labeling were to in sphingomyelin Cells were with for and in bovine PMA were as and to Lipid and the media were and the cells were with of cellular were the of and J. Biochem. PubMed Scopus Google Scholar). Lipids were and in of the was The TLC of acid for ceramides and for The plates were with Life to the signal and for for The major ceramide were by and standards was The to lipid was and the was by liquid cell to cells were with the internal standards and and with an and in 100 of were on the and from the with to the and internal standards were and the was based on the generated by an with the of the standards and an of the The were The from the were to and with the a of and ceramide with specific acid and were and to small interfering for glucosylceramide synthase was and by MCF-7 cells were with the or as as by the of to a of in HEK 293 but in MCF-7 novel translocation of classical PKC α and βII, to a of around the microtubule-organizing was observed in several cell types HEK and cells (1Becker K.P. Hannun Y.A. J. Biol. Chem. 2003; 278: 52747-52754Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). studies into the translocation of a PKC βII revealed that this novel juxtanuclear translocation was not in all cell of HEK 293 cells with 100 PMA for 60 min an in the of βII from the to the plasma membrane and to the juxtanuclear compartment (Fig. In similar stimulation of MCF-7 resulted in translocation of βII to the plasma membrane only with translocation (Fig. course in MCF-7 cells revealed that this the two cell lines was not of a in the of translocation with PMA for or not juxtanuclear translocation not results in the response of PKC to PMA the of Ceramide in MCF-7 Cells but in HEK 293 has been that the sphingolipid ceramide inhibit both the and translocation of classical PKC isoenzymes in both a and J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Hannun Y.A. Obeid L.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Luberto C. Hannun Y.A. J. 2001; PubMed Scopus Google Scholar). it was that PMA induce the of ceramide in the prostate cell cells but not in all prostate cancer cell lines (7Garzotto M. White-Jones M. Jiang Y. Ehleiter D. Liao W.C. Haimovitz-Friedman A. Fuks Z. Kolesnick R. Cancer Res. 1998; 58: 2260-2264PubMed Google Scholar). if stimulation to a in ceramide in MCF-7 cells but not in both cell types were with and for with 100 PMA. seen in 60 min of PMA to an in ceramide in MCF-7 cells, and this was not seen in HEK 293 cells. The of PMA on ceramide in MCF-7 cells were also The course studies revealed that ceramide was to of levels 30 min of and this ceramide sustained through of stimulation (Fig. seen in levels of all of the ceramide were under PMA and the of was with a induction at 60 as in and these of or ceramide levels PMA stimulation in MCF-7 cells are with was observed in the or ceramide by which to and results that PMA is to induce the formation of ceramide in MCF-7 cells but not in HEK of ceramide in MCF-7 in a ceramide in MCF-7 cells is by PKC and fumonisin of ceramide from MCF-7 cells with and with 100 myriocin or fumonisin B1 for Cells were with or 100 PMA for 60 were on a in acid ceramide by and PMA has also been to induce ceramide formation in LnCAP prostate cancer cells (7Garzotto M. White-Jones M. Jiang Y. Ehleiter D. Liao W.C. Haimovitz-Friedman A. Fuks Z. Kolesnick R. Cancer Res. 1998; 58: 2260-2264PubMed Google Scholar), and we could not juxtanuclear translocation of PKC in response to PMA in cells not results a ceramide formation and of juxtanuclear to if ceramide could inhibit translocation of PKC βII to the juxtanuclear HEK 293 cells with βII were with C6-ceramide for by 60 min of 100 PMA. In cells with PMA was translocation of βII to the juxtanuclear compartment (Fig. In of cells with C6-ceramide prevented the juxtanuclear translocation in response to PMA (Fig. endogenous ceramide could a similar as ceramide, HEK 293 cells with βII were for 30 min with 100 bacterial SMase by 60 min PMA. cells with PMA with bacterial SMase to an inhibition of juxtanuclear translocation (Fig. and show that endogenous ceramide inhibit translocation of PKC βII to the the that endogenous ceramide may be for the of the PMA in MCF-7 cells. PMA-induced Ceramide in MCF-7 Cells PKC and the but de is that the major cellular for PMA is PKC and that PMA many of its cellular through stimulation of PKC to PKC was for the formation of ceramide, cells were with and in the of the PKC In cells with was of ceramide (Fig. that the ceramide is by PKC and that this ceramide translocation of PKC. In previous studies in LnCAP cells, it was that PMA ceramide formation through activation of the de novo pathway based on studies with FB1, an of ceramide synthase (7Garzotto M. White-Jones M. Jiang Y. Ehleiter D. Liao W.C. Haimovitz-Friedman A. Fuks Z. Kolesnick R. Cancer Res. 1998; 58: 2260-2264PubMed Google Scholar). to the and mechanism involved in ceramide MCF-7 cells were with FB1, and the results showed that ceramide formation in response to PMA (Fig. Thus, the formation of ceramide in response to PMA requires of ceramide Because ceramide synthase on sphingosine by its to ceramide, the action of in inhibiting this not activation of the de novo pathway of sphingoid formation or the generation of sphingosine from of complex the salvage pathway (Fig. 1) Jr., A.H. K. J. Biol. Chem. Full Text PDF PubMed Google Scholar). We several to the two we myriocin which the initial of the de novo pathway, the condensation of serine and Cells with myriocin showed inhibition of ceramide in response to PMA (Fig. and the action of the salvage and not the de novo pathway. that 100 myriocin de novo ceramide in our MCF-7 cells were for with myriocin and with In a myriocin the of into MCF-7 cells with inhibition at 100 not these two the of PMA on palmitate labeling of ceramide were under a pulse a chase PMA de novo of ceramide, PMA the acute of palmitate into ceramide cells are with palmitate On the other hand, if PMA of sphingolipids in the formation of sphingosine as a which is to ceramide, PMA not the acute of In that labeling of sphingolipids with palmitate labeling of ceramide in response to PMA. MCF-7 cells were acutely with palmitate and with PMA these PMA not the of into ceramide (Fig. In cells were with palmitate to PMA ceramide formation 60 min of (Fig. that PMA the accumulation of ceramide from with ceramide PMA the accumulation of which primarily from of complex In which through the de novo pathway, was in response to PMA (Fig. of of for ceramide PMA Taken together, the results induction of the salvage pathway in response to PMA. Because the studies of complex we the of PMA on of sphingomyelin was observed as early as 15 min following PMA and a in the was seen at the of 60 min (Fig. small in sphingomyelin are the levels of sphingomyelin are those of the results induction of sphingomyelin as the of the Fumonisin B1 in MCF-7 inhibition of PMA-induced ceramide formation by that the absence of PKC translocation to the in MCF-7 cells may be MCF-7 cells with βII were with or for and 60 min of 100 PMA. In cells with was translocation of βII to the plasma membrane only (Fig. and with resulted in the of translocation of PKC βII to the (Fig. a role for ceramide as the endogenous of PKC However, ceramide may also to glucosylceramide through the action of glucosylceramide synthase and the glucosylceramide serves as a for the formation of complex for a role for glucosylceramide or in the inhibition of PKC βII juxtanuclear was by small interfering in PMA-induced translocation of βII to the plasma membrane was not by with for it formation of of not to be involved in the juxtanuclear translocation of PKC βII. the endogenous ceramide by PMA in the of juxtanuclear translocation of PKC in MCF-7 cells. The results from this define a negative feedback mechanism activated by PKC that results in inhibition of translocation of PKC βII to the juxtanuclear Thus, the results the activation of PKC by PMA in MCF-7 cells the formation of ceramide, which in appears to be the that the translocation of PKC βII to the juxtanuclear but not to the plasma the results from this on a novel mechanism of ceramide The results also provide evidence for a specific role of this pathway in negative regulation of PKC. the results a to the appreciation of of the formation of ceramide through the activation of or the de novo pathway has under are many as to the mechanisms that regulate these In many of the studies the de novo pathway have on the ability of to inhibit ceramide synthase, a this pathway. However, it be noted that ceramide synthase on or that are generated from activation of the de novo pathway or that are generated from hydrolysis of complex a pathway the pathway (Fig. 1) Jr., A.H. K. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The results from this show that PMA activates the salvage pathway and not the de novo pathway. This conclusion is based on the following 1) ceramide formation, a of SPT, not the response to PMA. is the first and in the de novo pathway, and therefore its inhibition the formation of sphingolipids 2) The of pulse and chase labeling was Thus, PMA not induce of palmitate into ceramide under pulse activation of de novo synthesis, PMA of into ceramide under chase the palmitate was into lipid 3) PMA an accumulation of sphingosine and not studies by Jr., A.H. K. J. Biol. Chem. Full Text PDF PubMed Google Scholar) have evidence that unlike primarily from the of complex sphingolipids and ceramide and not as an in the de novo pathway. This also activation of the salvage pathway by PMA. this we only as to the mechanisms by which PMA a in This is to inhibition of the de novo pathway as we not a in acute of palmitate into sphingolipids with PMA. is that PMA may activate ceramide synthase as a of activation of the salvage pathway. 4) the studies with labeling also an of PMA on sphingomyelin levels and activation of a the in sphingomyelin were it be noted that in MCF-7 cells a of ceramide a of only in sphingomyelin Taken together, these activation of the salvage pathway and not the de novo pathway. We also that these in these two pathways in other have noted of be noted that other studies have the and of the salvage pathway in lipid metabolism R. J. Z. A. Obeid L.M. Hannun Y.A. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar, M. M. K. A. S. S. R. T. 1998; PubMed Scopus Google Scholar, 1998; PubMed Scopus Google Scholar). The results from this show that PKC is for activation of this pathway in response to PMA, and this the first description of regulation of sphingolipid the results provide specific of activation of this pathway, inhibition of translocation of PKC βII to the juxtanuclear region. Thus, formation of endogenous ceramide by the action of bacterial SMase on membrane sphingomyelin was sufficient to inhibit the ability of PMA to induce juxtanuclear translocation of PKC in a cell that demonstrated this In this results with showed that endogenous ceramide in response to PMA was necessary for the of PKC translocation in cells. Thus, activation of the salvage pathway is both necessary and sufficient to inhibit juxtanuclear translocation of PKC. Most interestingly, this pathway is not activated by in most cell The mechanisms for this activation may of specific isoenzymes of PKC, and these are under The results also ceramide and not other key in this response. sphingosine levels with FB1, a role for of ceramide to sphingoid the response to PMA was not by of glucosylceramide synthase, a key role for or any of its However, at a role for other of ceramide as the of this response be it be noted that formation of ceramide is for inhibiting the PMA response of ceramide is the In the of these results to define specific pathways of ceramide generation with specific functions. previous studies have that de novo generated ceramide in response to stimulation of the in cells results in accumulation of Luberto C. M. H. de Hannun Y.A. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar), and this was implicated in the response of those cells S. H. Hannun Y.A. de J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). On the other hand, activation of neutral was to selectively induce the formation of and and evidence was for a role for this pathway in cell cycle regulation N. J. Luberto C. Obeid L.M. Hannun Y.A. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar) and in formation J. 2004; PubMed Scopus Google Scholar). The results provide a specific pathway of ceramide formation with its in regulating translocation of PKC. Previous studies have also of ceramide on PKC. Hannun Y.A. Obeid L.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) showed that prolonged action of ceramide results in and of and this was at least in to the activation of Thus, ceramide generated from pathways may on PKC. Taken together, these results define a novel feedback pathway by which PKC activates sphingolipid salvage in ceramide This in translocation of PKC βII to the juxtanuclear region. We for of the We the H. for the of and we also the at the of