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 ␤II (PKC ␤II) 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 ␤II with 4␤-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 ␤II. Addition of exogenous ceramides or formation of endogenous ceramide by the action of bacterial sphingomyelinase prevented PMA-induced translocation of PKC ␤II 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 ␤II in MCF-7 cells. Taken together, the results showed that endogenous ceramide is both necessary and sufficient for preventing juxtanuclear translocation of PKC ␤II 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 ␤II.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. This group consists of PKC ␣, PKC ␤I, PKC ␤II, and PKC ␥ 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 (2, 3).Activation of PKC requires the translocation of PKC to the plasma membrane in response to acutely generated DAG (3, 4). 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 (1), supported by the recent results from Hu and Exton (5), have demonstrated a novel translocation of PKC ␣ and ␤II 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 ...

The sphingolipid salvage pathway in ceramide metabolism and signaling

“…An increase in sphingosine has been shown to occur in response to TNF-α [61], serum starvation [62], interleukin-1β [63], bradykinin [64], phorbol ester [17], and Fas ligands [65]. The release of sphingosine is presumed to arise from enhanced degradation of complex sphingolipids, sphingomyelin or ceramide.…”

“…Recent studies have begun to implicate members of the PKC of Ser/Thr protein kinases as upstream regulators of the sphingoid base salvage pathway resulting in ceramide synthesis following PKC activation in human breast cancer cell line (MCF-7 cells) [17,23].Initially, it was shown that PKC activation by phorbol esters stimulated the formation of ceramide, and it was concluded that phorbol esters activated the de novo pathway [4], based on the sensitivity of ceramide formation to fumonisin B1. However, in a more recent study, it was shown that phorbol ester-induced ceramide was not myriocin-sensitive.…”

“…Moreover, mass spectrometric analysis demonstrated an elevation of free sphingosine following PKC activation, further suggesting enhanced degradation of sphingolipids or glycosphingolipids. In light of those results, it was suggested that PKC stimulates the sphingosine-salvage pathway for ceramide synthesis [17].…”

“…This latter pathway has been referred to as either sphingolipid recycling or the salvage pathway [16][17][18][19][20][21]. This pathway involves a number of key enzymes that include SMases, possibly glucocerebrosidase (acid-β-glucosidase), ceramidases, and (dihydro)ceramide synthases (Fig.…”

“…A growing body of evidence is starting to point toward roles for ceramide generated through the salvage pathway in many biological responses, such as growth arrest [18], apoptosis [22], cellular signaling [23] and trafficking [17]. Indeed, this may emerge as a commonly employed pathway in cell regulation that may even reconcile some of the previous seemingly contradictory results on activation of acid SMase vs the de novo pathway.…”