ACTIVATION OF THE SPHINGOMYELIN CYCLE BY BREFELDIN-A - EFFECTS OF BREFELDIN-A ON DIFFERENTIATION AND IMPLICATIONS FOR A ROLE FOR CERAMIDE IN REGULATION OF PROTEIN TRAFFICKING

The sphingomyelin (SM) cycle is an emerging pathway of signal transduc tion that plays a role in the control of cell growth, cell differentia tion, and apoptosis, During earlier investigation of SM pools hydrolyz ed in the SM cycle, we examined the effects of the fungal macrolide br efeldin A (BFA) on cellular levels of SM in HL-60 leukemia cells, We f ound that BFA induced up to 20-25% hydrolysis of SM. Here we show that this BFA-sensitive SM pool corresponds to the pool of SM hydrolyzed b y a previously discovered activator of the SM cycle, 1,25-dihydroxyvit amin D-3. BFA was also able to induce the biological end points of SM cycle activation: growth inhibition and differentiation, Reciprocally, ceramide inhibited the secretion of S-35-labeled proteins from HL-60 cells and induced a subset of effects of BFA on organelle morphology. Since a ceramide-activated protein phosphatase has been previously sug gested as a direct in vitro target of ceramide action, the effects of modulators of protein kinases and phosphatases were examined, Okadaic acid enhanced protein secretion and was able to oppose the effects of both ceramide and BFA on organelle morphology. Dioctanoylglycerol and phorbol myristate acetate, known activators of protein kinase C, were also found to oppose the inhibitory actions of ceramide on secretion. These studies identify BFA as an activator of the SM cycle, with ceram ide as a potential mediator of some of the effects of BFA. Additionall y, taken with the effects of the PKC activators, these studies suggest that constitutive protein secretion is not a default pathway but is s ubject to regulation by processes of signal transduction.