Prior studies demonstrated that conversion of
sphingomyelin to
ceramide via
sphingomyelinase action resulted in the generation of free sphingoid bases and inactivation of
protein kinase C in human
leukemia (HL-60) cells (Kolesnick, R. N. (1989) J. Biol. Chem. 264, 7617-7623). The present studies define the novel
phospholipid ceramide 1-phosphate in these cells and present evidence for formation of this compound by preferential utilization of
ceramide derived from spingomyelin. A
ceramide 1-phosphate standard, prepared enzymatically via
diacylglycerol kinase, was utilized for localization. In cells labeled to equilibrium with 32Pi to label the head group of the molecule, the basal
ceramide 1-phosphate level was 30 +/- 2 pmol/10(6) cells. Generation of
ceramide via the use of exogenous
sphingomyelinase resulted in time- and concentration-dependent formation of
ceramide 1-phosphate. As little as 3.8 x 10(-5) units/ml was effective and a 3-fold increase was observed with a maximal concentration of 3.8 x 10(-2) units/ml; ED50 approximately 2 x 10(-4) units/ml. This effect was observed by 5 min and maximal at 30 min. Similarly, in cells labeled with [3H]
serine to probe the sphingoid base backbone, the basal level of
ceramide 1-phosphate was 39 +/- 5 pmol/10(6) and increased 2.5-fold with
sphingomyelinase; ED 50 approximately 5 x 10(-5) units/ml. To determine the source of the
phosphate moiety, studies were performed with cells short term labeled with 32Pi and resuspended in medium without radiolabel. Under these conditions,
sphingomyelin was virtually unlabeled. Nevertheless,
sphingomyelin (3.8 x 10(-2) units/ml) induced a 12-fold increase in radiolabel incorporation, suggesting
ceramide 1-phosphate formation occurred via
ceramide phosphorylation. This event appeared specific for
ceramide derived from
sphingomyelin since
ceramide from
glycosphingolipids was not converted to
ceramide 1-phosphate. In sum, these studies demonstrate the novel
phospholipid ceramide 1-phosphate in HL-60 cells and suggest the possibility that a path exists from
sphingomyelin to
ceramide 1-phosphate via the phosphorylation of
ceramide.