DETECTION AND CHARACTERIZATION OF CERAMIDE-1-PHOSPHATE PHOSPHATASE-ACTIVITY IN RAT-LIVER PLASMA-MEMBRANE

A calcium-dependent ceramide (Cer) kinase was recently detected in human leukemia (HL-60) cells (Kolesnick, R. N., and Hemer, M. R. (1990) J. Biol. Chem. 265, 18803-18808) where it may function in terminating the regulatory effects of Cer, and in synaptic vesicles (Bajjalieh, S. M., Martin, T. F. J., and Floor, E. (1989) J. Biol. Chem. 264,14354-14360). We now demonstrate that the addition of both Cer-1-phosphate (Cer-1-P) and a short-acyl chain analog of Cer-1-P, N-hexanoylsphingosine-1-phosphate (C6-Cer-1-P) to cultured cells and a variety of subcellular fractions results in rapid degradation to Cer and C6-Cer, respectively. The Cer-1-P phosphatase activity is enriched in a rat liver plasma membrane fraction and appears to be distinct from the phosphatase that hydrolyzes phosphatidic acid (PA), PA phosphohydrolase, as shown by the difference in sensitivity of Cer-1-P and PA hydrolysis to propranolol, detergent, and heat treatment. Moreover, the K(m) of Cer-1-P hydrolysis is 10-fold lower than the K(m) of PA hydrolysis in plasma membrane. PA is a noncompetitive inhibitor of Cer-1-P hydrolysis, with an inhibition constant 1-1.5-fold higher than the K(m) of Cer-1-P hydrolysis. In contrast, Cer-1-P does not inhibit PA hydrolysis. Finally, we describe the synthesis of a novel analog of Cer-1-P which is not hydrolyzed in vitro and in vivo and is internalized in cultured cells by endocytosis. These results are discussed in relation to the possible roles of Cer-1-P in regulating intracellular levels of Cer.