Abstract
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 Km of Cer-1-P hydrolysis is 10-fold lower than the Km 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 Km 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.
Highlights
SinceCeris involved in cellregulation, itsintracellular liver plasma membrane fraction and appears to be levels must becarefully controlled
We demonstrate that Cer-1-P irsapidly hydrolyzed in cultured cells, and althoughhydrolyzed in a variety of subcellular fractionsfrom rat liver and brain,hydrolysis is enriched in liver plasma membrane (PM) and brain synaptosomes
Ce-NBD-Cer formed as a result of Ce-NBD-Cer-1-P hydrolysis was subsequently metabolized to Ce-NBD-glucosylceramideand Ce-NBD-SM.The following Ce-NBD-sphingolipids were used as TLC standards as indicated: Cer, glucosylceramide (GlcCer),Cer-1-P (CerP),and SM
Summary
Counter using Lumax:toluene (1:3, v/v) as scintillation fluid. The A calcium-dependent Cer kinase that phosphorylates Cer stock concentration of C6-NBD-labeledlipids was determined using a stock solution of Cs-NBD-Cer, prepared by dissolving 1 mg of CeNBD-Cer in 2 ml of ch1oroform:methanol (19:1, v/v). Stock concento Cer-1-P has been detected in rat brain synaptic vesicles (Bajjalieh et al, 1989) and HL-60 cells (Dressler and Kolesnick, 1990; Kolesnick and Hemer, 1990).We have examined whether Cer-1-P is hydrolyzed by these and other trations of Cs-NBD and other phospholipids were determined by tissues. SubcellulnrFractionation-Rat liver microsomes (Ehrenreich et al., 1973)and a purified PM fraction (Hubbardet al., 1983)were prepared as described (Futerman et al, 1990). Served inrat liver PM (44.4 iZ 0.2 nmol of C6-NBD-Cer. Synaptosomes from rat brain were prepared according to Dunkley et al (1988). 20 p1 of a C6-lipid.BSAcomplex (containing various amounts of the C6-lipid) were added to 50 p1 of buffer respectively, and thespecific activity of CHO cells (14.5 f 3.6 nmol/min/mg) was 6-fold higher than HL-60 cells (2.4 f 0.8 nmol/min/mg) (Fig. 1).In a rat liver homogenate, using either. The specific activity of Ce-["CICer-1-P was adjusted by the pH optima of 5.5-7.0 (not shown)
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