Diacylglycerol generated during sphingomyelin synthesis is involved in protein kinase C activation and cell proliferation in Madin-Darby canine kidney cells
Department of Biochemistry, Centro de Investigación y de Estudios Avanzados del Instituo Politénico Nacional, P.O. Box 14-740, México City, México.Biochemical Journal (Impact Factor: 4.4). 09/2003; 373(Pt 3):917-24. DOI: 10.1042/BJ20021732
We have investigated the effects of inhibiting sphingomyelin (SM) biosynthesis on cellular diacylglycerol (DAG) content and protein kinase C (PKC) activation during growth initiation in Madin-Darby canine kidney cells. We utilized beta-chloroalanine (BCA) to inactivate serine C -palmitoyltransferase, the first enzyme in the sphingolipid biosynthesis pathway. This inactivation prevented growth, but did not affect viability. When the inhibitor was replaced with fresh culture medium, the cells continued their proliferation in a normal way. BCA (2 mM) inhibited [(32)P]P(i), [(3)H]palmitic acid and [ methyl -(3)H]choline incorporation into SM, but did not influence the synthesis of other major phospholipids. SM synthesis and DAG generation were decreased by 51% and 47.6% respectively. Particulate PKC activity was not observed in cells incubated with BCA, in contrast with a 5-fold increase in control cells. BCA inhibited 75% of the [(3)H]thymidine incorporation, and the cells were arrested before the S phase of the cell cycle. Moreover, exogenous D-erythrosphingosine restored SM synthesis, DAG generation and cell proliferation. These data indicate that the contribution of DAG generated during SM synthesis plays an important role in PKC activation and cell proliferation.
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- " In the cell, DAG is generated from PA in an enzyme reaction involving phosphatidate phosphatase and from phosphatidylinositol 4,5-bisphosphate (PIP2) in an enzymatic reaction involving phospholipase C.    The produced DAG acts as a second messenger of protein kinase C to control cellular differentiation  and proliferation.  DAG also yields phosphatidic ethanolamine (PE) and PS for CDPethanolamine , phosphatidylcholine (PC) for cytidine diphosphatecholine (CDP-choline), and triacylglycerol (TAG) for acyl-CoA thioester.  These processes were investigated for lipidomics and proteomics analyses. "
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- "It can be seen (Table 3) that PKC activity in the particulate fraction was increased 3.6–9-fold in the control (first 3–6 h of incubation ), whereas in the myriocin-treated cells no increase was detected. This supports our proposal that DAG generated during sphingolipid synthesis participates in signaling (PKC activation) necessary for the progression in the cell cycle and proliferation (Cerbon and Lopez-Sanchez, 2003). "
ABSTRACT: The sphingolipids biosynthesis pathway generates bioactive molecules crucial to the regulation of physiological processes. We have recently reported that DAG (diacylglycerol) generated during sphingomyelin synthesis, plays an important role in PKC (protein kinase C) activation, necessary for the transit through the cell cycle (G1 to S transition) and cell proliferation (Cerbon and Lopez-Sanchez, 2003. Diacylglycerol generated during sphingomyelin synthesis is involved in protein kinase C activation and cell proliferation in Madin-Darby canine kidney cells. Biochem. J. 373, 917-924). Since pathogenic Entamoeba invadens synthesize the sphingolipids inositol-phosphate ceramide (IPC) and ethanolamine-phosphate ceramide (EPC) as well as sphingomyelin (SM), we decided to investigate when during growth initiation, the synthesis of sphingolipids takes place, DAG is generated and PKC is activated. We found that during the first 6h of incubation there was a significant increase in the synthesis of all three sphingolipids, accompanied by a progressive increment (up to 4-fold) in the level of DAG, and particulate PKC activity was increased 4-8 times. The enhanced DAG levels coincided with decrements in the levels of sphingoid bases, conditions adequate for the activation of PKC. Moreover, we found that inhibition of sphingolipid synthesis with myriocin, specific inhibitor of the synthesis of sphinganine, reduce DAG generation, PKC activation and cell proliferation. All these inhibitory processes were restored by metabolic complementation with exogenous D-erythrosphingosine, indicating that the DAG generated during sphingolipid synthesis was necessary for PKC activation and cell proliferation. Also, we show that PI (phosphatidylinositol), PE (phosphatidylethanolamine) and PC (phosphatidylcholine) are the precursors of their respective sphingolipids (IPC, EPC and SM), and therefore sources of DAG to activate PKC.Experimental Parasitology 03/2009; 122(2):106-11. DOI:10.1016/j.exppara.2009.02.011 · 1.64 Impact Factor
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