thermozymocidin and 3-chloroalanine

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.

Topics: Animals; beta-Alanine; Cell Proliferation; Diglycerides; Entamoeba; Enzyme Activation; Enzyme Inhibitors; Fatty Acids, Monounsaturated; Protein Kinase C; Signal Transduction; Sphingolipids; Sphingomyelins; Sphingosine; Time Factors

We recently reported that DAG (diacylglycerol) generated during sphingomyelin synthesis plays an important role in protein kinase C activation and cell proliferation in Madin-Darby canine kidney cells [Cerbon and Lopez-Sanchez (2003) Biochem. J. 373, 917-924]. In yeast cells, IPC (inositol phosphoceramide) synthase catalyses the transfer of phosphoinositol from phosphatidylinositol to ceramide to form IPC and generates DAG. In the present study, we found that, during the G1 to S transition after N2-starvation, there was a significant increase in the synthesis of IPC accompanied by a progressive increase (up to 6-fold) in the level of DAG. The increased DAG levels coincided with decrements in ceramide and sphingoid base levels, conditions that are adequate for the activation of putative protein kinase C required for the G1 to S transition and proliferation of yeast cells. To separate the role of DAG generated during IPC synthesis from that originating from other sources, we utilized beta-chloroalanine and myriocin, inhibitors of serine:palmitoyl-CoA transferase, the first committed step in sphingolipid synthesis, to avoid accumulation of sphingolipid intermediates. When the synthesis of sphingolipids was inhibited, DAG accumulation was significantly decreased and the G1 to S transition was blocked; such blockage was avoided by metabolic complementation with phytosphingosine. The DAG/ceramide ratio was 0.27 and it changed to 2.0 during growth re-initiation, suggesting that the synthesis of phosphosphingolipids could act to switch growth arrest (increased ceramide) to a mitogenic signal (increased DAG), and that this signalling process is preserved in yeast and mammalian cells.

Topics: beta-Alanine; Cell Proliferation; Ceramides; Depsipeptides; Diglycerides; Fatty Acids, Monounsaturated; G1 Phase; Hexosyltransferases; Phospholipids; Protein Kinase C; S Phase; Saccharomyces cerevisiae; Signal Transduction; Sphingolipids