The role of diacylglycerol in the stimulation of phosphatidylcholine synthesis in CHO cells
Abstract
The mechanism by which certain agents stimulate PC synthesis is poorly understood. It is known that in response to certain agents, elevation of cellular diacylglycerol levels occur. Diacylglycerol could stimulate PC synthesis by substrate availability, activation of one of the enzymes in the pathway, either choline kinase or CTP:phosphocholine cytidylyltransferase, or by activation of protein kinase C. Phospholipase C treatment, which generates diacylglycerol by the hydrolysis of PC, stimulated PC synthesis in two CHO cell lines, a wild-type cell line, WTB, and a mutant cell line, DTG 1-5-4, which is defective in endosome acidification. DTG 1-5-4 had a reduced phospholipase C stimulatory response as compared to WTB. Phospholipase C treatment resulted in the translocation of cytidylyltransferase in both cell lines. Subcellular localization of diacylglycerol determined that most of the diacylglycerol was localized at the plasma membrane in untreated cells. In the presence of phospholipase C, diacylglycerol was localized at both the plasma membrane and endoplasmic reticulum. More diacylglycerol was localized at the endoplasmic reticulum for WTB, whereas, DTG 1-5-4 accumulated diacylglycerol at the plasma membrane. These data suggested that diacylglycerol can be transported from the plasma membrane to the endoplasmic reticulum and that in DTG 1-5-4 the inefficient transport of diacylglycerol could result in the inhibition of PC synthesis by phospholipase C. Since diacylglycerol is a protein kinase C activator, the role of protein kinase C in PC synthesis was investigated. TPA, a protein kinase C activator, stimulated PC synthesis in both cell lines. The degree of stimulation was less in DTG 1-5-4. TPA treatment caused a slight translocation of cytidylyltransferase in WTB; no translocation of cytidylyltransferase was observed in DTG 1-5-4. It was concluded that phospholipase C does not activate PC synthesis by activating protein kinase C since phospholipase C was able to stimulate PC synthesis when protein kinase C had been down-regulated in both cell lines. The experiments in this thesis determined that diacylglycerol activated PC synthesis by substrate availability and translocation of cytidylyltransferase and not by activation of protein kinase C.
Degree
Ph.D.
Advisors
Kent, Purdue University.
Subject Area
Biochemistry
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