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An enzyme that catalyses the three-step methylation of phosphatidylethanolamine to phosphatidylcholine as well as the methylation of fatty acids and that uses S-adenosylmethionine as the methyl donor has been purified about 200-fold from rat liver. Irradiation of the purified enzyme with a short-wavelength u.v. light in the presence of [methyl-3H]8-azido-S-adenosylmethionine followed by electrophoresis results in the incorporation of radioactivity into a single protein band of about 25 kDa. It is concluded that a single catalytic subunit catalyses the conversion of phosphatidylethanolamine into phosphatidylcholine and fatty acid methylation.

Cot kinase is a protein serine/threonine kinase, classified as a mitogen-activated protein kinase kinase kinase, implicated in T lymphocyte activation. Here we show that an increase in Cot kinase expression promotes tumor necrosis factor-alpha (TNF-alpha) production in Jurkat T cells stimulated by soluble anti-CD3 or by low concentrations of phorbol 12,13-dibutyrate (PDBu) and calcium ionophore. Overexpression of Cot kinase in Jurkat cells activates TNF-alpha gene expression. Cot kinase promotes TNF-alpha promoter activation to a similar extent as calcium ionophore and PDBu or soluble anti-CD28 and PDBu. Neither phorbol esters nor calcium ionophore can replace Cot kinase on TNF-alpha promoter-driven transcription. Expression of a dominant negative form of Cot kinase inhibits TNF-alpha promoter activation induced by stimulation with either calcium ionophore and PDBu, soluble anti-CD28 and PDBu, or soluble anti-CD3 and PDBu. TNF-alpha promoter-driven transcription by Cot kinase is partially mediated by MAPK/ERK kinase and is cyclosporin A-resistant. Cot kinase increases at least the AP-1 and AP-2 response elements. These data indicate that Cot kinase plays a critical role in TNF-alpha production.

The addition of 1 microM-vasopressin or -angiotensin to isolated rat hepatocytes induced a fast transient inhibition of the rate of incorporation of [Me-3H]choline into phosphatidylcholine. The cationophore A23187 induced a similar inhibition of phosphatidylcholine synthesis. The addition of micromolar Ca2+ to rat liver microsomes inhibited the activity of CDP-choline: 1,2-diacylglycerol cholinephosphotransferase. This inhibition is due a decrease in the Vmax. of the enzyme without affecting the Km for CDP-choline. It is concluded that Ca2+ regulates phosphatidylcholine synthesis in rat liver.

In Dictyostelium discoideum, the chemoattractant cyclic AMP activates the enzyme guanylate cyclase, giving a brief up to 10-fold increase in the intracellular cyclic GMP content. The addition of physiological cyclic GMP concentrations to a homogenate of D. discoideum cells markedly increased the incorporation of the 3H-labeled methyl group from S-adenosyl-L-[methyl-3H]methionine into mono- and dimethylated phosphatidylethanolamine and phosphatidylcholine. Lipid methylation was inhibited by S-adenosyl-L-homocysteine, which inhibits transmethylation. When whole cells prelabeled with L-[methyl-3H]methionine were exposed to cyclic AMP, a rapid transient increase in the amount of [methyl-3H]phosphatidylcholine was observed. The time course of [methyl-3H]phosphatidylcholine formation agrees with its being mediated by the intracellular increase in cyclic GMP originating during chemotactic stimulation. Addition of the 8-Br derivative of cyclic GMP to whole cells also increased the levels of labeled phosphatidylcholine. It is therefore likely that cyclic GMP contributes to chemotaxis by regulating membrane function via phospholipid methylation.

The addition of micromolar Ca2+ in the presence of ATP (10 microM) to rat liver microsomes stimulates phospholipid methylation and the formation of fatty acid methyl esters. Our data indicate that calmodulin mediates the effect of Ca2+ on both phospholipid and fatty acid methylation. This evidence includes: 1) chlorpromazine, an inhibitor of Ca2+ effects mediated through calmodulin, prevented the activation by Ca2+ plus ATP of the methylation reactions; 2) isolation of the microsomes in the presence of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (5 mM), to remove endogenous calmodulin also prevented Ca2+ plus ATP stimulation of both phospholipid and fatty acid methylation; 3) the addition of exogenous calmodulin to microsomes isolated in the presence of 5 mM ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid restored Ca3+ plus ATP stimulation of both classes of lipids: and 4) pre-incubation of liver microsomes with an antiserum against calmodulin prevented the activation of lipid methylation by Ca2+ plus ATP.