Isabel Varela

Stimulation by vasopressin and angiotensin of phospholipid methyltransferase in isolated rat hepatocytes

Phosphatidylethanolamine and phosphatidylcholine are the two major phosptiolipid components of membranes from rat hepatocytes [1]. Phosphatidylcholine can be synthesized either by transcholination [2] or by transmethytation [3]. The transmethylation pathway involves the addition of 3 methyl groups to the polar head of phosphatidylethanolamine, the methyl donor being S-adenosyl-L-methionine. Two intermediates are formed during the synthesis of phosphatidylcholine by transmethylation: N-methyl. phosphatidylethanolamine and N,N-dimethyl-phosphatidylethanolamine [4]. In rat liver, phospholipid methyltransferase activity is associated with the microsomal fraction [5,6]. Although rat liver is one of the tissues were the specific activity of phospholipid methyltransferase is relatively high, the contribution of this pathway to the total synthesis of phosphatidylcholine is also minor (20-40%) in this tissue [7]. However, the finding that many signals acting on the cell surface, including hormones [8,9], immunoglobulins [ 10], attractants [ 11-13 ], and phagocytizable particles [ 14], modulate phospholipid methylation, suggest an important function for this process during signal transduction. We have shown that treatment of isolated hepatocytes with glucagon produces a time and dose-dependent activation of phospholipid methyltransferase, through a mechanism which seems to be dependent on cyclic AMP [9,15]. Rat hepatocytes possess at least 3 hormone receptors, a-adrenergic, vasopressin and angiotensin, in whose actions Ca 2÷ and not cyclic AMP, is involved [16-18]. This paper shows an activation of phospholipid methyltransferase by vasopressin and angiotensin in isolated rat hepatocytes which is dependent on Ca 2÷. Furthermore, the ionophore A23187 mimics the effect of these hormones.

Photoaffinity labelling shows that Escherichia coli isocitrate dehydrogenase kinase/phosphatase contains a single ATP-binding site

Ultraviolet irradiation of E.coli isocitrate dehydrogenase kinase/phosphatase in the presence of 8‐azidoATP resulted in parallel losses of its kinase and phosphatase activities, and in covalent attachment of the reagent to the protein at a single site. ATP and ADP protected the two activities to similar extents. The data suggest that the activation of the phosphatase by adenine nucleotides results from binding of the nucleotides to the active site of the kinase.

Phospholipid methyltransferase phosphorylation by intact hepatocytes: Effect of glucagon

We have obtained a rabbit antiserum that specifically immunoprecipitates the 50K and 25K proteins of rat liver phospholipid methyltransferase. Exposure of intact rat hepatocytes preincubated with [32P]phosphate to glucagon induces a time-dependent phosphorylation of the 50K protein of phospholipid methyltransferase. The incorporation of 32P into the 50K protein was only on phosphoserine. These data support the concept that the activation of rat liver phospholipid methyltransferase by glucagon is mediated by phosphorylation of the enzyme.

Modulation by the ratio S-adenosylmethionineS-adenosylhomocysteine of cyclic AMP-dependent phosphorylation of the 50 kDa protein of rat liver phospholipid methyltransferase

The present results show that the catalytic subunit of cyclic AMP-dependent protein kinase phosphorylates the 50 kDa protein of rat liver phospholipid methyltransferase at one single site on a serine residue. Phosphorylation of this site is stimulated 2- to 3-fold by S-adenosylmethionine. S-adenosylmethionine-dependent protein phosphorylation is time- and dose-dependent and occurs at physiological concentrations. S-adenosylhomocysteine has no effect on protein phosphorylation but inhibits S-adenosylmethionine-dependent protein phosphorylation. S-Adenosylmethionine/S-adenosylhomocysteine ratios varying from 0 to 5 produce a dose-dependent stimulation of the phosphorylation of the 50 kDa protein. In conclusion, these results show, for the first time, that the ratio S-adenosylmethionine/S-adenosylhomocysteine can modulate phosphorylation of a specific protein.

Vasopressin-stimulated phosphorylation of rat liver phospholipid methyltransferase in isolated hepatocytes

Addition of vasopressin (1μM) to isolated rat hepatocytes prelabeled with [32P]phosphate was accompanied by a 250% increase in the phosphorylation of phospholipid methyltransferase. Vasopressin‐stimulated phospholipid methyltransferase phosphorylation was time‐ and dose‐dependent. 32P‐labeled phospholipid methyltransferase was recovered by immunoprecipitation and SDS‐polyacrylamide gel electrophoresis. After electrophoresis, phospholipid methyltransferase was electroeluted from the polyacrylamide gel and subjected to tryptic digestion or HCl hydrolysis. Analysis of 32P‐labeled peptides reveals only one site of phosphorylation and the analysis of [32P]phosphoamino acids indicates that phosphoserine is the only labeled amino acid.

Role of Glycosyl-Phosphatidylinositols in Insulin Signalling

Insulin is one of the best studied hormones. The main actions of insulin are to stimulate the synthesis of glycogen, lipids and proteins, through the modulation of the metabolic pathways implicated in these processes, and the stimulation of cell growth. The physiological effects of insulin include the stimulation of the uptake of glucose, amino acids and ions; the regulation of the state of serine-and threonine-phosphorylation of a variety of proteins and rate-limiting enzymes like glycogen phosphorylase, hormone-sensitive lipase and ATP citrate lyase; and the regulation of the expression of the genes for several regulatory enzymes (review by Denton, 1986). All these effects, whose chronology vary from seconds to hours, are initiated by the interaction of the hormone with the insulin receptor, an integral membrane glycoprotein composed of two α (Mr about 130 kDa) and two s (95 kDa) subunits. The α subunits bind insulin and are linked by disulphide bonds to each other and to the s-subunits. Following insulin binding, the s-subunits are rapidly autophosphorylated predominantly at tyrosine residues. Direct evidence, indicating that insulin action depends on the protein tyrosine kinase activity of the insulin receptor, has been obtained by site-directed mutagenesis of the insulin receptor cDNA as well as with monoclonal antibodies to the insulin receptor kinase domain (review by Rosen, 1987).

Receptor mediated regulation of phospholipid methyltransferase activity

The biosynthesis of phosphatidylcholine(PC) by N-methylation of phosphatidylethanolamine(PE) is known for about 20 years (Bremer and Greenberg, 1961) however, only since recently we started to learn about the possible biological implications of this reaction. The finding that many signals acting on the cell surface, including hormones (Hirata et al. 1979a; Castano et al. 1980; Alemany et al. 1981), attractants (Pike et al. 1979; Hirata et al. 1979b; Mato and Marin Cao, 1979; Alemany et al. 1980), immunoglobulins (Ishizaka et al. 1980) and phagocytizable particles (Garcia Gil et al. 1981a) modulate phospholipid methylation, suggests an important function for this process during signal transduction. In most cases the experimental approach has been to measure the incorporation of (methyl-3H) groups into PC during stimulation in cells prelabeled with (methyl-3H)-methiomine. In a few cases phospholipid methyltransferase activity has been measured during stimulation. Results from these experiments indicate that the enzyme phospholipid methyltransferase exists in different interconvertible stages of activity and that the interaction ligand-receptor switches the enzyme from one stage of activity to another. This essay summarizes our results with two different experimental systems: rat hepatocytes and human polymorphonuclear leukocytes (PMN).