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Antibody prepared against the catalytic subunit of protein phosphatase-2A from rabbit skeletal muscle, could completely inhibit this enzyme, but did not significantly affect the activities of protein phosphatases-1, 2B and 2C. The antibody was used to establish the following points. The three forms of protein phosphatase-2A that can be resolved by ion-exchange chromatography, termed 2A0, 2A1, and 2A2, share the same catalytic subunit. The antigenic sites on the catalytic subunit of protein phosphatase-2A remain accessible to the antibody, when the catalytic subunit is complexed with the other subunits of protein phosphatases-2A0, 2A1 and 2A2. The catalytic subunits of protein phosphatase-2A from rabbit skeletal muscle and rabbit liver are very similar, as judged by immunotitration experiments. Protein phosphatase-1 and protein phosphatase-2A account for virtually all the phosphorylase phosphatase activity in dilute tissue extracts prepared from skeletal muscle, liver, heart, brain and kidney, and for essentially all the glycogen synthase phosphatase activity in dilute skeletal muscle and liver extracts. Protein phosphatase-2A is almost absent from the protein-glycogen complex prepared from skeletal muscle or liver extracts. Protein phosphatase-2A accounts for a major proportion of the phosphatase activity in dilute liver extracts towards 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, 6-phosphofructo-1-kinase, fructose 1,6-bisphosphatase, pyruvate kinase and phenylalanine hydroxylase, the major phosphorylated enzymes involved in the hormonal control of hepatic glycolysis and gluconeogenesis.

Rat liver S-adenosyl-L-methionine synthetase exists in two forms which are, respectively, a dimer and a tetramer of an Mr 48,500 subunit. The high-molecular-mass form is converted into the low-molecular-mass form by incubation with 1.4 M LiBr. The kinetic properties of the low-molecular-mass form obtained by LiBr treatment are the same as those obtained with the low-molecular-mass S-adenosyl-L-methionine synthetase form purified from rat liver cytosol. These results demonstrate that the differences in specific activity and regulatory properties of the high-molecular-mass and the low-molecular-mass S-adenosyl-L-methionine synthetase forms are due to their different polymeric states.

Inositol-phosphates, glucosamine and glucose-6-phosphate blocked the effects of insulin on target protein phosphorylation in adipocytes, but the unsubstituted or sulphated derivatives of inositol or of glucose, or N-acetyl-glucosamine were without effect. The insulin stimulated tyrosine phosphorylation of the insulin receptor was not affected. The sugar-phospates inositol-phosphate and glucose-6-phosphate did not enter into the cells. They also blocked the insulin-like effects of a potential second messenger of insulin, a phosphooligosaccharide (POS), which has previously been shown to mimick the effects of insulin on protein phosphorylation in intact cells.

Publisher Summary This chapter describes the purification of two forms of protein phosphatase- 1 (PP-I 1 and PP-l G ), and three forms of protein phosphatase-2A (PP-2A 0 , PP-2A 1 , and PP-2A 2 ) from skeletal muscle. Procedures for isolating the free catalytic subunits (termed PP-l c and PP-2A c ) are documented and the structures of these enzymes are summarized in the chapter. The free catalytic subunits are isolated by a modification of the procedure of Lee and co-workers. Skeletal muscle extracts prepared from 3000 g of muscle are adjusted to pH 7.2 with 10 M ammonium hydroxide, and 350 g of solid ammonium sulfate are added to each liter of solution to bring the degree of saturation to 55%. After standing for 30 min the suspension is centrifuged for 40 min at 4200 g and the supernatant discarded. The PP-2A c from poly(L-lysine)-Sepharose is purified in an identical manner to PP-I c .

A 2.5 kb clone containing the full-length coding sequence of a type-2A protein phosphatase catalytic subunit has been isolated from a rabbit skeletal muscle cDNA library constructed in λgt10. The sequence of the protein deduced from the cDNA contains 309 residues (35.58 kDa). A major mRNA species at 2.0 kb and a minor component at 2.8 kb were visualized by Northern blotting in both skeletal muscle and liver. The type-2A enzyme showed weak homology with mammalian alkaline phosphatases between residues 55 and 95. The protein sequence of the type-2A phosphatase from rabbit skeletal muscle differs from that reported for the bovine adrenal enzyme in three regions.

The mechanism of insulin action is only partly understood1,2. At one end of the signalling chain, the structure of the insulin receptor is known in detail3, and at the other end, insulin controls cellular metabolism by regulating the phosphorylation of serine and threonine residues in key target enzymes4,5. The molecular events linking the occupied receptor to changes in target enzyme phosphorylation have remained obscure. Recently, insulin was shown to promote the hydrolysis of a phosphatidylinositol glycan with release of its polar head-group6,7. The head group was reported to activate a high-affinity cyclic AMP-phosphodiesterase6 and pyruvate dehydrogenase8, to inhibit catecholamine-stimulated lipolysis9, and also to inhibit phospholipid methyltransferase10 and adenylate cyclase8. We report here that in intact adipocytes this head-group faithfully copies the insulin-directed effects on the phosphorylation and dephosphorylation of target proteins of the hormone.

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).

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.

The addition of oleoylanilide or linoleylanilide to human polymorphonuclear leukocytes induces a time- and dose-dependent generation of arachidonic acid. Half-maximal effect is caused by a dose of 0.2 mg linoleylanilide/ml. Fatty acid anilides also produce a time- and dose-dependent inhibition of the synthesis of triacylglycerol. Half-maximal effect is caused by 1 microgram linoleylanilide/ml. These results indicate that fatty acid anilides, which have been found in the illegal cooking oil which intoxicated thousands of Spaniards, alter lipid metabolism in human polymorphonuclear leukocytes.

Glucagon produces a time- and dose-dependent activation of phospholipid methyltransferase activity in isolated rat hepatocytes. Half-maximal effect is caused by a dose of glucagon of 1 x 10(-10) M. This activation is due to an increase of the Vmax value of the enzyme, without affecting the Km value for S-adenosylmethionine. Exogenous cyclic AMP added to isolated rat hepatocytes mimics the effect of glucagon, and the activation of phospholipid methyltransferase by a nonsaturating concentration of glucagon is spontaneously reversible within 40 min of incubation.

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.

Cot/Tpl-2 kinase, homologous to members of mitogen-activated protein kinase kinase kinase, was initially discovered by its capacity to promote cell transformation. Cot/Tpl-2 mRNA levels are increased during G0 to G1 phase progression in T lymphocytes, suggesting a role for this kinase later on in the cell cycle. The IL-2-dependent CTLL-2 cells were used to investigate the role of Cot kinase in G1 to S phase transition. Transient expression of Cot kinase in CTLL-2 cells increases DNA synthesis triggered by IL-2 and the transient expression of a dominant negative form of Cot kinase in CTLL-2 markedly reduces the DNA synthesis triggered by this cytokine. Cell cycle analysis of synchronized CTLL-2 stabling overexpressing Cot kinase indicates that this kinase contributes to the passage to S and G2-M phases of the cell cycle. Cot kinase reduces the levels of the cyclin kinase inhibitor p27kip, whereas bcl-xL expression is unaffected. Cot kinase also increases E2F transcriptional activity in a phosphatidylinositol 3 kinase-independent way and acts in synergy with this kinase. These data give evidence, for the first time, of the regulation of different G1 progression events by Cot kinase.

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.

We have measured the activity of S-adenosyl-L-methionine synthetase, the ratio between the high- and low-molecular-weight forms of this enzyme and the concentration of S-adenosyl-L-methionine in liver biopsies from a group of controls (n = 6) and in six cirrhotics (five posthepatitic and one alcoholic). The total activity of S-adenosyl-L-methionine synthetase was markedly reduced in cirrhosis (37.5% of that found in the control group). This was due to a specific reduction in the high-molecular-weight S-adenosyl-L-methionine synthetase in the group of cirrhotics (73.9 pmoles per min per mg protein) when compared with that observed in controls (460.3 pmoles per min per mg protein). Despite this reduction in the rate of synthesis of S-adenosyl-L-methionine (the high-molecular-weight form of the enzyme is 15 times more active than the low-molecular-weight form at physiological concentration of substrates), the concentration of this metabolite was the same in the control group (17.3 +/- 2.6 microM) and in the group of cirrhotics (17.8 +/- 3.1 microM). To explain these findings, it is postulated that in human liver, where the concentration of S-adenosyl-L-methionine is lower than the Km values of a variety of enzymes that use this metabolite (around 50 to 100 microM), a reduction in the synthesis of S-adenosyl-L-methionine is compensated by a reduction in the rate of utilization of this molecule without affecting the intrahepatic concentration of S-adenosyl-L-methionine.(ABSTRACT TRUNCATED AT 250 WORDS)