Protein kinase inhibition by omega-3 fatty acids

Recent data suggest that omega-3 fatty acids may be effective in epilepsy, cardiovascular disorders, arthritis, and as mood stabilizers for bipolar disorder; however, the mechanism of action of these compounds is unknown. Based on earlier studies implicating omega-3 fatty acids as inhibitors of protein kinase C activity in intact cells, we hypothesized that omega-3 fatty acids may act through direct inhibition of second messenger-regulated kinases and sought to determine whether the omega-3 double bond might uniquely confer pharmacologic efficacy and potency for fatty acids of this type. In our studies we observed that omega-3 fatty acids inhibited the in vitro activities of cAMP-dependent protein kinase, protein kinase C, Ca(2+)/calmodulin-dependent protein kinase II, and the mitogen-activated protein kinase (MAPK). Our results with a series of long-chain fatty acid structural homologs suggest an important role for the omega-3 double bond in conferring inhibitory efficacy. To assess whether omega-3 fatty acids were capable of inhibiting protein kinases in living neurons, we evaluated their effect on signal transduction pathways in the hippocampus. We found that omega-3 fatty acids could prevent serotonin receptor-induced MAPK activation in hippocampal slice preparations. In addition, we evaluated the effect of omega-3 fatty acids on hippocampal long-term potentiation, a form of synaptic plasticity known to be dependent on protein kinase activation. We observed that omega-3 fatty acids blocked long-term potentiation induction without inhibiting basal synaptic transmission. Overall, our results from both in vitro and live cell preparations suggest that inhibition of second messenger-regulated protein kinases is one locus of action of omega-3 fatty acids.     

Protein kinase C activation by cis-fatty acid in the absence of Ca2+ and phospholipids

Protein kinase C has been shown to be a phospholipid/Ca2+-dependent enzyme activated by diacylglycerol (Nishizuka, Y. (1984) Nature 308, 693-697; Nishizuka, Y. (1984) Science 225, 1365-1370). We have reported that unsaturated fatty acids (oleic acid and arachidonic acid) can activate protein kinase C independently of Ca2+ and phospholipid (Murakami, K., and Routtenberg, A. (1985) FEBS Lett. 192, 189-193). This study shows that other cis-fatty acids such as linoleic acid also fully activate protein kinase C in the same manner. None of the saturated fatty acids (C:4 to C:18) nor the detergents (sodium dodecyl sulfate and Triton X-100) tested here were as effective as oleic acid. Unlike oleic acid, these detergents strongly inhibited protein kinase C activity induced by Ca2+/phosphatidylserine (PS) and diacylglycerol. Lowering the critical micelle concentration of oleic acid by increasing ionic strength also strongly inhibited oleic acid activation of protein kinase C activity. Dioleoylphosphatidylserine activated protein kinase C effectively (Ka = 7.2 microM). On the other hand, dimyristoylphosphatidylserine, which contains saturated fatty acids at both acyl positions, failed to activate protein kinase C even in the presence of Ca2+. These observations suggest that: protein kinase C activation by free fatty acid is specific to the cis-form and is not due to their detergent-like action, cis-fatty acid activation is due to the direct interaction of protein kinase C with the monomeric form of cis-fatty acids and not with the micelles of fatty acids, and cis-fatty acids at acyl positions in PS are also important for Ca2+/PS activation of protein kinase C.     

Effect of cis-unsaturated fatty acids on aortic protein kinase C activity.

Long-chain cis-unsaturated fatty acids could substitute for phosphatidylserine and activate bovine aortic protein kinase C in assays with histone as substrate. The optimal concentration was 24-40 microM for oleic, linoleic and arachidonic acids. With arachidonic acid, the Ka for Ca2+ was 130 microM and kinase activity was maximal at 0.5 mM-Ca2+. Diolein only slightly activated the oleic acid-stimulated enzyme at low physiological Ca2+ concentrations (0.1 and 10 microM). Oleic acid also stimulated kinase C activity, determined with a Triton X-100 mixed-micellar assay. Under these conditions, the fatty acid activation was absolutely dependent on the presence of diolein, but a Ca2+ concentration of 0.5 mM was still required for maximum kinase C activity. The effect of fatty acids on protein kinase C activity was also investigated with the platelet protein P47 as a substrate, since the properties of kinase C can be influenced by the choice of substrate. In contrast with the results with histone, fatty acids did not stimulate the phosphorylation of P47 by the aortic protein kinase C. Activation of protein kinase C by fatty acids may allow the selective phosphorylation of substrates, but the physiological significance of fatty acid activation is questionable because of the requirement for high concentrations of Ca2+.     

Arachidonic and cis-unsaturated fatty acids induce selective platelet substrate phosphorylation through activation of cytosolic protein kinase C

The ability of arachidonic acid and other fatty acids to induce phosphorylation of endogenous substrates and the role of protein kinase C in mediating these effects were examined. In a cell-free cytosolic system derived from human platelets, arachidonic, oleic, and other cis-unsaturated fatty acids induced a dose-dependent phosphorylation of several endogenous substrates. These substrates form a subset of phorbol ester-induced phosphorylations. Multiple lines of evidence suggested the direct involvement of protein kinase C in mediating fatty acid-induced phosphorylations. These observations suggest that arachidonic acid and other unsaturated fatty acids are capable of activating protein kinase C in a physiologic environment resulting in the phosphorylation of multiple endogenous substrates.     

Activation of protein kinase C by cis- and trans-fatty acids and its potentiation by diacylglycerol

Both cis- and trans-unsaturated but not saturated fatty acids activated protein kinase C purified to apparent homogeneity from rat brain. Fatty-acid-induced enzyme activation was not more than additive with that by phospholipids and was potentiated by diacylglycerol. Recently, we demonstrated that cis- and trans-unsaturated fatty acids induced platelet aggregation and phosphorylation of specific proteins. Both events were potentiated by a cell-permeable diacylglycerol [(1987) Biochem. Biophys. Res. Commun. 149, 762-768]. Thus, trans-unsaturated fatty acids may provide useful experimental tools for the study of protein kinase C activation in vitro and in vivo. Our results suggest that fatty acids and diacylglycerol may synergistically be involved in hormonal stimulation of protein kinase C, as certain hormonal stimuli cause release of diacylglycerol and fatty acids from phospholipids by parallel activation of phospholipases C and A2.     

Activation of protein kinase C by cis- and trans-octadecadienoic acids in intact human platelets and its potentiation by diacylglycerol

Octadecadienoic acids (linoleic acid and linolelaidic acid) and the diacylglycerol, 1-oleoyl-2-acetyl-rac-glycerol (OAG) concentration-dependently induced activation of gel-filtered human platelets, i.e. aggregation and phosphorylation of 20 kDa and 47 kDa peptides. In contrast, octadecenoic acids (oleic and elaidic acid) and octadecanoic (stearic) acid were inactive. Octadecadienoic acid-induced platelet activation was suppressed by the protein kinase C inhibitor, polymyxin B, but not by the cyclooxygenase inhibitor, indomethacin. OAG-induced activation was potentiated by octadecadienoic acids present at non-stimulatory concentrations. Our data suggest that octadecadienoic acids and diacylglycerol synergistically induce platelet activation via protein kinase C. Furthermore, linolelaidic acid may provide a useful experimental tool to study fatty acid regulation of protein kinase C in intact cells.     

Increased Hippocampal 5-Lipoxygenase mRNA Content in Melatonin-Deficient, Pinealectomized Rats

Abstract: Tryptophan hydroxylase, the initial and rate-limiting enzyme in the biosynthesis of the neurotransmitter serotonin, is activated by protein kinase A and calcium/calmodulin-dependent protein kinase. One important aspect of the regulation of any enzyme by a phosphorylation-dephosphorylation cascade, and one that is lacking for tryptophan hydroxylase, lies in the identification of its site of phosphorylation by protein kinases. Recombinant forms of brain tryptophan hydroxylase were expressed as glutathione S -transferase fusion proteins and exposed to protein kinase A. This protein kinase phosphorylates and activates full-length tryptophan hydroxylase. The inactive regulatory domain of the enzyme (corresponding to amino acids 1–98) was also phosphorylated by protein kinase A. The catalytic core of the hydroxylase (amino acids 99–444), which expresses high levels of enzyme activity, was neither phosphorylated nor activated by protein kinase A. Conversion of serine-58 to arginine resulted in the expression of a full-length tryptophan hydroxylase mutant that, although remaining catalytically active, was neither phosphorylated nor activated by protein kinase A. These results indicate that the activation of tryptophan hydroxylase by protein kinase A is mediated by the phosphorylation of serine-58 within the regulatory domain of the enzyme.     

A novel yeast gene coding for a putative protein kinase

A yeast gene termed YKR2 coding for a putative protein kinase was isolated using the cloned cDNA for rabbit protein kinase C as a hybridization probe. The encoded protein YKR2, containing 677 amino acids, shows about 40% sequence identity in the kinase region to a family of serine/threonine-specific protein kinases from various species.     

Arachidonic acid and related methyl ester mediate protein kinase C activation in intact platelets through the arachidonate metabolism pathways

Unlike unsaturated fatty acids, which almost fully activated purified brain protein kinase C in a phosphatidylserine- and Ca2(+)-free reaction, related methyl esters were poorly active in vitro. In contrast, methyl arachidonate was revealed to be as potent as arachidonic acid in activating protein kinase C in intact platelets. Arachidonic acid-mediated activation peaked at 20 s while methyl arachidonate-mediated activation plateaued at 2 min when both lipids were added at 50 microM. At concentrations higher than 0.3 mM, all tested unsaturated fatty acids and related methyl esters were weak activators of the enzyme, with the exception of linolenic acid and methyl linolenate which evoked strong enzyme activation. However, inhibitors of arachidonate metabolism blocked both arachidonic-acid and methyl-arachidonate-induced responses. At 5 microM arachidonic acid and methyl arachidonate, protein kinase C activation was due to a cyclooxygenase product(s) whereas at 50 microM the lipoxygenase pathway was mostly involved in the reaction. Therefore, arachidonic acid and its methyl ester activate protein kinase C in platelets mainly through action of their metabolites and eicosanoid synthesis. It is suggested that such indirect protein kinase C activation may account for the tumor-promoting activity of unsaturated fatty acids and related methyl esters.     

Regulation of glycogen synthesis by amino acids in cultured human muscle cells

Insulin and a number of metabolic factors stimulate glycogen synthesis and the enzyme glycogen synthase. Using human muscle cells we find that glycogen synthesis is stimulated by treatment of the cells with lithium ions, which inhibit glycogen synthase kinase 3. Insulin further stimulates glycogen synthesis in the presence of lithium ions, an effect abolished by wortmannin and rapamycin. We report also that amino acids stimulate glycogen synthesis and glycogen synthase, these effects also being blocked by rapamycin and wortmannin. Amino acids stimulate p70(s6k) and transiently inhibit glycogen synthase kinase 3 without effects on the activity of protein kinase B or the mitogen-activated protein kinase pathway. Thus, the work reported here demonstrates that amino acid availability can regulate glycogen synthesis. Furthermore, it demonstrates that glycogen synthase kinase 3 can be inactivated within cells independent of activation of protein kinase B and p90(rsk).     

cDNA cloning and characterization of eck, an epithelial cell receptor protein-tyrosine kinase in the eph/elk family of protein kinases.

A human epithelial (HeLa) cDNA library was screened with degenerate oligonucleotides designed to hybridize to highly conserved regions of protein-tyrosine kinases. One cDNA from this screen was shown to contain a putative protein-tyrosine kinase catalytic domain and subsequently used to isolate another cDNA from a human keratinocyte library that encompasses the entire coding region of a 976-amino-acid polypeptide. The predicted protein has an external domain of 534 amino acids with a presumptive N-terminal signal peptide, a transmembrane domain, and a cytoplasmic domain of 418 amino acids that includes a canonical protein-tyrosine kinase catalytic domain. Molecular phylogeny indicates that this protein kinase is closely related to eph and elk and that this receptor family is more closely related to the non-receptor protein-tyrosine kinase families than to other receptor protein-tyrosine kinases. Antibodies raised against a TrpE fusion protein immunoprecipitated a 130-kDa protein that became phosphorylated on tyrosine in immune complex kinase assays, indicating that this protein is a bona fide protein-tyrosine kinase. Analysis of RNA from 13 adult rat organs showed that the eck gene is expressed most highly in tissues that contain a high proportion of epithelial cells, e.g., skin, intestine, lung, and ovary. Several cell lines of epithelial origin were found to express the eck protein kinase at the protein and RNA levels. Immunohistochemical analysis of several rat organs also showed staining in epithelial cells. These observations prompted us to name this protein kinase eck, for epithelial cell kinase.     

Signalling by amino acid nutrients

It is clear that mTORC1 (mammalian target of rapamycin complex 1) is regulated by the presence of ambient amino acid nutrients. However, the mechanism by which amino acids regulate mTORC1 is still open to question, despite extensive efforts. Our recent work has revealed that PR61?, a B56 family regulatory subunit of PP2A (protein phosphatase 2A), associates with and regulates the activity of MAP4K3 (mitogen-activated protein kinase kinase kinase kinase 3), a protein kinase regulated by amino acid sufficiency that acts upstream of mTORC1. In searching for a physiological process regulated by amino acids, we have demonstrated recently that arginine plays a role in the activation of LPS (lipopolysaccharide)-induced MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase]/ERK signalling in macrophages. PP2A similarly associates with the upstream regulator of MEK in this signalling pathway, TPL-2 (tumour progression locus-2), in response to arginine availability. Thus PP2A is a negative regulator of both MAP4K3 and TPL-2 in both mTORC1 and MEK/ERK signalling pathways.     

A novel transmembrane serine/threonine protein kinase gene from a rifamycin SV-producing amycolatopsis mediterranei U32.

Genomic DNA sequencing in the vicinity of methylmalonyl-CoA mutase gene (mutAB) from a rifamycin SV-producing Amycolatopsis mediterranei U32 allowed us to clone, sequence, and identify a gene encoding a novel serine/threonine protein kinase (amk). The sequence contains a complete ORF of 1821 base pairs encoding a predicted protein of 606 amino acids in length. The N-terminal domain of the protein shows significant homology to the catalytic domain of other protein kinases from both prokaryotic and eukaryotic sources. It also contains all the structural features that are highly conserved in active protein kinases, including the Gly-X-Gly-X-X-Gly motif of ATP-binding and the essential amino acids known to be important for the recognition of the correct hydroxyamino acid in serine/threonine protein kinase. This protein kinase gene was expressed in Escherichia coli and was shown to have the ability of autophosphorylation. The autophosphorylated site was found to be the threonine at position 164 by labeled phosphoamino acid analysis and site-directed mutagenesis. The C-terminal half of protein kinase was found to contain strong transmembrane structures by PhoA fusion protein analysis, suggesting that Amk protein kinase is a transmembrane protein. A Southern hybridization experiment showed that this type of protein kinase is distributed ubiquitously and might play significant physiological roles in the various species of streptomycetes. However, overexpression of amk gene in Streptomyces cinnamonensis showed no effect on methylmalonyl-CoA mutase activity, monensin production and the hyphae morphology. Although its biological role is still unknown, Amk protein kinase is the first transmembrane serine/threonine protein kinase described for genus Amycolatopsis.     

Substitution by fatty acids for phosphatidylserine in a reconstitution of phorbol ester binding to protein kinase C.

1. Fatty acids can be substituted or phosphatidylserine in a reconstitution of phorbol ester binding to protein kinase C. 2. Phorbol ester, however, does not seem to be effectively utilized for the activation of the enzyme. 3. It is suggested that fatty acids play a role on the activation of protein kinase C in the abnormal conditions such as ischemia, while the phospholipid-dependent activation has a physiological significance in normal conditions.     

A calcium and free fatty acid-modulated protein kinase as putative effector of the fusicoccin 14-3-3 receptor.

A protein kinase that is activated by calcium and cis-unsaturated fatty acids has been characterized from oat (Avena sativa L.) root plasma membranes. The kinase phosphorylates a synthetic peptide with a motif (-R-T-L-S-) that can be phosphorylated by both protein kinase C (PKC) and calcium-dependent protein kinase (CDPK)-type kinases. Calphostin C and chelerythrine, two PKC inhibitors, completely inhibited the kinase activity with values of inhibitor concentration for 50% inhibition of 0.7 and 30 microns, respectively. At low Ca2+ concentrations cis-unsaturated fatty acids (linolenic acid, linoleic acid, arachidonic acid, and oleic acid) stimulated the kinase activity almost 10-fold. The two inhibitors of the kinase, calphostin C and chelerythrin, strongly reduced the fusicoccin (FC)-induced H+ extrusion, and the activators of the kinase, the cis-unsaturated fatty acids, prevented [3H]FC binding to the FC 14-3-3 receptor. CDPK antibodies cross-reacted with a 43-kD band in the plasma membrane and in a purified FC receptor fraction. A polypeptide with the same apparent molecular mass was recognized by a synthetic peptide that has a sequence homologous to the annexin-like domain from barely 14-3-3. The possibility of the involvement of a kinase, with properties from both CDPK and PKC, and a phospholipase A2 in the FC Signal transduction pathway is discussed.     

Glucose stimulates protein synthesis in skeletal muscle of neonatal pigs through an AMPK- and mTOR-independent process

Skeletal muscle protein synthesis is elevated in neonates in part due to an enhanced response to the rise in insulin and amino acids after eating. In vitro studies suggest that glucose plays a role in protein synthesis regulation. To determine whether glucose, independently of insulin and amino acids, is involved in the postprandial rise in skeletal muscle protein synthesis, pancreatic-substrate clamps were performed in neonatal pigs. Insulin secretion was inhibited with somatostatin and insulin was infused to reproduce fasting or fed levels, while glucose and amino acids were clamped at fasting or fed levels. Fractional protein synthesis rates and translational control mechanisms were examined. Raising glucose alone increased protein synthesis in fast-twitch glycolytic muscles but not in other tissues. The response in muscle was associated with increased phosphorylation of protein kinase B (PKB) and enhanced formation of the active eIF4E.eIF4G complex but no change in phosphorylation of AMP-activated protein kinase (AMPK), tuberous sclerosis complex 2 (TSC2), mammalian target of rapamycin (mTOR), 4E-binding protein-1 (4E-BP1), ribosomal protein S6 kinase (S6K1), or eukaryotic elongation factor 2 (eEF2). Raising glucose, insulin, and amino acids increased protein synthesis in most tissues. The response in muscle was associated with phosphorylation of PKB, mTOR, S6K1, and 4E-BP1 and enhanced eIF4E.eIF4G formation. The results suggest that the postprandial rise in glucose, independently of insulin and amino acids, stimulates protein synthesis in neonates, and this response is specific to fast-twitch glycolytic muscle and occurs by AMPK- and mTOR-independent pathways.     

Fatty-acid-induced activation of NADPH oxidase in plasma membranes of human neutrophils depends on neutrophil cytosol and is potentiated by stable guanine nucleotides

Both cis and trans unsaturated fatty acids and sodium dodecyl sulfate activated NADPH oxidase in plasma membranes of human neutrophils in the presence of neutrophil cytosol. In contrast, 5,8,11,14-icosatetraynoic acid, saturated fatty acids, esters, peroxides and 4 beta-phorbol 12-myristate 13-acetate, a potent activator of protein kinase C, were inactive. 5,8,11,14-icosatetraynoic acid inhibited superoxide formation elicited by fatty acids. Guanosine 5'[gamma-thio]triphosphate (GTP[gamma S]), a potent activator of guanine-nucleotide-binding proteins (N-proteins) enhanced superoxide formation elicited by fatty acids up to fourfold, supporting our previous suggestion that NADPH oxidase is regulated by an N-protein [Seifert, R. et al. (1986) FEBS Lett. 205, 161-165]. Cytosols from various tissues, soybean lipoxygenase and protein kinase C, purified from chicken stomach, did not substitute neutrophil cytosol. The activity of neutrophil cytosol was destroyed by heating at 95 degrees C. Superoxide formation was not affected by the inhibitor of protein kinase C 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7). Removal of cytosolic ATP by preincubation with hexokinase and glucose, dialysis of neutrophil cytosol or chelation of calcium with EGTA did not abolish the stimulatory effect of arachidonic acid and GTP[gamma S]. Thus, the cytosolic cofactor appears to be a neutrophil-specific and heat-labile protein, which is neither a lipoxygenase nor protein kinase C.     

Effect of nicotinic acid on chicken liver protein kinase activity and cAMP content

The biosynthesis of fatty acids in the chicken liver was stimulated by feeding up chickens with high-carbon products. After fasting the cAMP content and protein kinase activity in chicken fall considerably as compared to the control. After administration of nicotinic acid to chicken under experiment the content of cAMP and the protein kinase activity in the liver tissue rise to the highest extent, returning to initial values by the end of the day. The maximal increase in the cAMP content and protein kinase activity coincides in time with the maximum of the acetyl-CoA-carboxylase activity decrease. An assumption is advanced that biosynthesis of fatty acids in the liver tissue of chickens is regulated by a change in the degree of acetyl-CoA-carboxylase phosphorylation with the participation of adenylate cyclase system.     

Cloning and Analyses of a Dual Specific Serine􊄯Thronine Protein Kinase Gene with High Conservative and Constitutive Expression in Oryza (OsSTK)

A cytoplasmic serine􊄯thronine protein kinase gene (OsSTK), had been cloned from Oryza genus. It was found high conservative and constitutive expression in Oryza. OsSTK gene had two exons, separated by 114 bp short intron. The open reading frame of OsSTK gene that predicted encoded a 419 amino acids protein . The amino acid sequence of OsSTK had low identities ( less than 53% ) with any other known protein kinase . The phylogenetic tree based on the partial DNA sequences of OsSTK from different species and types of wild rice and cultivated rice, was close to the taxation system ofrice . Interestingly , OsSTK had a serine, including basic amino acids and charged amino acids abundant polypeptide with a “GDGDGDGDG”sequence at N- terminal that had not been found in any other genes. OsSTK may play dual specificity that phosphorylates both serine􊄯thronine and tyrosone, because the amino acids module of VIb , VIII and XI catalytic domain have both the serine􊄯thronine and tyrosine kinase characters .