Inmunosenescencia prematura en ratones con una deficiencia en la síntesis de catecolaminas. Efecto del ambiente social / Premature immunosenescence in catecholamines syntesis deficient mice. Effect of social environment

Introducción. La salud depende del buen funcionamiento de los sistemas homeostáticos (el nervioso, endocrino e inmunitario) y de la adecuada comunicación entre ellos. Se ha comprobado que el estado funcional y redox del sistema inmunitario es un excelente marcador de salud, y que una inmunosenescencia prematura supone una menor esperanza de vida. Dado que las catecolaminas modulan la funcionalidad de las células inmunitarias, la alteración en su síntesis podría contribuir a esa inmunosenescencia. Entre las estrategias que se pueden utilizar para controlarla está el ambiente social. Objetivo. Comprobar si una haploinsuficiencia de la tirosina hidroxilasa (TH), enzima limitante de la síntesis de catecolaminas, generaría una inmunosenescencia prematura, y si es posible la modulación de esta por el ambiente social. Material y métodos. Se usaron ratones machos ICR-CD1 adultos (9±1 meses) hemizigotos (HZ) para la tirosina hidroxilasa (TH-HZ) y controles (WT), que fueron distribuidos en cuatro subgrupos: WT>50% (en la jaula, la proporción de WT fue mayor al 50%), WT<50%, TH-HZ<50% y TH-HZ>50%. En leucocitos peritoneales se valoró la fagocitosis, quimiotaxis y linfoproliferación en presencia de lipopolisacárido. También, la actividad de las enzimas antioxidantes glutatión reductasa y glutatión peroxidasa, y el cociente glutatión oxidado/glutatión reducido. Resultados. Los TH-HZ>50% presentaron, en leucocitos, una funcionalidad y estado redox deteriorados respecto a WT>50 y similar a ratones viejos. Sin embargo, los TH-HZ<50% mostraron valores similares a los WT<50%. Conclusión. Una haploinsuficiencia de la enzima TH provoca una inmunosenescencia prematura, la cual puede ser compensada por la convivencia con un número apropiado de animales WT (AU)

Introduction. Healthy state depends on the appropriate function of the homeostatic systems (nervous, endocrine and immune systems) and the correct communication between them. The functional and redox state of the immune system is an excellent marker of health, and animals with premature immunosenescence show a shorter lifespan. Since catecholamines modulate the function of immune cells, the alteration in their synthesis could provoke immunosenescence. The social environment could be a strategy for modulating this immunosenescence. Aim. To determine if an haploinsufficiency of tyrosine hydroxylase (TH), the limiting enzyme of synthesis of catecholamines, may produce a premature immunosenescence and if this immunosenescence could be modulated by the social environment. Materials and methods. Adult (9±1 months) male ICR-CD1 mice with deletion of a single allele (hemi-zygotic: HZ) of the tyrosine hydroxylase enzyme (TH-HZ) and wild-type (WT) mice were used. Animals were housed in four subgroups: WT>50% (in the cage, the proportion of WT mice was higher than 50% in relation to TH-HZ), WT<50%, TH-HZ<50% and TH-HZ>50%. Peritoneal leukocytes were collected and phagocytosis, chemotaxis and proliferation of lymphocytes in the presence of lipopolysaccharide were analyzed. Glutathione reductase and glutathione peroxidase activities as well as oxidized/reduced glutathione ratio were studied. Results. TH-HZ>50% mice showed a deteriorated function and redox state in leukocytes respect to WT>50% and similar to old mice. However, TH-HZ<50% animals had similar values to those found in WT<50% mice. Conclusion. The haploinsufficiency of TH generates premature immunosenescence, which appears to be compensated by living together with an appropriate number of WT animals (AU)

Targeted toxicants to dopaminergic neuronal cell death.

Parkinson's disease (PD ) is mainly characterized by a progressive degeneration of dopaminergic neurons in the substantia nigra resulting in chronic deficits in motor functions. Administration of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP ) produces PD symptoms and recapitulates the main features of PD in human and animal models. MPTP is converted to 1-methyl-4-phenylpyridine (MPP+ ), which is the active toxic compound that selectively destroys dopaminergic neurons. Here, we describe methods and protocols to evaluate MPTP/MPP+-induced dopaminergic neurodegeneration in both murine primary mesencephalic cultures and animal models. The ability of MPTP/MPP+ to cause dopaminergic neuronal cell death is assessed by immunostaining of tyrosine hydroxylase (TH).

Expression and purification of recombinant human tyrosine hydroxylase as a fusion protein in Escherichia coli.

Tyrosine hydroxylase is the rate-limiting step in the synthesis of dopamine and is tightly regulated. Previous studies have shown it to be covalently modified and potently inhibited by 3,4-dihydroxyphenylacetaldehyde (DOPAL), an endogenous neurotoxin via dopamine catabolism which is relevant to Parkinson's disease. In order to elucidate the mechanism of enzyme inhibition, a source of pure, active tyrosine hydroxylase was necessary. The cloning and novel purification of human recombinant TH from Escherichia coli is described here. This procedure led to the recovery of ~23 mg of pure, active and stable enzyme exhibiting a specific activity of ~17 nmol/min/mg. The enzyme produced with this procedure can be used to delineate the tyrosine hydroxylase inhibition by DOPAL and its relationship to Parkinson's disease. This procedure improves upon previous methods because the fusion protein gives rise to high expression and convenient affinity-capture, and the cleaved and highly purified hTH makes the product useful for a wider variety of applications.

Cloning and characterization of a novel enzyme: tyrosine hydroxylase from Schistosoma japonicum.

Catecholamines, such as dopamine and noradrenaline, play important roles as neuromuscular transmitters and modulators in all parasitic helminthes, including Schistosoma japonicum. S. japonicum tyrosine hydroxylase (SjTH) was amplified by rapid amplification of cDNA ends polymerase chain reaction that shows strong homology to Schistosoma mansoni tyrosine hydroxylase, the enzyme that catalyzes the first and rate-limiting step in the biosynthesis of catecholamines. The SjTH transcripts encoded the protein of 463 amino acids and a predicted size of 54 kDa. Purified recombinant SjTH as an N-terminal histidine fusion protein expressed in Escherichia coli showed catalytic activity that was confirmed with (3)H tyrosine uptake. The purified enzyme was found to have the same absolute requirement for a tetrahydrobiopterin cofactor and similar sensitivity to be inhibited by high concentration of the substrate, tyrosine, as the mammalian enzyme. Also, purified SjTH showed characteristic inhibition by catecholamine products. The phosphorylated peptide from SjTH could interact with Sj14-3-3 signal protein. This evidence indicates that SjTH encodes a functional tyrosine hydroxylase that has catalytic properties similar to those of the mammalian hosts' enzyme, and its catalytic activity could be regulated by a phosphorylated or dephosphorylated form. This demonstration of SjTH further suggests that the parasites have the enzymatic capacity to synthesize catecholamines endogenously.

Characterization of tyrosine hydroxylase from Manduca sexta.

In insects, 3,4-dihydroxyphenylalanine (DOPA) is required for tanning of newly formed cuticle and the production of melanin during some types of immune responses. DOPA is produced by the hydroxylation of tyrosine, and this reaction can be catalyzed by two types of enzymes: tyrosine hydroxylase (TH) and phenoloxidase (PO). TH is required for cuticle tanning in Drosophila melanogaster and for cuticle pigmentation in other insect species, but additional functions of TH have been uncertain. In contrast, an immune function for PO has been well documented. The goal of this study was to characterize TH from Manduca sexta with a focus on its possible contribution to cuticle tanning and immune-associated melanization. We cloned a full-length TH cDNA, purified recombinant TH, and confirmed that MsTH and MsPO have tyrosine hydroxylating activity. To determine possible functions, we analyzed TH expression profiles. TH mRNA and protein were present in eggs at the stage when the pharate larval cuticle begins to tan and also in the integument of molting larvae. The amount of TH in the integument was correlated with the degree of cuticle tanning. Unlike PO, which was found to be constitutively expressed by hemocytes and was present in plasma, TH was upregulated in hemocytes and the fat body in response to an immune challenge and remained intracellular. These data suggest that TH is required for cuticle tanning and immunity in M. sexta. Based on the collective information from many studies, we propose a model in which TH is a major producer of the DOPA required for both cuticle tanning and immune-associated melanization.

A tyrosinase with an abnormally high tyrosine hydroxylase/dopa oxidase ratio.

The sequencing of the genome of Ralstonia solanacearum[Salanoubat M, Genin S, Artiguenave F, et al. (2002) Nature 415, 497-502] revealed several genes that putatively code for polyphenol oxidases (PPOs). This soil-borne pathogenic bacterium withers a wide range of plants. We detected the expression of two PPO genes (accession numbers NP_518458 and NP_519622) with high similarity to tyrosinases, both containing the six conserved histidines required to bind the pair of type-3 copper ions at the active site. Generation of null mutants in those genes by homologous recombination mutagenesis and protein purification allowed us to correlate each gene with its enzymatic activity. In contrast with all tyrosinases so far studied, the enzyme NP_518458 shows higher monophenolase than o-diphenolase activity and its initial activity does not depend on the presence of l-dopa cofactor. On the other hand, protein NP_519622 is an enzyme with a clear preference to oxidize o-diphenols and only residual monophenolase activity, behaving as a catechol oxidase. These catalytic characteristics are discussed in relation to two other characteristics apart from the six conserved histidines. One is the putative presence of a seventh histidine which interacts with the carboxy group on the substrate and controls the preference for carboxylated and decarboxylated substrates. The second is the size of the residue isosteric with the aromatic F261 reported in sweet potato catechol oxidase which acts as a gate to control accessibility to CuA at the active site.

[Purification of recombinant Schistosoma japonicum signaling protein 14-3-3 and antibody preparation].

OBJECTIVE: To prepare and characterize the polyclonal and monoclonal antibodies(McAbs) against Schistosoma japonicum(Sj) signaling protein 14-3-3. METHODS: Recombinant Sj14-3-3 protein was expressed and the gel slices were lyophilized. Rabbits were immunized with the ground gel powder to prepare polyclonal antibodies against Sj14-3-3. BALB/c mice were immunized with recombinant Sj14-3-3 purified by electroelution. The anti-rSj14-3-3 monoclonal antibody was obtained by using hybridoma technique. Characterization of the antibodies was performed by enzyme linked immunosorbent assay (ELISA) and Western blotting analysis. RESULTS: Purified recombinant Sj14-3-3 protein was obtained. The titers of immune sera from the animals were 1:8-1:64. The monoclonal antibody(McAb) against 14-3-3 antigen was obtained and its subclass was found to be IgG1. The McAb reacted strongly and specifically with the expressed Sj14-3-3 protein. CONCLUSION: The anti-14-3-3 sera were obtained and the hybridoma cell lines which secreted stable monoclonal antibodies against Sj14-3-3 were established, which provides a new approach to study the role of Sj14-3-3 in the signal transduction of S. japonicum.

The neurotoxic phospholipase A2 associates, through a non-phosphorylated binding motif, with 14-3-3 protein gamma and epsilon isoforms.

Two novel acceptors for ammodytoxin C, a presynaptically neurotoxic phospholipase A(2) from snake venom, have been purified from porcine cerebral cortex by a toxin-affinity-based procedure. Using tandem mass spectrometry, the isolated acceptors were identified as 14-3-3 gamma and epsilon isoforms, highly conserved cytoplasmic proteins involved in the regulation of numerous physiological processes. The interaction between ammodytoxin C and 14-3-3 proteins is direct and not mediated by calmodulin, a high-affinity acceptor for both ammodytoxin C and 14-3-3 proteins, as demonstrated in pull-down experiments and by surface plasmon resonance. The latter technique gave an apparent dissociation constant of 1.0+/-0.2 microM for the interaction between chip-immobilized 14-3-3 and ammodytoxin C. 14-3-3 usually interacts with proteins through specific phospho-Ser/Thr motifs. Ammodytoxin C is not a phospho-protein, therefore the interaction must occur through a non-phosphorylated binding site, most probably the KEESEK sequence at its C-terminal end. The interaction we describe suggests an explanation for the pathophysiological effects evoked by some secreted phospholipases A(2), such as the inhibition of protein phosphorylation, of terminal ion currents, and of neurotransmission, as well as the initiation of neuronal cell death, all processes regulated by 14-3-3 proteins.

Akt phosphorylates the Yes-associated protein, YAP, to induce interaction with 14-3-3 and attenuation of p73-mediated apoptosis.

We have used an affinity purification method to identify substrates of protein kinase B/Akt. One protein that associates with 14-3-3 in an Akt-dependent manner is shown here to be the Yes-associated protein (YAP), which is phosphorylated by Akt at serine 127, leading to binding to 14-3-3. Akt promotes YAP localization to the cytoplasm, resulting in loss from the nucleus where it functions as a coactivator of transcription factors including p73. p73-mediated induction of Bax expression following DNA damage requires YAP function and is attenuated by Akt phosphorylation of YAP. YAP overexpression increases, while YAP depletion decreases, p73-mediated apoptosis following DNA damage, in an Akt inhibitable manner. Akt phosphorylation of YAP may thus suppress the induction of the proapoptotic gene expression response following cellular damage.

Purification and characterization of the active form of tyrosine hydroxylase from mesangial cells in culture.

The capacity of mesangial cells (MC) to produce catecholamines (CAs) has been investigated in our laboratory. To study the CA cascade, it is necessary to examine some steps in their metabolic pathway. Tyrosine hydroxylase (TH) catalyzes the rate-limiting step in the biosynthesis of these biogenic amines (dopamine (DA), norepinephrine (NE), and epinephrine (EPI)). Since the glomerular mesangium is their target in the regulation of renal sodium transport and renin secretion, the aim of the study was to determine the presence of TH in these cells in culture. The CA levels were detected in immortalized MC by high-performance liquid chromatography with electrochemical detection. The following concentrations were found in the intracellular region and in the medium, respectively: NE = 284 +/- 31 and 134 +/- 22, EPI = 75 +/- 14 and 22 +/- 5, and DA = 42 +/- 14, 40 +/- 20 pg/mg cell protein. The enzymatic activity of the cell lysate and medium was measured based on L-dopa formation. In the presence of o-phenanthroline, both samples presented 39% inhibition. The biopterin was detected in the intracellular and in the medium (64.87 and 631.99 pmol/mg protein, respectively) using high-performance liquid chromatography with ultraviolet detection. The cell lysate was submitted to a DEAE-Sephacel column, followed by gel filtration, and Heparin-Sepharose. TH was purified 613.16-fold with a specific activity of 466.0 pg/mg cell protein. Immunoblotting using monoclonal antibody revealed the presence of TH in the different purification steps. Purified TH was sequenced, presenting an alignment with amino-terminal sequence of mouse enzyme. Our results demonstrated the presence of active TH in MC, suggesting that these cells are able to produce CA "in vivo", and establishing a convenient purification method for TH that can be applied to the study of the molecular properties of the enzyme modified "in vivo" by different physiological and pathophysiological stimuli.

Affinity purification of diverse plant and human 14-3-3-binding partners.

Many proteins that bind to a 14-3-3 column in competition with a 14-3-3-binding phosphopeptide have been purified from plant and mammalian cells and tissues. New 14-3-3 targets include enzymes of biosynthetic metabolism, vesicle trafficking, cell signalling and chromatin function. These findings indicate central regulatory roles for 14-3-3s in partitioning carbon among the pathways of sugar, amino acid, nucleotide and protein biosynthesis in plants. Our results also suggest that the current perception that 14-3-3s bind predominantly to signalling proteins in mammalian cells is incorrect, and has probably arisen because of the intensity of research on mammalian signalling and for technical reasons.

Isolation and structure of the mouse 14-3-3 eta chain gene and the distribution of 14-3-3 eta mRNA in the mouse brain.

14-3-3 protein is a brain-specific protein discovered by Moore and Perez, but at present is thought to be a multifunctional protein. To clarify the brain-specific function of the protein, we intend constructing a 14-3-3 eta gene knock-out mouse. As the first step of this process, we isolated the mouse 14-3-3 eta chain gene and determined its structure. The mouse gene is about 10 kb long and composed of two exons separated by a long intron. The transcription start site was identified and the polyadenylation signals (AATAAA) were found in exon 2 of the mouse gene. In the 5'-upstream sequence, we found several cis elements including a CRE sequence, a TATA box-like sequence, and a C/EBP element. Furthermore, the distribution of 14-3-3 eta mRNA in the mouse brain was examined by in situ hybridization histochemistry. The highest signals were found in the Purkinje cells of the cerebellum, the pyramidal cells of the hippocampus and the olfactory bulb neurons of the adult mouse. Neuronal expression of 14-3-3 eta in these regions mRNA may generally increase during postnatal brain development. The distribution of protein kinase C gamma in the mouse brain was also examined by immunohistochemistry. From the distribution of 14-3-3 eta mRNA and protein kinase C gamma in the mouse brain, the involvement of these compounds in the induction and maintenance of LTP was discussed.

Subcellular distribution of 14-3-3 proteins in the unicellular green alga Chlamydomonas reinhardtii.

A polyclonal antibody was raised against a recombinant Chlamydomonas 14-3-3-beta-galactosidase (beta-Gal) fusion protein and characterized for its epitope specificity towards the corresponding Chlamydomonas 14-3-3 protein by scan-peptide analysis. This antibody recognized four Chlamydomonas polypeptides with apparent molecular masses 32, 30, 27, and 24 kDa, which also reacted with the antiserum depleted of anti-(Escherichia coli beta-Gal) IgG, but not with the corresponding preimmune serum or the antiserum preincubated with purified 14-3-3 proteins. Western-blot analyses performed with the antibody depleted of anti-(beta-Gal) IgG revealed that more or less pronounced levels of 14-3-3 proteins were present in all subcellular fractions of Chlamydomonas reinhardtii except the nuclei. The highest levels of 14-3-3 protein were observed in the cytosol and microsomal fraction. The 30-kDa isoform was predominant in the cytosol, whereas the 27-kDa isoform was prevalent in the microsomes. When microsomal membranes were separated by sucrose-density-gradient centrifugation, Western-blot analysis revealed distinct patterns of 14-3-3 isoforms in the endoplasmic reticulum, dictyosome, and plasma membrane fractions identified by marker enzyme activities. These findings indicate that the four 14-3-3 proteins of C. reinhardtii differentially interact with endoplasmic reticulum, dictyosomes, and plasma membrane.

Isolation and purification of tyrosine hydroxylase from callus cultures of Portulaca grandiflora.

Tyrosine hydroxylase was separated from polyphenol oxidase activity and was highly purified from betacyanin producing callus cultures of Portulaca grandiflora. The purified enzyme catalyzed the formation of DOPA (L-3,4-dihydroxyphenylalanine) from tyrosine and required the pterin compounds (6-methyl-5,6,7,8-tetrahydropterin; 5,6,7,8-tetrahydrobiopterin; 6,7-dimethyl-5,6,7,8-tetrahydropterin) as coenzyme. The K(m) values for tyrosine and 6-methyl-5,6,7,8-tetrahydropterin were 0.5 mM and 0.15 mM, respectively. This enzyme was activated by Fe(2+) and Mn(2+), and inhibited by metal chelating agents.

An immunoblotting method for high-resolution isoelectric focusing of protein isoforms on immobilized pH gradients.

Post-translational modifications such as phosphorylation and acetylation are important elements for regulating the activity of enzymes or structural proteins. These modifications give rise to isoforms that are often not resolved by separation methods relying on the size of proteins. Here, we optimized an isoelectric focusing (IEF)-immunoblotting method suitable for analyzing protein isoforms in total cell extracts. The separations were carried out in parallel on commercially available immobilized pH gradient slab gels (IPG). The buffer used for separation contained urea, thiourea, dithiothreitol, as well as the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS), and was designed to match those used in two-dimensional polyacrylamide gel electrophoresis (PAGE) separations where efficient solubilization is required. Proteins were transferred to membranes by passive diffusion in the presence of 4 M guanidinium chloride using protocols optimized for several protein classes (tubulin, stathmin, 14-3-3 proteins) some of which required removal of CHAPS prior to transfer. In conjunction with narrow-range pH gradient gels, excellent resolution of isoforms differing by phosphorylation or acetylation was achieved. The usefulness of pI and titration curve calculations for predicting the pI shifts expected for post-translational modifications of proteins with known amino acid composition was demonstrated. Using stathmin--which contains four phosphorylation sites--as an example, the effects on the pI-shifts were well predicted. This sensitive and widely applicable IEF-blotting technology is expected to be especially suited for analyzing protein isoforms first detected by two-dimensional electrophoresis.

Large scale expression, purification and 2D crystallization of recombinant plant plasma membrane H+-ATPase.

P-type ATPases convert chemical energy into electrochemical gradients that are used to energize secondary active transport. Analysis of the structure and function of P-type ATPases has been limited by the lack of active recombinant ATPases in quantities suitable for crystallographic studies aiming at solving their three-dimensional structure. We have expressed Arabidopsis thaliana plasma membrane H+-ATPase isoform AHA2, equipped with a His(6)-tag, in the yeast Saccharomyces cerevisiae. The H+-ATPase could be purified both in the presence and in the absence of regulatory 14-3-3 protein depending on the presence of the diterpene fusicoccin which specifically induces formation of the H+-ATPase/14-3-3 protein complex. Amino acid analysis of the purified complex suggested a stoichiometry of two 14-3-3 proteins per H+-ATPase polypeptide. The purified H(+)-ATPase readily formed two-dimensional crystals following reconstitution into lipid vesicles. Electron cryo-microscopy of the crystals yielded a projection map at approximately 8 A resolution, the p22(1)2(1) symmetry of which suggests a dimeric protein complex. Three distinct regions of density of approximately equal size are apparent and may reflect different domains in individual molecules of AHA2.

Rethinking the role of phosducin: light-regulated binding of phosducin to 14-3-3 in rod inner segments.

Phosducin (Pd), a small protein found abundantly in photoreceptors, is widely assumed to regulate light sensitivity in the rod outer segment through interaction with the heterotrimeric G protein transducin. But, based on histochemistry and Western blot analysis, Pd is found almost entirely in the inner segment in both light and dark, most abundantly near the rod synapse. We report a second small protein, 14-3-3, in the rod with a similar distribution. By immunoprecipitation, phospho-Pd is found to interact with 14-3-3 in material from dark-adapted retina, and this interaction is markedly diminished by light, which dephosphorylates Pd. Conversely, unphosphorylated Pd binds to inner segment G protein(s) in the light. From these results and reported functions of 14-3-3, we have constructed a hypothesis for the regulation of light sensitivity at the level of rod synapse. By dissociating the Pd/14-3-3 complex, light enables both proteins to function in this role.

Identification of a rat 30-kDa protein recognized by the antibodies to a recombinant rat cutaneous fatty acid-binding protein as a 14-3-3 protein.

Immunoblot analysis with polyclonal antibodies raised against a recombinant rat cutaneous fatty acid-binding protein revealed a 30-kDa protein other than the 15-kDa fatty acid-binding protein in rat skin cytosol. This protein was present in a number of rat organs and in mouse 3T3 L1 cells. The amino acid sequences of the enzymatic peptides of the 30-kDa protein extracted from SDS-PAGE gels suggested that it was a mixture of the subunits of the eukaryotic signaling molecule, 14-3-3 protein. Glutathione S-transferase fusion proteins of 14-3-3 protein subunits were examined for cross-reaction by Western blotting, and the epsilon-subunit alone was found to be immunoreactive, so far as tested. It is likely that the 30-kDa protein detected in the rat tissues by the antibodies is the 14-3-3 protein epsilon-subunit. Although there is no apparent sequence similarity between the fatty acid-binding protein and the 14-3-3 protein subunit, they appear to share a common structural element recognized by the antibodies. Since 14-3-3 proteins and fatty acid-binding proteins are known to interact with a wide variety of cellular proteins, the presence of a common local structure might mutually modulate such interactions.

Glucocorticoid receptor interaction with 14-3-3 and Raf-1, a proposed mechanism for cross-talk of two signal transduction pathways.

The glucocorticoid receptor (GR) functions as a ligand-dependent transcription factor. In the present study we describe a specific immunoaffinity chromatography purification of GR from liver cytosol from adrenalectomized rats that may be used to identify hitherto unknown cytosolic GR interacting proteins. We have identified the ubiquitously expressed 14-3-3 as well as Raf-1, a downstream effector of Ras, as GR co-purifying proteins. In our semi-quantitative analysis liganded/activated GR showed the strongest interaction with 14-3-3 and Raf-1, but 14-3-3 was also found to co-purify with GR in a nonliganded/nonactivated state. By extensive salt washes we were also able to demonstrate that the glucocorticoid induced interaction between GR, 14-3-3, and Raf-1, respectively, is remarkably stable and withstood 2.4 m salt. The interaction between GR and 14-3-3 was also verified by 14-3-3 co-immunoprecipitation studies. Our observations that GR and Raf-1 are found within the same protein complex ("receptosome") in the cytoplasm of rat liver cells could provide a mechanistic explanation for glucocorticoid effects on the Raf-1-Ras signaling pathway.

14-3-3 proteins and a 13-lipoxygenase form associations in a phosphorylation-dependent manner.

Recently, we have demonstrated by two different methods that lipoxgenases (LOXs) and 14-3-3 proteins form interactions in barley embryos [Holtman, Roberts, Oppedijk, Testerink, van Zeijl and Wang (2000) FEBS Lett. 474, 48-52]. It was shown by both co-immunoprecipitations and surface-plasmon resonance experiments that 13-LOX, but not 9-LOX, forms interactions with 14-3-3 proteins. In the present report we show that the presence of 13-LOX and 14-3-3 proteins was established in high-molecular-mass complexes. Amounts of 13-LOX and 14-3-3 proteins in high-molecular-mass fractions increased during germination, but were reduced after dephosphorylation of protein extracts or competition with the 14-3-3-binding peptide P-Raf-259, indicating that 13-LOX and 14-3-3 proteins interact in a phosphorylation-dependent manner.