Multiple complexes involved in tyrosine hydroxylase mRNA stability in rat adrenal medulla, after reserpine stimulation.

A 28-nucleotide sequence within the 3'-untranslated region (3'UTR) of tyrosine hydroxylase (TH) mRNA has been suggested to influence the turnover rate of the TH messenger in vitro (W. R. Paulding and M. F. Czyzyk-Krzeska, 1999, J. Biol. Chem. 274, 2532-2538). In this study, we show that treatment with reserpine, a catecholamine-depleting drug which increases the stability of TH mRNA, allows the binding of a cytosolic protein to this 28-mer site in the TH 3'UTR in the rat adrenal medulla. An ex vivo kinetic analysis shows that the resulting 54-kDa ribonucleoprotein is early induced by reserpine. However, the formation of this complex is not coupled with the upregulation of TH mRNA, indicating that this 54-kDa complex could not be the unique factor accountable for the long-term stabilization of the TH messenger. Following this result we found that several other cis-acting elements, located in single-stranded stem loops within the secondary structure of TH 3'UTR, formed multiple complexes (43, 54, and 105 kDa) with cytosolic, polysome-associated, and also nuclear proteins. Our findings demonstrate that the messenger stability does not depend solely on the formation of a unique RNA-protein complex, but involves mechanisms with higher complexity implicating the interactions between posttranscriptional, nuclear RNA export, and translational processes.

Human tyrosine hydroxylase isoforms. Inhibition by excess tetrahydropterin and unusual behavior of isoform 3 after camp-dependent protein kinase phosphorylation.

Human tyrosine hydroxylase exists as four isoforms (hTH1-4), generated by alternative splicing of pre-mRNA, with tissue-specific distribution. Unphosphorylated hTH3 and hTH1 were produced in large amounts in Escherichia coli and purified to homogeneity. The phosphorylation sites were determined after labeling with [32P]phosphate in the presence of cAMP-dependent protein kinase (PKA) and calmodulin-dependent protein kinase II (CaM-PKII). Ser40 was phosphorylated by PKA, and both Ser19 and Ser40 were phosphorylated by CaM-PKII. The enzyme kinetics of hTH3 were determined in the presence of various concentrations of the natural co-substrate (6R)-tetrahydrobiopterin and compared with those of recombinant hTH1 (similar to rat TH). We show that, under initial velocity conditions, excess (6R)-tetrahydrobiopterin inhibits hTH3 and hTH1. The TH catalytic constants (kcat) were determined for each of the two isoenzymes: hTH3 is about five times more active than hTH1. Phosphorylation by CaM-PKII did not affect the kinetic parameters of hTH3. The classical activation of TH by PKA phosphorylation, demonstrated for hTH1, was not observed with hTH3. Furthermore, hTH3 escapes activity regulation by phosphorylation and is always more active than phosphorylated hTH1. The properties of the hTH3 enzyme may be relevant to diseases affecting dopaminergic cells.

Phosphorylation and activation of human tyrosine hydroxylase in vitro by mitogen-activated protein (MAP) kinase and MAP-kinase-activated kinases 1 and 2.

Mitogen-activated protein-kinase (MAP) kinase-activated protein kinases 1 and 2 (MAPKAP kinase-1, MAPKAP kinase-2), were found to phosphorylate bacterially expressed human tyrosine hydroxylase in vitro at comparable rates to other proteins thought to be physiological substrates of these protein kinases. The phosphorylation of all four alternatively spliced forms of human tyrosine hydroxylase by MAPKAP kinases-1 and -2 reached plateau values at 1 mol/mol subunit and 2 mol/mol subunit, respectively; the sites of phosphorylation were identified as Ser40 (MAPKAP kinase-1) and Ser19 and Ser40 (MAPKAP kinase-2). In contrast to calmodulin-dependent protein kinase-II, which phosphorylates Ser19 faster than Ser40, MAPKAP kinase-2 phosphorylated Ser40 about twice as fast as Ser19. The maximal activation of tyrosine hydroxylase by MAPKAP kinase-1 or-2 was about 3-fold, and activation by MAPKAP kinases-1 and -2 or calmodulin-dependent protein kinase-II correlated with the extent of phosphorylation of Ser40. The four alternatively spliced forms of human tyrosine hydroxylase were phosphorylated at Ser31 by MAP kinase, but at markedly different rates (3 = 4 > 1 >> 2). Forms 3 and 4 were phosphorylated rapidly and stoichiometrically by MAP kinase doubling the activity, while phosphorylation of form 1 by MAP kinase to 0.4 mol/mol subunit increased activity by 40%. The effect on activity of phosphorylating both Ser31 and Ser40 was not additive. The possible roles of MAPKAP kinase-1, MAPKAP kinase-2 and MAP kinase in the regulation of tyrosine hydroxylase in vivo are discussed.

A mammalian tryptophanyl-tRNA synthetase shows little homology to prokaryotic synthetases but near identity with mammalian peptide chain release factor.

Determination of the amino acid sequence of beef pancreas tryptophanyl-tRNA synthetase was undertaken through both cDNA and direct peptide sequencing. A full-length cDNA clone containing a 475 amino acid open reading frame was obtained. The molecular mass of the corresponding peptide chain, 53,728 Da, was in agreement with that of beef tryptophanyl-tRNA synthetase, as determined by physicochemical methods (54 kDa). Expression of this clone in Escherichia coli led to tryptophanyl-tRNA synthetase activity in cell extracts. The open reading frame included two sequences analogous to the consensus sequences, HIGH and KMSKS, found in class I aminoacyl-tRNA synthetases. The homology with prokaryotic and yeast mitochondrial tryptophanyl-tRNA synthetases was low and was limited to the regions of the consensus sequences. However, a 90% homology was observed with the recently described rabbit peptide chain release factor (eRF) [Lee et al. (1990) Proc. Natl. Acad. Sci. 87, 3508-3512]. Such a strong homology may reveal a new group of genes deriving from a common ancestor, the products of which could be involved in tRNA aminoacylation (tryptophanyl-tRNA synthetase) or translation termination (eRF).

Localization of acidic fibroblast growth factor (aFGF) mRNA in mouse and bovine retina by in situ hybridization.

Acidic fibroblast growth factor (aFGF) mRNA has been detected in adult mouse or bovine retina by in situ hybridization with bovine aFGF cDNA clones. It is localized on ganglion cell layer, inner nuclear layer, photoreceptors and slightly on pigmented epithelium. This synthesis of aFGF in highly specialized retinal cell types is discussed in the framework on current views about the role of FGF in retinal cell biology.

Basic fibroblast growth factor expression in human omental microvascular endothelial cells and the effect of phorbol ester.

The human omentum contains a potent, not yet identified angiogenic activity. The omentum is very vascularized. Therefore, we investigated whether human omental microvascular endothelial cells (HOME cells) express the angiogenic peptide basic fibroblast growth factor (bFGF). Cytosol prepared from HOME cells stimulated DNA synthesis in bovine epithelial lens cells (BEL cells). The mitogenic activity could be neutralized by an anti-bFGF antibody. Basic FGF-like material from the HOME cell cytosol was bound onto a heparin-Sepharose column at 0.6 M and was eluted at 3 M NaCl. The 3 M NaCl eluted material reacted with the specific anti-bFGF antibody in an ELISA and stimulated DNA synthesis. It did not react with a specific anti-acidic fibroblast growth factor (aFGF) antibody. Western blotting experiments using the same bFGF antibody showed the presence of a major band of 17 Kd and a doublet of 20-22 Kd. Northern blotting of non-stimulated HOME cells using a specific 1.4 kb bFGF probe showed the presence of 5 molecular species of 6.6, 3.7, 2.2, 2.0, and 1.0 kb. No aFGF mRNA was detected with a specific previously characterized 4.04 kb probe. 12-O-tetradecanoylphorbol 13-acetate (TPA) did not influence significantly the expression of bFGF at the protein and mRNA level in HOME cells. Thus, protein kinase C activation by TPA did not appear to modulate significantly the expression of bFGF for that cell type. Contrastingly, human umbilical vein endothelial cells (HUVE cells), which expressed no bFGF and aFGF mRNA at a basal level, were induced to express bFGF but not aFGF mRNA when stimulated by TPA. These results suggest that the described angiogenic activity could be the bFGF-like mitogen contained in HOME cells and that these cells are different from endothelial cells derived from large vessels (HUVE cells) regarding the expression of bFGF.

Expression of phosphoglycerate mutase mRNA in differentiating rat satellite cell cultures.

Poly(A)+ mRNA was isolated from rat satellite cell cultures and analyzed by Northern blot analyses for mRNA content of phosphoglycerate mutase (PGAM) isozymes. In non-differentiating satellite cells only PGAM-B mRNA was detected, but when cells were differentiated into myotubes, which undergo spontaneous contraction, mRNA for PGAM-M muscle-specific isozyme was also detected. This finding is in perfect concordance with the transition of PGAM isozymes encountered in the same cell cultures, and strongly supports a transcriptional control of PGAM expression throughout myogenesis independently of nerve influence.

Acidic and basic fibroblast growth factor mRNAs are expressed by skeletal muscle satellite cells.

We postulated that Fibroblast Growth Factor (FGF) involved in fetal or regenerative morphogenesis of skeletal muscle originated from this tissue. Using a bovine retina cDNA probe encoding acidic FGF, we showed that growing muscles from bovine fetuses express this mRNA, but that this expression is reduced in neonate muscles. Cultures of proliferating satellite cells isolated from adult rat muscles expressed aFGF mRNA strongly but bFGF mRNA weakly; these mRNAs disappeared in cells differentiated into myotubes. 10(-7)M 12-O-tetradecanoyl phorbol -13-acetate (TPA) increased aFGF mRNA expression in both proliferating and differentiated satellite cells. Contrastingly, proliferating L6 myogenic cells only expressed aFGF mRNA significantly under TPA treatment. Therefore, the satellite cells did seem to be a possible source for FGF, especially aFGF, which might regulate the myogenic process.

Increased sensitivity of various genes to endogenous DNase activity in terminal differentiating chick lens fibers.

In the lens, epithelial cells from the equatorial zone differentiate into postmitotic elongated fibers. One aspect of this differentiation is nuclear shape transformation and DNA degradation. This process is controlled by DNase activity which in fiber nuclei increases with development. DNase activity is also present in the epithelial cell nuclei which appears to be non-functional but could be activated in vitro by exogenous addition of Ca2+. We have analyzed the possible selective action of endogenous DNase on 3 genes involved in lens terminal differentiation, namely delta-crystallin, beta-tubulin and vimentin, and on 1 gene not thought to participate in this process, ovalbumin. We have compared restriction DNA patterns of these genes in nuclei isolated from 11-day-old chick embryos and incubated in Ca2+-free medium or in fresh epithelial and fiber lens tissue at 11 and 18 days of development. During incubation in vitro of 11-day fiber nuclei, there is a net increase in the sensitivity of the delta-crystallin, beta-tubulin, ovalbumin and vimentin chromatin to the endogenous DNase. The vimentin gene appears to be more stable than the beta-tubulin and delta-crystallin genes indicating a degree of specificity of the endogenous DNase activity. In the epithelial nuclei, the lens-specific genes appear to be more stable but paradoxically there is a net degradation of the ovalbumin gene. In freshly isolated tissues the 4 genes were detected in epithelial and fiber cells at 11 and 18 days. Furthermore, in the mature fibers in which the nuclei were degenerating, the latter genes were still not completely digested.

Characterization of a bovine acidic FGF cDNA clone and its expression in brain and retina.

A cDNA encoding the acidic eye-derived growth factor (EDGF II) similar to the acidic fibroblast growth factor (aFGF), a potent cell mitogen, has been isolated from a bovine retinal cDNA library. The cDNA, 4.1 kb in size, has a sequence coding for the 155 amino acids of bovine aFGF, and shows similarity with human aFGF (87% identity). The coding sequence is flanked by a 5'-untranslated region of 0.8 kb and a 3'-untranslated end of 3.0 kb. Northern blot analysis of bovine brain and retina poly(A+) RNAs showed the existence of four aFGF mRNA species. Two of these species are 9.9 and 6.0 kb in size, not abundant and could represent premessengers. The other two species, 4.2 and 2.5 kb, are abundant.

S-antigen is coded by [poly A+] mRNA from bovine retina.

Total RNA, [poly (A)-] mRNA and [poly (A)+] mRNA purified from bovine retina were translated in vitro in a rabbit reticulocyte lysate system. Immunoprecipitation of translation products with antibodies to the retinal S-antigen (a photoreceptor specific protein involved in autoimmune retinal disease) revealed this protein as a 50,000 daltons band comigrating with purified S-antigen. This indicates that the S-antigen is synthesized in the retina and is not a maturation or degradation product of a larger protein. Its messenger RNA is the polyadenylated RNA, as for some other proteins expressed in nervous tissue.