Previously we have shown that the CCA end of a P-tRNA can be crosslinked with the RPL36AL protein of the large subunit of mammalian ribosomes; it belongs to the L44e protein family present in all eukaryotic and archaeal ribosomes. Here we confirm and extend this finding and demonstrate that: 1) this crosslink is specific for a tRNA at the P/E hybrid site, as a tRNA in all other tRNA positions of pre-translocational ribosomes could not be crosslinked with a ribosomal protein, 2) the crosslink was formed most efficiently with C74 and C75 of P/E-tRNA, but could also connect the ultimate A of this tRNA with Lys53 of protein RPL36AL, 3) this protein contains seven monomethylated residues (three lysyl and three arginyl residues, as well as glutaminyl residue 51), 4) Q51 is part of a conserved GGQ motif in the L44e proteins in eukaryotic 80S ribosomes that is identical to the universally conserved motif of release factors implicated in promoting peptidyl-tRNA hydrolysis, and 5) the large number of modifications, in which some of the residues were methylated to about 50 %, might indicate that protein RPL36AL is a preferential target for regulation.

This work describes the in vitro properties of fulllength CDC25 Mm (1262 amino acid residues), a GDP/GTP exchange factor (GEF) of H-ras p21. CDC25 Mm , isolated as a recombinant protein in Escherichia coli and purified by various chromatographic methods, could stimulate the H-ras p21⅐GDP dissociation rate; however, its specific activity was 25 times lower than that of the isolated catalytic domain comprising the last C-terminal 285 residues (C-CDC25 Mm285 ) and 5 times lower than the activity of the C-terminal half-molecule (631 residues). This reveals a negative regulation of the catalytic domain by other domains of the molecule. Accordingly, the GEF activity of CDC25 Mm was increased severalfold by the Ca 2؉ -dependent protease calpain that cleaves around a PEST-like region (residues 798 -853), producing C-terminal fragments of 43-56 kDa. In agreement with the presence of an IQ motif on CDC25 Mm (residues 202-229), calmodulin interacted functionally with the exchange factor. Depending on the calmodulin concentration an inhibition up to 50% of the CDC25 Mm -induced nucleotide exchange activity on H-ras p21 was observed, an effect requiring Ca 2؉ ions. Calmodulin also inhibited C-CDC25 Mm285 but with a ϳ100 times higher IC 50 than in the case of CDC25 Mm (ϳ10 M versus 0.1 M, respectively). Together, these results emphasize the role of the other domains of CDC25 Mm in controlling the activity of the catalytic domain and support the involvement of calmodulin and calpain in the in vivo regulation of the CDC25 Mm activity.The mouse CDC25 Mm 1 protein is a guanine nucleotide exchange factor (GEF) regenerating the active form of H-ras p21, the complex with GTP (1-3). Homologous products were found in rat (p140-rasGRF) (4) and human (H-GRF) (5, 6). These rasGEFs have been described to be specific for the central nervous system (4 -9). Some evidence has also been reported for the existence of full-length and truncated forms of these exchange factors in other tissues (10,11). Experiments in vivo suggest that the upstream connection of this GEF involves G-protein-coupled receptors (9, 12, 13) and not hormone-receptor-bound tyrosine kinases via the adaptor protein GRB2, as has been found for SOS, a ubiquitous rasGEF (14 -16). CDC25 Mm contains in the N-terminal moiety two domains of pleckstrin homology (PH1 and PH2), one of DBL homology (DH) and a coiled-coil region that follows the PH1 domain (cf. (21) and the DH is a domain sharing similarity with a GDP/GTP exchange factor of members of the Rho family (2,4,22,23). Farnsworth et al. (24) reported that in vivo the activity of the homologous p140-rasGRF from rat brain is enhanced by raising the calcium concentration, an effect associated with the binding of calmodulin, and that p140-rasGRF and calmodulin form a stable complex. A direct action of calmodulin was supported by the presence in the N-terminal region of CDC25 Mm of an IQ domain, a sequence frequently found in proteins interacting with calmodulin (25, 26). Very recent experiments in vivo have indicated that PH1, ...

We have demonstrated previously that the E-site specific protein RPL36AL present in human ribosomes can be crosslinked with the CCA-end of a P-tRNA in situ. Here we report the following: (i) We modeled RPL36AL into the structure of the archaeal ortholog RPL44E extracted from the known X-ray structure of the 50S subunit of Haloarcula marismortui. Superimposing the obtained RPL36AL structure with that of P/E tRNA observed in eukaryotic 80S ribosomes suggested that RPL36AL might in addition to its CCA neighbourhood interact with the inner site of the tRNA elbow similar to an interaction pattern known from tRNA•synthetase pairs. (ii) Accordingly, we detected that the isolated recombinant protein RPL36AL can form a tight binary complex with deacylated tRNA, and even tRNA fragments truncated at their CCA end showed a high affinity in the nanomolar range supporting a strong interaction outside the CCA end. (iii) We constructed programmed 80S complexes containing the termination factor eRF1 (stop codon UAA at the A-site) and a 2’,3’-dialdehyde tRNA (tRNAox) analog at the P-site. Surprisingly, we observed a crosslinked ternary complex containing the tRNA, eRF1 and RPL36AL crosslinked both to the aldehyde groups of tRNAox at the 2’- and 3’-positions of the ultimate A. We also demonstrated that, upon binding to the ribosomal A-site, eRF1 induces an alternative conformation of the ribosome and/or the tRNA, leading to a novel crosslink of tRNAox to another large-subunit ribosomal protein (namely L37) rather than to RPL36AL, both ribosomal proteins being labeled in a mutually exclusive fashion. Since the human 80S ribosome in complex with P-site bound tRNAox and A-site bound eRF1 corresponds to the post-termination state of the ribosome, the results represent the first biochemical evidence for the positioning of the CCA-arm of the P-tRNA in close proximity to both RPL36AL and eRF1 at the end of the translation process.

This work characterizes several aspects of the interaction between H-ras p21 and the catalytic domain of the mammalian GDP/GTP exchange factor (GEF) CDC25Mm (C-CDC25Mm), isolated in pure form as recombinant protein from Escherichia coli. Formation of a complex with nucleotide-free H-ras p21 could be analyzed on native gel electrophoresis by combining C-CDC25Mm and p21.GDP, as the result of the fast separation of GDP from p21. Therefore, in order to obtain highly purified heterodimer in preparative amounts, p21.GDP and C-CDC25Mm were exposed to an electric field and the complex purified by anionic chromatography. The rate of the C-CDC25Mm-mediated nucleotide exchange on p21 depended on the nature of the bound nucleotide (6 times faster for p21.GDP than p21.GTP) but was uninfluenced by the nature of the free nucleotide acting as second substrate. No saturating concentration of p21.GDP could be determined by measuring the C-CDC25Mm-mediated increase in the nucleotide exchange rate with a nitrocellulose binding assay. On this basis the Km and the kcat of the reaction were calculated to be> 11 microM and > 0.8 s-1, respectively. The dramatic increase in the nucleotide exchange rate was found to be almost exclusively based on the strong stimulation of the "off-rate". The "on-rate" of the nucleotide on the p21.C-CDC25Mm complex was similar for GDP and GTP and was little increased vs that on p21 alone. The observation that the apparent affinity of both nucleotides for the p21.C-CDC25Mm complex was lower than for p21 alone stressed the great affinity of this complex, whose Kd was calculated to be approximately 3 pM. These results are discussed and compared with the properties of other GEFs, from which C-CDC25Mm differs for a number of specific properties.

No abstract

Lung myofibroblasts play a major role in the pathophysiology of asthma, contributing not only to tissue remodelling but also to airway inflammation. Nevertheless, only recently, attention has been focused on these cells as potential targets for anti-allergic drugs. Herein, we analysed the pharmacological response of lung myofibroblasts to beta2-agonists associated or not to inhaled corticosteroids, investigating their effects on (i) the constitutive and transforming growth factor-beta (TGF-beta)-induced expression of alpha-smooth muscle actin (alpha-SMA), the main functional marker of myofibroblastic differentiation and contractility; (ii) isometric contraction and (iii) tumour necrosis factor-alpha (TNF-alpha)-induced nuclear translocation of the pro-inflammatory transcription factor nuclear factor-kappaB (NF-kappaB). The beta2-agonist salmeterol (SMl) has on human lung myofibroblasts new direct anti-contractile/anti-inflammatory effects that are amplified by the combined use of low concentrations of the glucocorticoid fluticasone propionate (FP). First, SMl and/or FP (10(-12) M) inhibits the constitutive and TGF-beta-induced expression of alpha-SMA. Second, the two drugs block the TNF-alpha-induced nuclear translocation of the pro-inflammatory transcription factor NF-kappaB. Finally, SMl decreases TNF- alpha-induced production of the inflammatory cytokine IL-6. The complementary anti-inflammatory/ anti-contractile effects displayed by SMl and FP on lung myofibroblasts in vitro may be related to the improvement in lung function and symptom control obtained in vivo by the early use of low doses of glucocorticoids in combination with long-acting beta2-agonists.

Activation of the neuronal Ras GDP/GTP exchange factor (GEF) CDC25Mm/GRF1 is known to be associated with phosphorylation of serine/threonine. To increase our knowledge of the mechanism involved, we have analyzed the ability of several serine/threonine kinases to phosphorylate CDC25Mm in vivo and in vitro. We could demonstrate the involvement of cAMP-dependent protein kinase (PKA) in the phosphorylation of CDC25Mm in fibroblasts overexpressing this RasGEF as well as in mouse brain synaptosomal membranes. In vitro, PKA was found to phosphorylate multiple sites on purified CDC25Mm, in contrast to protein kinase C, calmodulin kinase II, and casein kinase II, which were virtually inactive. Eight phosphorylated serines and one threonine were identified by mass spectrometry and Edman degradation. Most of them were clustered around the Ras exchanger motif/PEST motifs situated in the C-terminal moiety (residues 631-978) preceding the catalytic domain. Ser 745 and Ser 822 were the most heavily phosphorylated residues and the only ones coinciding with PKA consensus sequences. Substitutions S745D and S822D showed that the latter mutation strongly inhibited the exchange activity of CDC25Mm on Ha-Ras. The multiple PKA-dependent phosphorylation sites on CDC25Mm suggest a complex regulatory picture of this RasGEF. The results are discussed in the light of structural and/or functional similarities with other members of this RasGEF family.

In this report, we have used periodate-oxidized tRNA (tRNAox) as an affinity laleling reagent to demonstrate that: (i) the bL12 protein contacts the CCA-arm of P-site bound tRNA on the Escherichia coli 70S ribosomes; (ii) the stoichiometry of labelling is one molecule of tRNAox bound to one polypeptide chain of endogenous bL12; (iii) cross-linking in situ of bL12 with tRNAox on the ribosomes provokes the loss of activity; (iv) intact tRNA protects bL12 in the 70S ribosomes against cross-linking with tRNAox; (v) both tRNAox and pyridoxal 5'-phosphate (PLP) compete for the same or for proximal cross-linking site(s) on bL12 inside the ribosome; (vi) the stoichiometry of cross-linking of PLP to the recombinant E. coli bL12 protein is one molecule of PLP covalently bound per polypeptide chain; (vii) the amino acid residue of recombinant bL12 cross-linked with PLP is Lys-65; (viii) Lys-65 of E. coli bL12 corresponds to Lys-53 of eL42 which was previously shown to cross-link with P-site bound tRNAox on human 80S ribosomes in situ; (ix) finally, E. coli bL12 and human eL42 proteins display significant primary structure similarities, which argues for evolutionary conservation of these two proteins located at the tRNA-CCA binding site on eubacterial and eukaryal ribosomes.