Interaction between protein S and complement C4b-binding protein (C4BP). Affinity studies using chimeras containing c4bp beta-chain short consensus repeats.
(57/4818)
Human C4b-binding protein (C4BP) is a regulator of the complement system and plays an important role in the regulation of the anticoagulant protein C pathway. C4BP can bind anticoagulant protein S, resulting in a decreased cofactor function of protein S for activated protein C. C4BP is a multimeric protein containing several identical alpha-chains and a single beta-chain (C4BPbeta), each chain being composed of short consensus repeats (SCRs). Previous studies have localized the protein S binding site to the NH2-terminal SCR (SCR-1) of C4BPbeta. To further localize the protein S binding site, we constructed chimeras containing C4BPbeta SCR-1, SCR-2, SCR-3, SCR-1+2, SCR-1+3, and SCR-2+3 fused to tissue-type plasminogen activator. Binding assays of protein S with these chimeras indicated that SCR-2 contributes to the interaction of protein S with SCR-1, since the affinity of protein S for SCR-1+2 was up to 5-fold higher compared with SCR-1 and SCR-1+3. Using an assay that measures protein S cofactor activity, we showed that cofactor activity was decreased due to binding to constructs that contain SCR-1. SCR-1+2 inhibited more potently than SCR-1 and SCR-1+3. SCR-3 had no additional effect on SCR-1, and therefore the effect of SCR-2 was specific. In conclusion, beta-chain SCR-2 contributes to the interaction of C4BP with protein S. (+info)
Cloning and studies of the mouse cDNA encoding Smad3.
(58/4818)
Following stimulation by the transforming growth factor-beta (TGF-beta) family in the cytoplasm, the Smad family is phosphorylated and translocated to the nucleus and activates several gene transcriptions. In this study, the mouse Smad3 cDNA including the open reading frame (ORF) was cloned from the mouse brain using a RACE (rapid amplification of cDNA ends) technique, and its expression pattern was analyzed in mouse tissue using northern blot. The predicted amino acid (aa) sequences of mouse Smad3 showed a high homology with human Smad3 (99.3%) and mouse Smad2 (85.4%). It revealed that this protein may be highly conserved in different species of mammals. Northern blot analyses revealed that Smad3 was highly expressed in the brain and ovary, and that the size of major transcript was about 5.7 kb. In situ hybridization analyses revealed the high expression of Smad3 was detected in the pyramidal cells of the hippocampus, the granule cells of the dentate gyrus, the granular cells of the cerebral cortex and the granulosa cells of the ovary. Smad3 may be essential transducer of signals from TGF-beta and activin in these cells. (+info)
Two distinct SECIS structures capable of directing selenocysteine incorporation in eukaryotes.
(59/4818)
Translation of UGA as selenocysteine requires specific RNA secondary structures in the mRNAs of selenoproteins. These elements differ in sequence, structure, and location in the mRNA, that is, coding versus 3' untranslated region, in prokaryotes, eukaryotes, and archaea. Analyses of eukaryotic selenocysteine insertion sequence (SECIS) elements via computer folding programs, mutagenesis studies, and chemical and enzymatic probing has led to the derivation of a predicted consensus structural model for these elements. This model consists of a stem-loop or hairpin, with conserved nucleotides in the loop and in a non-Watson-Crick motif at the base of the stem. However, the sequences of a number of SECIS elements predict that they would diverge from the consensus structure in the loop region. Using site-directed mutagenesis to introduce mutations predicted to either disrupt or restore structure, or to manipulate loop size or stem length, we show that eukaryotic SECIS elements fall into two distinct classes, termed forms 1 and 2. Form 2 elements have additional secondary structures not present in form 1 elements. By either insertion or deletion of the sequences and structures distinguishing the two classes of elements while maintaining appropriate loop size, conversion of a form 1 element to a functional form 2-like element and of a form 2 to a functional form 1-like element was achieved. These results suggest commonality of function of the two classes. The information obtained regarding the existence of two classes of SECIS elements and the tolerances for manipulations of stem length and loop size should facilitate designing RNA molecules for obtaining high-resolution structural information about these elements. (+info)
The A26G replacement in the consensus sequence A-X-X-X-X-G-K-[T,S] of the guanine nucleotide binding site activates the intrinsic GTPase of the elongation factor 2 from the archaeon Sulfolobus solfataricus.
(60/4818)
A recombinant form of the elongation factor 2 from the archaeon Sulfolobus solfataricus (SsEF-2), carrying the A26G substitution, has been produced and characterized. The amino acid replacement converted the guanine nucleotide binding consensus sequences A-X-X-X-X-G-K-[T,S] of the elongation factors EF-G or EF-2 into the corresponding G-X-X-X-X-G-K-[T,S] motif which is present in all the other GTP-binding proteins. The rate of poly(U)-directed poly(Phe) synthesis and the ribosome-dependent GTPase activity of A26GSsEF-2 were decreased compared to SsEF-2, thus indicating that the A26G replacement partially affected the function of SsEF-2 during translocation. In contrast, the A26G substitution enhanced the catalytic efficiency of the intrinsic SsEF-2 GTPase triggered by ethylene glycol [Raimo, G., Masullo, M., Scarano, G., & Bocchini, V. (1997) Biochimie 78, 832-837]. Surprisingly, A26GSsEF-2 was able to hydrolyse GTP even in the absence of ethylene glycol; furthermore, the alcohol increased the affinity for GTP without modifying the catalytic constant of A26GSsEF-2 GTPase. Compared to SsEF-2, the affinity of A26GSsEF-2 for [3H]GDP was significantly reduced. These findings suggest that A26 is a regulator of the biochemical functions of SsEF-2. The involvement of this alanine residue in the guanine nucleotide-binding pocket of EF-2 or EF-G is discussed. (+info)
Substrate sequestration by a proteolytically inactive Lon mutant.
(61/4818)
Lon protein of Escherichia coli is an ATP-dependent protease responsible for the rapid turnover of both abnormal and naturally unstable proteins, including SulA, a cell division inhibitor made after DNA damage, and RcsA, a positive regulator of transcription. Lon is a multimer of identical 94-kDa subunits, each containing a consensus ATPase motif and a serine active site. We found that overexpressing Lon, which is mutated for the serine active site (LonS679A) and is therefore devoid of proteolytic activity, unexpectedly led to complementation of the UV sensitivity and capsule overproduction of a lon deletion mutant. SulA was not degraded by LonS679A, but rather was completely protected by the Lon mutant from degradation by other cellular proteases. We interpret these results to mean that the mutant LonS679A binds but does not degrade Lon substrates, resulting in sequestration of the substrate proteins and interference with their activities, resulting in apparent complementation. Lon that carried a mutation in the consensus ATPase site, either with or without the active site serine, was no longer able to complement a Deltalon mutant. These in vivo results suggest that the pathway of degradation by Lon couples ATP-dependent unfolding with movement of the substrate into protected chambers within Lon, where it is held until degradation proceeds. In the absence of degradation the substrate remains sequestered. Comparison of our results with those from a number of other systems suggest that proteins related to the regulatory portions of energy-dependent proteases act as energy-dependent sequestration proteins. (+info)
Insulin stimulates phosphorylation of the forkhead transcription factor FKHR on serine 253 through a Wortmannin-sensitive pathway.
(62/4818)
In the nematode Caenorhabditis elegans, mutations of the insulin/insulin-like growth factor-1 receptor homologue Daf-2 gene cause developmental arrest at the dauer stage. The effect of Daf-2 mutations is counteracted by mutations in the Daf-16 gene, suggesting that Daf-16 is required for signaling by Daf-2. Daf-16 encodes a forkhead transcription factor. Based on sequence similarity, the FKHR genes are the likeliest mammalian Daf-16 homologues. FKHR proteins contain potential sites for phosphorylation by the serine/threonine kinase Akt. Because Akt is phosphorylated in response to insulin and has been implicated in a variety of insulin effects, we investigated whether insulin affects phosphorylation of FKHR. Insulin stimulated phosphorylation of endogenous FKHR and of a recombinant c-Myc/FKHR fusion protein transiently expressed in murine SV40-transformed hepatocytes. The effect of insulin was inhibited by wortmannin treatment, suggesting that PI 3-kinase activity is required for FKHR phosphorylation. Mutation of serine 253, located in a consensus Akt phosphorylation site at the carboxyl-terminal end of the forkhead domain, abolished the effect of insulin on FKHR phosphorylation. In contrast, mutation of two additional Akt phosphorylation sites, at amino acids threonine 24 or serine 316, did not abolish insulin-induced phosphorylation. These data indicate that FKHR may represent a distal effector of insulin action. (+info)
A potential role for Elf-1 in CD4 promoter function.
(63/4818)
The control of CD4 gene expression is believed to be linked directly to the signaling events that mediate T cell development and is directly dependent on the CD4 promoter. We have previously determined that this promoter contains four factor-binding sites important for its function. One of these sites, referred to as the P4 site, contains an Ets consensus recognition sequence. Using functional and biochemical analyses, we determine that Elf-1 binds to this site and specifically activates the CD4 promoter, indicating that Elf-1 is playing an important role in CD4 promoter function. In addition, a second nuclear factor binds to this region. Although there are consensus recognition sites for other factors, we demonstrate that none of these factors binds to the P4 site, nor do other known members of the Ets family. Thus, a novel transcription factor may bind to the CD4 promoter and help mediate its function. (+info)
Cell cycle expression and transcriptional regulation of DNA topoisomerase IV genes in caulobacter.
(64/4818)
DNA replication and differentiation are closely coupled during the Caulobacter crescentus cell cycle. We have previously shown that DNA topoisomerase IV (topo IV), which is encoded by the parE and parC genes, is required for chromosomal partitioning, cell division, and differentiation in this bacterium (D. Ward and A. Newton, Mol. Microbiol. 26:897-910, 1997). We have examined the cell cycle regulation of parE and parC and report here that transcription of these topo IV genes is induced during the swarmer-to-stalked-cell transition when cells prepare for initiation of DNA synthesis. The regulation of parE and parC expression is not strictly coordinated, however. The rate of parE transcription increases ca. 20-fold during the G1-to-S-phase transition and in this respect, its pattern of regulation is similar to those of several other genes required for chromosome duplication. Transcription from the parC promoter, by contrast, is induced only two- to threefold during this cell cycle period. Steady-state ParE levels are also regulated, increasing ca. twofold from low levels in swarmer cells to a maximum immediately prior to cell division, while differences in ParC levels during the cell cycle could not be detected. These results suggest that topo IV activity may be regulated primarily through parE expression. The presumptive promoters of the topo IV genes display striking similarities to, as well as differences from, the consensus promoter recognized by the major Caulobacter sigma factor sigma73. We also present evidence that a conserved 8-mer sequence motif located in the spacers between the -10 and -35 elements of the parE and parC promoters is required for maximum levels of parE transcription, which raises the possibility that it may function as a positive regulatory element. The pattern of parE transcription and the parE and parC promoter architecture suggest that the topo IV genes belong to a specialized subset of cell cycle-regulated genes required for chromosome replication. (+info)