Phosphorylation of the cap-binding protein eukaryotic translation initiation factor 4E by protein kinase Mnk1 in vivo.
(1/2412)
Eukaryotic translation initiation factor 4E (eIF4E) binds to the mRNA 5' cap and brings the mRNA into a complex with other protein synthesis initiation factors and ribosomes. The activity of mammalian eIF4E is important for the translation of capped mRNAs and is thought to be regulated by two mechanisms. First, eIF4E is sequestered by binding proteins, such as 4EBP1, in quiescent cells. Mitogens induce the release of eIF4E by stimulating the phosphorylation of 4EBP1. Second, mitogens and stresses induce the phosphorylation of eIF4E at Ser 209, increasing the affinity of eIF4E for capped mRNA and for an associated scaffolding protein, eIF4G. We previously showed that a mitogen- and stress-activated kinase, Mnk1, phosphorylates eIF4E in vitro at the physiological site. Here we show that Mnk1 regulates eIF4E phosphorylation in vivo. Mnk1 binds directly to eIF4G and copurifies with eIF4G and eIF4E. We identified activating phosphorylation sites in Mnk1 and developed dominant-negative and activated mutants. Expression of dominant-negative Mnk1 reduces mitogen-induced eIF4E phosphorylation, while expression of activated Mnk1 increases basal eIF4E phosphorylation. Activated mutant Mnk1 also induces extensive phosphorylation of eIF4E in cells overexpressing 4EBP1. This suggests that phosphorylation of eIF4E is catalyzed by Mnk1 or a very similar kinase in cells and is independent of other mitogenic signals that release eIF4E from 4EBP1. (+info)
Analysis of a ubiquitous promoter element in a primitive eukaryote: early evolution of the initiator element.
(2/2412)
Typical metazoan core promoter elements, such as TATA boxes and Inr motifs, have yet to be identified in early-evolving eukaryotes, underscoring the extensive divergence of these organisms. Towards the identification of core promoters in protists, we have studied transcription of protein-encoding genes in one of the earliest-diverging lineages of Eukaryota, that represented by the parasitic protist Trichomonas vaginalis. A highly conserved element, comprised of a motif similar to a metazoan initiator (Inr) element, surrounds the start site of transcription in all examined T. vaginalis genes. In contrast, a metazoan-like TATA element appears to be absent in trichomonad promoters. We demonstrate that the conserved motif found in T. vaginalis protein-encoding genes is an Inr promoter element. This trichomonad Inr is essential for transcription, responsible for accurate start site selection, and interchangeable between genes, demonstrating its role as a core promoter element. The sequence requirements of the trichomonad Inr are similar to metazoan Inrs and can be replaced by a mammalian Inr. These studies show that the Inr is a ubiquitous, core promoter element for protein-encoding genes in an early-evolving eukaryote. Functional and structural similarities between this protist Inr and the metazoan Inr strongly indicate that the Inr promoter element evolved early in eukaryotic evolution. (+info)
An evaluation of elongation factor 1 alpha as a phylogenetic marker for eukaryotes.
(3/2412)
Elongation factor 1 alpha (EF-1 alpha) is a highly conserved ubiquitous protein involved in translation that has been suggested to have desirable properties for phylogenetic inference. To examine the utility of EF-1 alpha as a phylogenetic marker for eukaryotes, we studied three properties of EF-1 alpha trees: congruency with other phyogenetic markers, the impact of species sampling, and the degree of substitutional saturation occurring between taxa. Our analyses indicate that the EF-1 alpha tree is congruent with some other molecular phylogenies in identifying both the deepest branches and some recent relationships in the eukaryotic line of descent. However, the topology of the intermediate portion of the EF-1 alpha tree, occupied by most of the protist lineages, differs for different phylogenetic methods, and bootstrap values for branches are low. Most problematic in this region is the failure of all phylogenetic methods to resolve the monophyly of two higher-order protistan taxa, the Ciliophora and the Alveolata. JACKMONO analyses indicated that the impact of species sampling on bootstrap support for most internal nodes of the eukaryotic EF-1 alpha tree is extreme. Furthermore, a comparison of observed versus inferred numbers of substitutions indicates that multiple overlapping substitutions have occurred, especially on the branch separating the Eukaryota from the Archaebacteria, suggesting that the rooting of the eukaryotic tree on the diplomonad lineage should be treated with caution. Overall, these results suggest that the phylogenies obtained from EF-1 alpha are congruent with other molecular phylogenies in recovering the monophyly of groups such as the Metazoa, Fungi, Magnoliophyta, and Euglenozoa. However, the interrelationships between these and other protist lineages are not well resolved. This lack of resolution may result from the combined effects of poor taxonomic sampling, relatively few informative positions, large numbers of overlapping substitutions that obscure phylogenetic signal, and lineage-specific rate increases in the EF-1 alpha data set. It is also consistent with the nearly simultaneous diversification of major eukaryotic lineages implied by the "big-bang" hypothesis of eukaryote evolution. (+info)
Unusually high evolutionary rate of the elongation factor 1 alpha genes from the Ciliophora and its impact on the phylogeny of eukaryotes.
(4/2412)
The elongation factor 1 alpha (EF-1 alpha) has become widely employed as a phylogenetic marker for studying eukaryotic evolution. However, a disturbing problem, the artifactual polyphyly of ciliates, is always observed. It has been suggested that the addition of new sequences will help to circumvent this problem. Thus, we have determined 15 new ciliate EF-1 alpha sequences, providing for a more comprehensive taxonomic sampling of this phylum. These sequences have been analyzed together with a representation of eukaryotic sequences using distance-, parsimony-, and likelihood-based phylogenetic methods. Such analyses again failed to recover the monophyly of Ciliophora. A study of the substitution rate showed that ciliate EF-1 alpha genes exhibit a high evolutionary rate, produced in part by an increased number of variable positions. This acceleration could be related to alterations of the accessory functions acquired by this protein, likely to those involving interactions with the cytoskeleton, which is very modified in the Ciliophora. The high evolutionary rate of these sequences leads to an artificial basal emergence of some ciliates in the eukaryotic tree by effecting a long-branch attraction artifact that produces an asymmetric topology for the basal region of the tree. The use of a maximum-likelihood phylogenetic method (which is less sensitive to long-branch attraction) and the addition of sequences to break long branches allow retrieval of more symmetric topologies, which suggests that the asymmetric part of the tree is most likely artifactual. Therefore, the sole reliable part of the tree appears to correspond to the apical symmetric region. These kinds of observations suggest that the general eukaryotic evolution might have consisted of a massive radiation followed by an increase in the evolutionary rates of certain groups that emerge artificially as early branches in the asymmetric base of the tree. Ciliates in the case of the EF-1 alpha genes would offer clear evidence for this hypothesis. (+info)
Cdc42: An essential Rho-type GTPase controlling eukaryotic cell polarity.
(5/2412)
Cdc42p is an essential GTPase that belongs to the Rho/Rac subfamily of Ras-like GTPases. These proteins act as molecular switches by responding to exogenous and/or endogenous signals and relaying those signals to activate downstream components of a biological pathway. The 11 current members of the Cdc42p family display between 75 and 100% amino acid identity and are functional as well as structural homologs. Cdc42p transduces signals to the actin cytoskeleton to initiate and maintain polarized gorwth and to mitogen-activated protein morphogenesis. In the budding yeast Saccharomyces cerevisiae, Cdc42p plays an important role in multiple actin-dependent morphogenetic events such as bud emergence, mating-projection formation, and pseudohyphal growth. In mammalian cells, Cdc42p regulates a variety of actin-dependent events and induces the JNK/SAPK protein kinase cascade, which leads to the activation of transcription factors within the nucleus. Cdc42p mediates these processes through interactions with a myriad of downstream effectors, whose number and regulation we are just starting to understand. In addition, Cdc42p has been implicated in a number of human diseases through interactions with its regulators and downstream effectors. While much is known about Cdc42p structure and functional interactions, little is known about the mechanism(s) by which it transduces signals within the cell. Future research should focus on this question as well as on the detailed analysis of the interactions of Cdc42p with its regulators and downstream effectors. (+info)
EDS1, an essential component of R gene-mediated disease resistance in Arabidopsis has homology to eukaryotic lipases.
(6/2412)
A major class of plant disease resistance (R) genes encodes leucine-rich-repeat proteins that possess a nucleotide binding site and amino-terminal similarity to the cytoplasmic domains of the Drosophila Toll and human IL-1 receptors. In Arabidopsis thaliana, EDS1 is indispensable for the function of these R genes. The EDS1 gene was cloned by targeted transposon tagging and found to encode a protein that has similarity in its amino-terminal portion to the catalytic site of eukaryotic lipases. Thus, hydrolase activity, possibly on a lipid-based substrate, is anticipated to be central to EDS1 function. The predicted EDS1 carboxyl terminus has no significant sequence homologies, although analysis of eight defective eds1 alleles reveals it to be essential for EDS1 function. Two plant defense pathways have been defined previously that depend on salicylic acid, a phenolic compound, or jasmonic acid, a lipid-derived molecule. We examined the expression of EDS1 mRNA and marker mRNAs (PR1 and PDF1.2, respectively) for these two pathways in wild-type and eds1 mutant plants after different challenges. The results suggest that EDS1 functions upstream of salicylic acid-dependent PR1 mRNA accumulation and is not required for jasmonic acid-induced PDF1.2 mRNA expression. (+info)
Evolutionary relationships of Metazoa within the eukaryotes based on molecular data from Porifera.
(7/2412)
Recent molecular data provide strong support for the view that all metazoan phyla, including Porifera, are of monophyletic origin. The relationship of Metazoa, including the Porifera, to Plantae, Fungi and unicellular eukaryotes has only rarely been studied by using cDNAs coding for proteins. Sequence data from rDNA suggested a relationship of Porifera to unicellular eukaryotes (choanoflagellates). However, ultrastructural studies of choanocytes did not support these findings. In the present study, we compared amino acid sequences that are found in a variety of metazoans (including sponges) with those of Plantae, Fungi and unicellular eukaryotes, to obtain an answer to this question. We used the four sequences from 70 kDa heat-shock proteins, the serine-threonine kinase domain found in protein kinases, beta-tubulin and calmodulin. The latter two sequences were deduced from cDNAs, isolated from the sponge Geodia cydonium for the phylogenetic analyses presented. These revealed that the sponge molecules were grouped into the same branch as the Metazoa, which is statistically (significantly) separated from those branches that comprise the sequences from Fungi, Plantae and unicellular eukaryotes. From our molecular data it seems evident that the unicellular eukaryotes existed at an earlier stage of evolution, and the Plantae and especially the Fungi and the Metazoa only appeared later. (+info)
Cleavage of eukaryotic translation initiation factor 4G by exogenously added hybrid proteins containing poliovirus 2Apro in HeLa cells: effects on gene expression.
(8/2412)
Efficient cleavage of both forms of eukaryotic initiation factor 4G (eIF4G-1 and eIF4G-2) has been achieved in HeLa cells by incubation with hybrid proteins containing poliovirus 2Apro. Entry of these proteins into cells is promoted by adenovirus particles. Substantial levels of ongoing translation on preexisting cellular mRNAs still continue for several hours after eIF4G degradation. Treatment of control HeLa cells with hypertonic medium causes an inhibition of translation that is reversed upon restoration of cells to normal medium. Protein synthesis is not restored in cells lacking intact eIF4G after hypertonic treatment. Notably, induction of synthesis of heat shock proteins still occurs in cells pretreated with poliovirus 2Apro, suggesting that transcription and translation of these mRNAs takes place even in the presence of cleaved eIF4G. Finally, the synthesis of luciferase was examined in a HeLa cell line bearing the luciferase gene under control of a tetracycline-regulated promoter. Transcription of the luciferase gene and transport of the mRNA to the cytoplasm occurs at control levels in eIF4G-deficient cells. However, luciferase synthesis is strongly inhibited in these cells. These findings indicate that intact eIF4G is necessary for the translation of mRNAs not engaged in translation with the exception of heat shock mRNAs but is not necessary for the translation of mRNAs that are being translated. (+info)