Role In Protein Translation Research Articles

KAI1/CD82 decreases Rac1 expression and cell proliferation through PI3K/Akt/mTOR pathway in H1299 lung carcinoma cells

Although the KAI1/CD82 protein has been reported to inhibit cell metastasis in many studies, its mechanism of action has not yet been fully elucidated. In the present study, we investigated the possible effects of KAI1/CD82 on the metastatic phenotype in H1299 lung carcinoma cells. These studies were based on the pivotal role that the acquisition of motile phenotype plays on the initial steps of metastasis. KAI1/CD82-mediated morphological changes were observed using phase contrast microscopy. We report here, that a KAI1/CD82-induced phenotypic change was involved in the decrease of Rac1 expression and GTPase activity. However, we found that KAI1/CD82 did not regulate Rac1 mRNA levels. This suggests the existence of another regulatory mechanism of Rac1 protein maturation or activation. To identify the signaling pathway of Rac1 regulation, we investigated the PI3K/Akt/mTOR pathway, since the PI3K/Akt pathway regulates Rac1 activation and mTOR is known to play a regulatory role in protein translation. H1299/CD82-transfectants showed lower mTOR expression and cell growth than the control group. The data obtained from this study suggested that KAI1/CD82 decreased the metastatic phenotype of H1299 lung carcinoma cells by down-regulating Rac1 expression through the PI3K/Akt/mTOR pathway.

Gab2 Is a Key Determinant of Mononuclear Phagocyte Development and a Regulator of Macrophage Recruitment in Acute Inflammation

Colony Stimulating Factor-1 (CSF-1) acts through the CSF-1R to regulate proliferation, survival and differentiation of mononuclear phagocytes (MNPs). GAB2, a scaffolding protein, modulates signals from numerous receptors, through recruitment of phosphatidylinositol 3-kinase (PI3K) and SHP2 phosphatase. Previously we reported at this meeting (Lee et al., Blood 110:2197, 2007) that under steady conditions, GAB2−/− mice showed a significant reduction in the number of bone marrow (BM) tissue macrophages (Mφs) and dramatic decreases in the number of CSF-1 dependent colony forming units (CFU-Cs) from total BM and from BM cells that have been induced to commit to the MNP lineage. We also showed by retroviral transduction that GAB2 relies on the recruitment of PI3K and an unidentified pathway to promote CFU-C formation. These findings raised additional questions:What is the identity of the BM progenitor that GAB2 regulates?What is the identity of the second pathway?Does GAB2 have a role in MNP recruitment under in vivo situations of increased MNP demands, e.g. in acute inflammation?To address these questions, we developed methods to isolate BM progenitors committed to the MNP lineage and flow cytometric assays to monitor intracellular phosphorylation in complex BM populations. Based on our CFU-C results, we predicted that GAB2 would have a major role in the expansion of the CD31highLy6C− subset, which should contain CSF-1 responsive early progenitors. We enriched for these cells from ex vivo expanded day 2 BM cultures by magnetic bead depletion of lymphocytes, granulocytes and erythroid cells. The LIN*−CD31highLy6C− subset showed a ≈ 30-fold enrichment of CFU-Cs compared to total BM. Those from Gab2−/− mice gave rise to ≈ 4-fold fewer CFU-Cs and exhibited increased apoptosis and diminished cell cycle progression. To verify that our findings also applied to MNP progenitors isolated directly from the BM, we purified early myeloid progenitors that can proliferate and differentiate in response to CSF- 1. These are lineage depleted (LIN−) cKit+CD31highLy6C− (LK31C) cells and they showed a 51-fold enrichment of CFU-Cs compared to total BM. LK31C cells from GAB2−/− mice also showed a 9-fold reduction in CFU-Cs, relative to GAB2+/+ cells. By CFSE tracking, LK31C cells from GAB2−/− mice demonstrated diminished proliferation. Thus, GAB2 is required for CSF-1 dependent expansion of CD31highLy6C− BM progenitors. To biochemically identify GAB2-regulated signaling pathways that promote expansion of CSF-1 responsive progenitors, we utilized flow cytometry to assay intracellular phosphorylation in day 2 LIN*− cells. We found that the percentage of responding cells as well as the level of phosphorylation of Akt, Erk and S6 were significantly diminished in the LIN*−CD31highLy6C− subset from GAB2−/− cells. We also found that optimal S6 phosphorylation was dependent on both the PI3K and Erk pathway, indicating that Gab2 could play an important role in protein translation in CSF-1 responsive progenitors. Since GAB2's recruitment of SHP2 may be important for optimal Erk activation, we investigated the role of GAB2-SHP2 in CFU-C formation. When a GAB2 mutant that cannot recruit SHP2 was overexpressed in GAB2−/− day 2 BM cells, the number of CFU-Cs was only 40% of that obtained with wildtype GAB2. Together with our previous findings, they indicate that both the PI3K and SHP2-Erk pathways recruited by GAB2 were required for optimal CFU-C formation. Since Gab2−/− mice have a significant defect in CSF-1 dependent MNP development, we reasoned that this deficit could be exacerbated in inflammatory conditions with a prominent MNP infiltration. To test this hypothesis, we used a well-established sterile peritonitis model. GAB2−/− mice showed a 75% (p=0.003) and 67% (p=0.005) reduction in recruited Mφs at 24h and 72h respectively after thioglycollate (TG) challenge. Moreover, there was a 68% increase in CFU-Cs from GAB2+/+ BM cells at 24h (p=0.0001) post TG challenge, whereas GAB2−/− BM cells showed no change. These findings indicate that GAB2−/− mice are much less able to elicit a MNP response after an inflammatory challenge. Altogether, our results reveal that GAB2 utilizes both PI3K-Akt and SHP2-Erk to maximally promote CSF-1 mediated expansion along the MNP lineage by targeting the lineage depleted CD31highLy6C− subset. In the absence of GAB2, the BM is unable to respond maximally to inflammatory challenges.

Altered protein synthesis is a trigger for long-term memory formation

There is ongoing debate concerning whether new protein synthesis is necessary for, or even contributes to, memory formation and storage. This review summarizes a contemporary model proposing a role for altered protein synthesis in memory formation and its subsequent stabilization. One defining aspect of the model is that altered protein synthesis serves as a trigger for memory consolidation. Thus, we propose that specific alterations in the pattern of neuronal protein translation serve as an initial event in long-term memory formation. These specific alterations in protein readout result in the formation of a protein complex that then serves as a nidus for subsequent perpetuating reinforcement by a positive feedback mechanism. The model proposes this scenario as a minimal but requisite component for long-term memory formation. Our description specifies three aspects of prevailing scenarios for the role of altered protein synthesis in memory that we feel will help clarify what, precisely, is typically proposed as the role for protein translation in memory formation. First, that a relatively short initial time window exists wherein specific alterations in the pattern of proteins translated (not overall protein synthesis) is involved in initializing the engram. Second, that a self-perpetuating positive feedback mechanism maintains the altered pattern of protein expression (synthesis or recruitment) locally. Third, that other than the formation and subsequent perpetuation of the unique initializing proteins, ongoing constitutive protein synthesis is all that is minimally necessary for formation and maintenance of the engram. We feel that a clear delineation of these three principles will assist in interpreting the available experimental data, and propose that the available data are consistent with a role for protein synthesis in memory.

The ins and outs of tRNA transport

Transfer RNAs (tRNAs) are involved in many cellular functions distributed throughout the cellular space. In addition to their role in protein translation, these molecules are engaged in tasks as diverse as the regulation of gene expression, amino‐acid synthesis, protein degradation, cell‐wall synthesis, porphyrin biosynthesis, priming of replication, RNA interference and the transport of macromolecules. The nucleus and the mitochondria of eukaryotic cells, as well as the chloroplasts in plants, contain their own genome that encodes a range of proteins and nucleic acids. Early evidence suggested that some of the RNA content of mitochondria could be transcribed from non‐mitochondrial DNA (Suyama, 1967). It is now well established that a variable number of tRNA species present in the mitochondria are indeed nucleus‐encoded (Fig 1). The phenomenon of mitochondrial tRNA import has been reported in plants, marsupials, the yeast Saccharomyces cerevisiae , and the protozoa Leishmania , Trypanosoma and Tetrahymena . When certain tRNA species are not encoded by the mitochondrial genome, there is a predictable requirement for the import of nucleus‐encoded tRNAs into the mitochondria (Schneider & Marechal‐Drouard, 2000). This is the case, for example, for the mitochondria of Leishmania , which are completely devoid of tRNA‐encoding genes. However, there are a few examples of tRNA import into mitochondria that already have a complete set of mitochondria‐encoded tRNAs. Other tRNA transport systems have also been identified (Fig 1), which include their retrograde transport from the cytoplasm to the nucleus (Takano et al , 2005; Shaheen & Hopper, 2005), the export of mitochondria‐encoded tRNA to the cytoplasm (Maniataki & Mourelatos, 2005), and the packaging of tRNAs into retroviruses (Waters & Mullin, 1977). The roles of some of these transport systems have yet to be defined. Figure 1. The travels of transfer RNA. …

Binding of Elongation Factor eEF1A2 to Phosphatidylinositol 4-Kinase β Stimulates Lipid Kinase Activity and Phosphatidylinositol 4-Phosphate Generation

Eukaryotic protein translation elongation factor 1 alpha 2 (eEF1A2) is an oncogene that transforms mammalian cell lines and increases their tumorigenicity in nude mice. Increased expression of eEF1A2 occurs during the development of breast, ovarian, and lung cancer. Here, we report that eEF1A2 directly binds to and activates phosphatidylinositol 4-kinase III beta (PI4KIIIbeta), an enzyme that converts phosphatidylinositol to phosphatidylinositol 4-phosphate. Purified recombinant eEF1A2 increases PI4KIIIbeta lipid kinase activity in vitro, and expression of eEF1A2 in rat and human cells is sufficient to increase overall cellular phosphatidylinositol 4-kinase activity and intracellular phosphatidylinositol 4-phosphate abundance. siRNA-mediated reduction in eEF1A2 expression concomitantly reduces phosphatidylinositol 4-kinase activity. This identifies a physical and functional relationship between eEF1A2 and PI4KIIIbeta.

Investigations of the protein synthesis dependency of mGluR-induced long-term depression in the dentate gyrus of freely moving rats

Hippocampal long-term depression (LTD) comprises an activity-dependent weakening of synaptic strength. In this study we compared persistent LTD induced by the group I mGluR agonist, DHPG, or the group III mGluR agonist, AP4, in the dentate gyrus of freely moving rats. The role of protein translation, using the translation inhibitors, anisomycin and emetine, was also investigated. Potentials were evoked from medial perforant path–dentate gyrus granule cell synapses of male Wistar rats by means of chronically implanted electrodes. Immediately after intracerebral (ventricular) application of DHPG or AP4 robust LTD (>24 h) occurred. Paired-pulse analysis during LTD, and application of mGluR antagonists after stabilisation of depression, supported that LTD genuinely occurred and that the depression was not a consequence of persistence of the agonists at the synapse. Application of a protein synthesis inhibitor 2 h prior to DHPG injection inhibited the expression of LTD (from ca. 6 h post-injection) but did not affect LTD induced by AP4. These data highlight differences in chemical LTD elicited by group I and group III mGluRs. Whereas AP4-induced LTD may arise as a result of modulation of presynaptic glutamate release mechanisms, the protein synthesis dependency of DHPG-induced LTD suggests an additional postsynaptic expression mechanism for this phenomenon.

Leishmania donovani engages in regulatory interference by targeting macrophage protein tyrosine phosphatase SHP-1

Protozoan parasites of the genus leishmania are obligate intracellular parasites of monocytes and macrophages. These pathogens have evolved to invade the mammalian immune system and typically survive for long periods of time. Leishmania have developed a variety of remarkable strategies to prevent their elimination by both innate and acquired immune effector mechanisms. One particular strategy of interest involves manipulation of host cell regulatory pathways so as to prevent macrophage activation required for efficient microbicidal activity. These interference mechanisms are the main focus of this review. Several lines of evidence have been developed to show that the Src homology-2 domain containing tyrosine phosphatase-1 (SHP-1) becomes activated in leishmania-infected cells and that this contributes to disease pathogenesis. Recent studies aimed at understanding the mechanism responsible for the change in activation state of SHP-1 led to the identification of leishmania EF-1α as an SHP-1 binding protein and SHP-1 activator. This was a surprising finding given that this ubiquitous and highly conserved protein plays an essential role in protein translation in both prokaryotic and eukaryotic cells. The role of leishmania EF-1α as an SHP-1 activator and its contribution to pathogenesis are reviewed with particular attention to the properties that distinguish it from host EF-1α.

The transformation suppressor protein Pdcd4 shuttles between nucleus and cytoplasm and binds RNA.

The Pdcd4 gene has originally been isolated in a search for genes that are activated in cells undergoing apoptosis. Independent of these studies, the Pdcd4 gene has been implicated in the suppression of tumor-promoter-mediated transformation of keratinocytes and as a downstream target of Myb in hematopoietic cells. The Pdcd4 protein has weak homology to the eucaryotic translation initiation factor eIF4G and has been shown to interact with certain translation initiation factors. To explore the molecular function of the Pdcd4 protein, we have studied its subcellular localization. We show that the Pdcd4 protein is a predominantly nuclear protein under normal growth conditions and that it is exported from the nucleus by a leptomycin B-sensitive mechanism upon serum withdrawal. The protein contains two nuclear export signals, one of which is very potent. In addition, we demonstrate that the Pdcd4 protein has RNA-binding activity and that the sequences involved in RNA-binding are located in the amino-terminal part of the protein. Taken together, our data raise the possibility that Pdcd4 is involved in some aspect of nuclear RNA metabolism in addition to its suspected role in protein translation.

X‐ray structure of Saccharomyces cerevisiae homologous mitochondrial matrix factor 1 (Hmf1)

X‐ray structure of <i>Saccharomyces cerevisiae</i> homologous mitochondrial matrix factor 1 (Hmf1)

Mapping kidney development

How does a complex organ like a kidney develop? An insight into the step-by-step process of development is reported in PNAS, 1 May. The researchers, lead by Sanjay Nigam, employed microarrays of 8740 genes to study the development of rat kidneys. Five discrete patterns of gene expression were observed. The pattern of expression is largely understandable, however, there were some surprises. For example, one group of genes that were highly-expressed in the early embryonic kidney have a large role in protein translation and DNA replication. Another group contained genes that steadily increased in relative levels of expression throughout development, including many genes involved in energy metabolism and transport. DM

Changes in global gene expression patterns during development and maturation of the rat kidney.

We set out to define patterns of gene expression during kidney organogenesis by using high-density DNA array technology. Expression analysis of 8,740 rat genes revealed five discrete patterns or groups of gene expression during nephrogenesis. Group 1 consisted of genes with very high expression in the early embryonic kidney, many with roles in protein translation and DNA replication. Group 2 consisted of genes that peaked in midembryogenesis and contained many transcripts specifying proteins of the extracellular matrix. Many additional transcripts allied with groups 1 and 2 had known or proposed roles in kidney development and included LIM1, POD1, GFRA1, WT1, BCL2, Homeobox protein A11, timeless, pleiotrophin, HGF, HNF3, BMP4, TGF-alpha, TGF-beta2, IGF-II, met, FGF7, BMP4, and ganglioside-GD3. Group 3 consisted of transcripts that peaked in the neonatal period and contained a number of retrotransposon RNAs. Group 4 contained genes that steadily increased in relative expression levels throughout development, including many genes involved in energy metabolism and transport. Group 5 consisted of genes with relatively low levels of expression throughout embryogenesis but with markedly higher levels in the adult kidney; this group included a heterogeneous mix of transporters, detoxification enzymes, and oxidative stress genes. The data suggest that the embryonic kidney is committed to cellular proliferation and morphogenesis early on, followed sequentially by extracellular matrix deposition and acquisition of markers of terminal differentiation. The neonatal burst of retrotransposon mRNA was unexpected and may play a role in a stress response associated with birth. Custom analytical tools were developed including "The Equalizer" and "eBlot," which contain improved methods for data normalization, significance testing, and data mining.

Activation of Phosphatidylinositol 3-Kinase, Protein Kinase B, and p70 S6 Kinases in Lipopolysaccharide-Stimulated Raw 264.7 Cells: Differential Effects of Rapamycin, Ly294002, and Wortmannin on Nitric Oxide Production

Phosphatidylinositol 3-kinase (PI 3-kinase) and protein kinase B are critical players in cell proliferation and survival. Their downstream effector protein kinase, p70 S6 kinase, has an established role in protein translation. The mechanism by which bacterial LPS induces production of nitric oxide (NO) in murine macrophages is incompletely understood, and a role for PI 3-kinase/p70 S6 kinase pathway had not been previously investigated. In this study we demonstrate that LPS induced a fivefold activation of p70 S6 kinase and a twofold stimulation of PI 3-kinase. Pretreatment of Raw 264.7 cells with either rapamycin or Ly290042 completely blocked LPS-induced activation of p70 S6 kinase. Protein kinase B was also activated (twofold) by LPS and was only minimally affected by these inhibitors. PI 3-kinase activity was inhibited by both Ly294002 and wortmannin. The effects on NO production by these agents were strikingly different. While both rapamycin and Ly294002 resulted in almost complete inhibition of NO production, wortmannin was ineffective. Surprisingly, none of the inhibitors reduced the production of the inducible nitric oxide synthase protein (iNOS) as determined by immunoprecipitation. In vivo labeling studies revealed that the iNOS protein was phosphorylated in concordance with the production of NO. We conclude that LPS-mediated NO production occurs via a PI 3-kinase-independent, but FKBP12-rapamycin-associated protein-dependent, pathway in RAW cells by a mechanism probably involving phosphorylation of iNOS.

Branched-chain Amino Acids Are Essential in the Regulation of PHAS-I and p70 S6 Kinase by Pancreatic β-Cells: A POSSIBLE ROLE IN PROTEIN TRANSLATION AND MITOGENIC SIGNALING

Amino acids have been identified as important signaling molecules involved in pancreatic beta-cell proliferation, although the cellular mechanism responsible for this effect is not well defined. We previously reported that amino acids are required for glucose or exogenous insulin to stimulate phosphorylation of PHAS-I (phosphorylated heat- and acid-stable protein regulated by insulin), a recently discovered regulator of translation initiation during cell mitogenesis. Here we demonstrate that essential amino acids, in particular branched-chain amino acids (leucine, valine, and isoleucine), are largely responsible for mediating this effect. The transamination product of leucine, alpha-ketoisocaproic acid, also stimulates PHAS-I phosphorylation although the transamination products of isoleucine and valine are ineffective. Since amino acids are secretagogues for insulin secretion by beta-cells, we investigated whether endogenous insulin secreted by beta-cells is involved. Interestingly, branched-chain amino acids stimulate phosphorylation of PHAS-I independent of endogenous insulin secretion since genistein (10 microM) and herbimycin A (1 microM), two tyrosine kinase inhibitors in the insulin signaling pathway, exert no effect on amino acid-induced phosphorylation of PHAS-I. Furthermore, branched-chain amino acids retain their ability to induce phosphorylation of PHAS-I under conditions that block insulin secretion from beta-cells. In exploring the signaling pathway responsible for these effects, we find that rapamycin (25 nM) inhibits the ability of branched-chain amino acids to stimulate the phosphorylation of PHAS-I and p70(s6) kinase, suggesting that the mammalian target of rapamycin signaling pathway is involved. The branched-chain amino acid, leucine, also exerts similar effects on PHAS-I phosphorylation in isolated pancreatic islets. In addition, we find that amino acids are necessary for insulin-like growth factor (IGF-I) to stimulate the phosphorylation of PHAS-I indicating that a requirement for amino acids may be essential for other beta-cell growth factors in addition to insulin and IGF-I to activate this signaling pathway. We propose that amino acids, in particular branched-chain amino acids, may promote beta-cell proliferation either by stimulating phosphorylation of PHAS-I and p70(s6k) via the mammalian target of rapamycin pathway and/or by facilitating the proliferative effect mediated by growth factors such as insulin and IGF-I.

Elongation factor‐1α stabilizes microtubules in a calcium/calmodulin‐dependent manner

Elongation factor-1α (EF-1α), a highly conserved protein named for its role in protein translation, is also a microtubule-associated protein (MAP). We used high-resolution differential interference contrast microscopy to quantify the effect of substoichiometric amounts of EF-1α (isolated from Daucus carota) on the dynamic instability of microtubules assembled in vitro from either animal or plant tubulin. EF-1α modulates the dynamic behavior of microtubules assembled from either tubulin source, resulting in longer and more persistent microtubules. EF-1α, at a 1:20 molar ratio to tubulin, significantly (P < 0.05) reduces the frequency of catastrophe threefold and decreases shortening velocities almost twofold for microtubules assembled from animal tubulin. For microtubules assembled from plant tubulin, substoichiometric amounts of EF-1α significantly (P < 0.05) suppress the frequency of catastrophe greater than twofold and causes an almost threefold reduction in shortening velocities. Elongation velocities increase almost twofold and rescues, which are not observed in the absence of EF-1α, occur. In addition, calcium/calmodulin (Ca2+/CaM), which regulates the ability of EF-1α to bundle taxol-stabilized microtubules in vitro, also modulates the effect of EF-1α on the dynamic behavior of microtubules assembled in vitro from animal tubulin. Microtubule severing in the presence of EF-1α was never observed. These data support the hypothesis that EF-1α modulates the dynamic behavior of microtubules assembled in vitro in a Ca2+/CaM-dependent manner. Cell Motil. Cytoskeleton 41:168–180, 1998. © 1998 Wiley-Liss, Inc.

Elongation factor-1alpha stabilizes microtubules in a calcium/calmodulin-dependent manner.

Elongation factor-1alpha (EF-1alpha), a highly conserved protein named for its role in protein translation, is also a microtubule-associated protein (MAP). We used high-resolution differential interference contrast microscopy to quantify the effect of substoichiometric amounts of EF-1alpha (isolated from Daucus carota) on the dynamic instability of microtubules assembled in vitro from either animal or plant tubulin. EF-1alpha modulates the dynamic behavior of microtubules assembled from either tubulin source, resulting in longer and more persistent microtubules. EF-1alpha, at a 1:20 molar ratio to tubulin, significantly (P < 0.05) reduces the frequency of catastrophe threefold and decreases shortening velocities almost twofold for microtubules assembled from animal tubulin. For microtubules assembled from plant tubulin, substoichiometric amounts of EF-1alpha significantly (P < 0.05) suppress the frequency of catastrophe greater than twofold and causes an almost threefold reduction in shortening velocities. Elongation velocities increase almost twofold and rescues, which are not observed in the absence of EF-1alpha, occur. In addition, calcium/calmodulin (Ca2+/CaM), which regulates the ability of EF-1alpha to bundle taxol-stabilized microtubules in vitro, also modulates the effect of EF-1alpha on the dynamic behavior of microtubules assembled in vitro from animal tubulin. Microtubule severing in the presence of EF-1alpha was never observed. These data support the hypothesis that EF-1alpha modulates the dynamic behavior of microtubules assembled in vitro in a Ca2+/CaM-dependent manner.

Functional Constraints and rbcL Evidence for Land Plant Phylogeny

Although the proportion of «functional» DNA in eukaryotic genomes is both debatable and subject to definition, most sequences gathered for phylogenetic purposes are indisputably functional. For example, patterns of variation are likely to be strongly constrained in ribosomal RNAs because of their structural and catalytic roles in protein translation, and in protein-coding genes, because of protein function itself. Although seemingly obvious, these concerns are usually ignored by workers producing gene trees. We have examined the extent of functional constraints in land-plant rbcL sequences. Not only do rbcL sequences appear to change with essentially clocklike regularity, but nucleotide-based cladograms imply tbat approximately 97.5% of codon changes on internal branches are functionally neutral (i.e., synonymous or functionally labile)