FUS contributes to mTOR-dependent inhibition of translation

The amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD)–linked RNA-binding protein called FUS (fused in sarcoma) has been implicated in several aspects of RNA regulation, including mRNA translation. The mechanism by which FUS affects the translation of polyribosomes has not been established. Here we show that FUS can associate with stalled polyribosomes and that this association is sensitive to mTOR (mammalian target of rapamycin) kinase activity. Specifically, we show that FUS association with polyribosomes is increased by Torin1 treatment or when cells are cultured in nutrient-deficient media, but not when cells are treated with rapamycin, the allosteric inhibitor of mTORC1. Moreover, we report that FUS is necessary for efficient stalling of translation because deficient cells are refractory to the inhibition of mTOR-dependent signaling by Torin1. We also show that ALS-linked FUS mutants R521G and P525L associate abundantly with polyribosomes and decrease global protein synthesis. Importantly, the inhibitory effect on translation by FUS is impaired by mutations that reduce its RNA-binding affinity. These findings demonstrate that FUS is an important RNA-binding protein that mediates translational repression through mTOR-dependent signaling and that ALS-linked FUS mutants can cause a toxic gain of function in the cytoplasm by repressing the translation of mRNA at polyribosomes.     

Structure-related protein tyrosine phosphatase 1B inhibition by naringenin derivatives

Naturally occurring flavonoids co-exist as glycoside conjugates, which dominate aglycones in their content. To unveil the structure-activity relationship of a naturally occurring flavonoid, we investigated the effects of the glycosylation of naringenin on the inhibition of enzyme systems related to diabetes (protein tyrosine phosphatase 1B (PTP1B) and α-glycosidase) and on glucose uptake in the insulin-resistant state. Among the tested naringenin derivatives, prunin, a single-glucose-containing flavanone glycoside, potently inhibited PTP1B with an IC₅₀ value of 17.5 ± 2.6 µM. Naringenin, which lacks a sugar molecule, was the weakest inhibitor compared to the reference compound, ursolic acid (IC₅₀: 5.4 ± 0.30 µM). In addition, prunin significantly enhanced glucose uptake in a dose-dependent manner in insulin-resistant HepG2 cells. Regarding the inhibition of α-glucosidase, naringenin exhibited more potent inhibitory activity (IC₅₀: 10.6 ± 0.49 µM) than its glycosylated forms and the reference inhibitor, acarbose (IC₅₀: 178.0 ± 0.27 µM). Among the glycosides, only prunin (IC₅₀: 106.5 ± 4.1 µM) was more potent than the positive control. A molecular docking study revealed that prunin had lower binding energy and higher binding affinity than glycosides with higher numbers of H-bonds, suggesting that prunin is the best fit to the PTP1B active site cavity. Therefore, in addition to the number of H-bonds present, possible factors affecting the protein binding and PTP1B inhibition of flavanones include their fit to the active site, hydrogen-bonding affinity, Van der Waals interactions, H-bond distance, and H-bond stability. Furthermore, this study clearly depicted the association of the intensity of bioactivity with the arrangement and characterization of the sugar moiety on the flavonoid skeleton.     

Allosteric inhibition of protein tyrosine phosphatase 1B

Obesity and type II diabetes are closely linked metabolic syndromes that afflict >100 million people worldwide. Although protein tyrosine phosphatase 1B (PTP1B) has emerged as a promising target for the treatment of both syndromes, the discovery of pharmaceutically acceptable inhibitors that bind at the active site remains a substantial challenge. Here we describe the discovery of an allosteric site in PTP1B. Crystal structures of PTP1B in complex with allosteric inhibitors reveal a novel site located approximately 20 A from the catalytic site. We show that allosteric inhibitors prevent formation of the active form of the enzyme by blocking mobility of the catalytic loop, thereby exploiting a general mechanism used by tyrosine phosphatases. Notably, these inhibitors exhibit selectivity for PTP1B and enhance insulin signaling in cells. Allosteric inhibition is a promising strategy for targeting PTP1B and constitutes a mechanism that may be applicable to other tyrosine phosphatases.     

Overexpression of budding yeast protein phosphatase Ppz1 impairs translation

The Ser/Thr protein phosphatase Ppz1 from Saccharomyces cerevisiae is the best characterized member of a family of enzymes only found in fungi. Ppz1 is regulated in vivo by two inhibitory subunits, Hal3 and Vhs3, which are moonlighting proteins also involved in the decarboxylation of the 4-phosphopantothenoylcysteine (PPC) intermediate required for coenzyme A biosynthesis. It has been reported that, when overexpressed, Ppz1 is the most toxic protein in yeast. However, the reasons for such toxicity have not been elucidated. Here we show that the detrimental effect of excessive Ppz1 expression is due to an increase in its phosphatase activity and not to a plausible down-titration of the PPC decarboxylase components. We have identified several genes encoding ribosomal proteins and ribosome assembly factors as mild high-copy suppressors of the toxic Ppz1 effect. Ppz1 binds to ribosomes engaged in translation and copurifies with diverse ribosomal proteins and translation factors. Ppz1 overexpression results in Gcn2-dependent increased phosphorylation of eIF2α at Ser-51. Consistently, deletion of GCN2 partially suppresses the growth defect of a Ppz1 overexpressing strain. We propose that the deleterious effects of Ppz1 overexpression are in part due to alteration in normal protein synthesis.     

Repression of mitogen-activated protein kinases ERK1/ERK2 activity by a protein tyrosine phosphatase in rat fibroblasts transformed by upstream oncoproteins

The observation that mitogen-activated protein (MAP) kinases ERK1 and ERK2 are constitutively activated in a number of oncogene-transformed cell lines has led to the hypothesis that prolonged activation of these enzymes is required for the transformation process. To investigate this question, we have examined the regulation of the ERK pathway in Rat1 fibroblasts transformed with activated c-Raf-1 (Raf22W), v-Ha-Ras, and v-Src. Expression of these oncoproteins had no effect on the enzymatic activity of ERK1 and ERK2 in either serum-starved or exponentially growing cells. Moreover, the stimulatory effect of serum on ERK1/ERK2 activity was substantially reduced or abrogated in these cells; this impairment was associated with a strong attenuation of c-fos gene induction. In contrast, expression of Raf22w, v-Ha-Ras, or v-Src resulted in the constitutive activation of the upstream kinases MEK1 and MEK2. Treatment of the cells with vanadate completely restored the activation of ERK1/ERK2 in oncogene-transformed cells, suggesting the involvement of a vanadate-sensitive tyrosine phosphatase. Northern blot analysis of VH1-like dual-specificity MAP kinase phosphatases did not reveal any significant difference in the mRNA expression pattern of these genes between parental and transformed Rat1 cells. Phosphoamino acid analysis indicated that ERK1 is phosphorylated on threonine, but not on tyrosine, in oncogene-transformed cells and that vanadate treatment restores tyrosine phosphorylation. We conclude from these results that ERK1/ERK2 activity is repressed by a single-specificity tyrosine phosphatase in oncogene-transformed rat fibroblasts. J. Cell. Physiol. 174:35–47, 1998. © 1998 Wiley-Liss, Inc.     

Mxi2 sustains ERK1/2 phosphorylation in the nucleus by preventing ERK1/2 binding to phosphatases

ERK1/2 (extracellular-signal-regulated kinase 1/2) MAPKs (mitogen-activated protein kinases) are tightly regulated by the cellular microenvironment in which they operate. Mxi2 is a p38α splice isoform capable of binding to ERK1/2 and ensuring their translocation to the nucleus. Therein Mxi2 sustains ERK1/2 phosphorylation levels and, as a consequence, ERK1/2 nuclear signals are enhanced. However, the molecular mechanisms underlying this process are still unclear. In the present study, we show that Mxi2 prevents nuclear but not cytoplasmic phosphatases from binding to and dephosphorylating ERK1/2, disclosing an unprecedented mechanism for the spatial regulation of ERK1/2 activation. We also demonstrate that the kinetics of ERK1/2 extranuclear signals can be significantly altered by artificially tethering Mxi2 to the cytoplasm. In this case, Mxi2 abolishes ERK1/2 inactivation by cytoplasmic phosphatases and potentiates ERK1/2 functions at this compartment. These results highlight Mxi2 as a key spatial regulator of ERK1/2 functions, playing a pivotal role in the balance between ERK1/2 nuclear and cytoplasmic signals.     

Mitogen-activated protein kinase phosphatase 1/dual specificity phosphatase 1 mediates glucocorticoid inhibition of osteoblast proliferation

Steroid-induced osteoporosis is a common side effect of long-term treatment with glucocorticoid (GC) drugs. GCs have multiple systemic effects that may influence bone metabolism but also directly affect osteoblasts by decreasing proliferation. This may be beneficial at low concentrations, enhancing differentiation. However, high-dose treatment produces a severe deficit in the proliferative osteoblastic compartment. We provide causal evidence that this effect of GC is mediated by induction of the dual-specificity MAPK phosphatase, MKP-1/DUSP1. Excessive MKP-1 production is both necessary and sufficient to account for the impaired osteoblastic response to mitogens. Overexpression of MKP-1 after either GC treatment or transfection ablates the mitogenic response in osteoblasts. Knockdown of MKP-1 using either immunodepletion of MKP-1 before in vitro dephosphorylation assay or short interference RNA transfection prevents inactivation of ERK by GCs. Neither c-jun N-terminal kinase nor p38 MAPK is activated by the mitogenic cocktail in 20% fetal calf serum, but their activation by a DNA-damaging agent (UV irradiation) was inhibited by either GC treatment or overexpression of MKP-1, indicating regulation of all three MAPKs by MKP-1 in osteoblasts. However, an inhibitor of the MAPK/ERK kinase-ERK pathway inhibited osteoblast proliferation whereas inhibitors of c-jun N-terminal kinase or p38 MAPK had no effect, suggesting that ERK is the MAPK that controls osteoblast proliferation. Regulation of ERK by MKP-1 provides a novel mechanism for control of osteoblast proliferation by GCs.     

The ERK mitogen-activated protein kinase pathway contributes to Ebola virus glycoprotein-induced cytotoxicity

Ebola virus is a highly lethal pathogen that causes hemorrhagic fever in humans and nonhuman primates. Among the seven known viral gene products, the envelope glycoprotein (GP) alone induces cell rounding and detachment that ultimately leads to cell death. Cellular cytoxicity is not seen with comparable levels of expression of a mutant form of GP lacking a mucin-like domain (GPDeltamuc). GP-induced cell death is nonapoptotic and is preceded by downmodulation of cell surface molecules involved in signaling pathways, including certain integrins and epidermal growth factor receptor. To investigate the mechanism of GP-induced cellular toxicity, we analyzed the activation of several signal transduction pathways involved in cell growth and survival. The active form of extracellular signal-regulated kinases types 1 and 2 (ERK1/2), phospho-ERK1/2, was reduced in cells expressing GP compared to those expressing GPDeltamuc as determined by flow cytometry, in contrast to the case for several other signaling proteins. Subsequent analysis of the activation states and kinase activities of related kinases revealed a more pronounced effect on the ERK2 kinase isoform. Disruption of ERK2 activity by a dominant negative ERK or by small interfering RNA-mediated ERK2 knockdown potentiated the decrease in alphaV integrin expression associated with toxicity. Conversely, activation of the pathway through the expression of a constitutively active form of ERK2 significantly protected against this effect. These results indicate that the ERK signaling cascade mediates GP-mediated cytotoxicity and plays a role in pathogenicity induced by this gene product.     

Eukaryotic translation termination factor 1 associates with protein phosphatase 2A and targets it to ribosomes

Purification of type 2A protein phosphatase (PP2A) from rabbit skeletal muscle resulted in the isolation of a trimeric phosphatase which is composed of a catalytic (PP2Ac), a structural (PR65alpha/Aalpha), and a regulatory (PR55alpha/Balpha) subunit, together with translation termination factor 1 (eRF1) and another protein of 55 kD (EMBO J., 15, 101-112). Yeast two-hybrid system analysis demonstrated that the eRF1 interacted with PP2Acalpha but not with PR65alpha/Aalpha or PR55alpha/Balpha. The N-terminal region of PP2Acalpha, comprising 50 amino acid residues, and the C-terminal part of eRF1, corresponding to an internal region between amino acids 338-381, were found to be necessary for eRF1--PP2Acalpha interaction in yeast. Immunoprecipitations using 12CA5 antibodies and extracts from COS1 cells transiently transfected with eRF1 tagged with 9-amino acid epitope from influenza hemagglutinin (HA) demonstrated the presence of eRF1--PP2Acalpha--PR65alpha/Aalpha complex in these cells. In addition, polysomes obtained from COS1 cells overexpressing HA--eRF1 displayed several-fold higher PP2A activity than control polysomes. No effect of either PP2Ac or dimeric and trimeric PP2A holoenzymes on the rate of translation termination was detected using an in vitro reconstituted translation termination assay. In summary, eRF1 appears to represent a novel PP2A-targeting subunit that brings this phosphatase in contact with putative ribosomal substrate(s). It remains to be established whether termination of translation requires dephosphorylation of participating protein factor(s).     

Molecular mechanism of ERK dephosphorylation by striatal‐enriched protein tyrosine phosphatase

Striatal‐enriched tyrosine phosphatase (STEP) is an important regulator of neuronal synaptic plasticity, and its abnormal level or activity contributes to cognitive disorders. One crucial downstream effector and direct substrate of STEP is extracellular signal‐regulated protein kinase (ERK), which has important functions in spine stabilisation and action potential transmission. The inhibition of STEP activity toward phospho‐ERK has the potential to treat neuronal diseases, but the detailed mechanism underlying the dephosphorylation of phospho‐ERK by STEP is not known. Therefore, we examined STEP activity toward para‐nitrophenyl phosphate, phospho‐tyrosine‐containing peptides, and the full‐length phospho‐ERK protein using STEP mutants with different structural features. STEP was found to be a highly efficient ERK tyrosine phosphatase that required both its N‐terminal regulatory region and key residues in its active site. Specifically, both kinase interaction motif (KIM) and kinase‐specific sequence of STEP were required for ERK interaction. In addition to the N‐terminal kinase‐specific sequence region, S245, hydrophobic residues L249/L251, and basic residues R242/R243 located in the KIM region were important in controlling STEP activity toward phospho‐ERK. Further kinetic experiments revealed subtle structural differences between STEP and HePTP that affected the interactions of their KIMs with ERK. Moreover, STEP recognised specific positions of a phospho‐ERK peptide sequence through its active site, and the contact of STEP F311 with phospho‐ERK V205 and T207 were crucial interactions. Taken together, our results not only provide the information for interactions between ERK and STEP, but will also help in the development of specific strategies to target STEP‐ERK recognition, which could serve as a potential therapy for neurological disorders.

     

Adenovirus late protein synthesis is resistant to the inhibition of translation induced by poliovirus

Inhibition of host protein synthesis after poliovirus infection has been suggested to be a consequence of the proteolytic degradation of a p220 polypeptide necessary to translate capped mRNAs. However, the synthesis of several adenovirus late proteins on capped mRNAs was resistant to poliovirus inhibition. Thus, the hexon protein was still made 8 h after poliovirus superinfection. The synthesis of other adenovirus proteins such as the fiber was much more sensitive to poliovirus-induced inhibition than the hexon, either in the absence or in the presence of guanidine. Detailed densitometric analyses clearly showed the differential behavior of several adenovirus late mRNAs to poliovirus shut-off of translation. This is striking in view of the fact that a common leader sequence in the 5' termini is present in the adenovirus late mRNAs. The use of 3-methyl quercetin, an inhibitor of poliovirus RNA synthesis (Castrillo, J. L., Vanden Berghe, D., and Carrasco, L. (1986) Virology 152, 219-227), showed that translation of several capped adenovirus mRNAs took place in poliovirus-infected cells after the synthesis of host proteins had ceased. The poliovirus mRNA and the adenovirus mRNA coding for the hexon protein are very efficient mRNAs and have a leader sequence of more than 740 and 250 nucleotides, respectively, with very rich secondary structures making it difficult to predict how the scanning model will operate on these two mRNAs.     

The protein tyrosine phosphatase SHP-2 regulates interleukin-1-induced ERK activation in fibroblasts

Focal adhesion complexes are actin-rich, cytoskeletal structures that mediate cell adhesion to the substratum and also selectively regulate signal transduction pathways required for interleukin (IL)-1beta signaling to the MAP kinase, ERK. IL-1-induced ERK activation is markedly diminished in fibroblasts deprived of focal adhesions whereas activation of p38 and JNK is unaffected. While IL-1 signaling is known to involve the activity of protein and lipid kinases including MAP kinases, FAK, and PI3K, little is known about the role of phosphatases in the regulation of IL-1 signal generation and attenuation. Here we demonstrate that SHP-2, a protein tyrosine phosphatase present in focal adhesions, modulates IL-1-induced ERK activation and the transient actin stress fiber disorganization that occurs following IL-1 treatment in human gingival fibroblasts. Using a combination of immunoblotting, immunoprecipitation, and immunostaining we show that SHP-2 is present in nascent focal adhesions and undergoes phosphorylation on tyrosine 542 in response to IL-1 stimulation. Blocking anti-SHP-2 antibodies, electoporated into the cytosol of fibroblasts, inhibited IL-1-induced ERK activation, actin filament assembly, and cell contraction, indicating a role for SHP-2 in these processes. In summary, our data indicate that SHP-2, a focal adhesion-associated protein, participates in IL-1-induced ERK activation likely via an adaptor function.     

The P body protein LSm1 contributes to stimulation of hepatitis C virus translation,but not replication,by microRNA-122

The P body protein LSm1 stimulates translation and replication of hepatitis C virus (HCV). As the liver-specific microRNA-122 (miR-122) is required for HCV replication and is associated with P bodies, we investigated whether regulation of HCV by LSm1 involves miR-122. Here, we demonstrate that LSm1 contributes to activation of HCV internal ribosome entry site (IRES)-driven translation by miR-122. This role for LSm1 is specialized for miR-122 translation activation, as LSm1 depletion does not affect the repressive function of miR-122 at 3′ untranslated region (UTR) sites, or miR-122–mediated cleavage at a perfectly complementary site. We find that LSm1 does not influence recruitment of the microRNA (miRNA)-induced silencing complex to the HCV 5′UTR, implying that it regulates miR-122 function subsequent to target binding. In contrast to the interplay between miR-122 and LSm1 in translation, we find that LSm1 is not required for miR-122 to stimulate HCV replication, suggesting that miR-122 regulation of HCV translation and replication have different requirements. For the first time, we have identified a protein factor that specifically contributes to activation of HCV IRES-driven translation by miR-122, but not to other activities of the miRNA. Our results enhance understanding of the mechanisms by which miR-122 and LSm1 regulate HCV.     

Dephosphorylation of cardiac myofibril C-protein by protein phosphatase 1 and protein phosphatase 2A

C-protein purified from chicken cardiac myofibrils was phosphorylated with the catalytic subunit of cAMP-dependent protein kinase to nearly 3 mol [32P]phosphate/mol C protein. Digestion of 32P-labeled C-protein with trypsin revealed that the radioactivity was nearly equally distributed in three tryptic peptides which were separated by reversed-phase HPLC. Fragmentation of 32P-labeled C-protein with CNBr showed that the isotope was incorporated at different ratios in three CNBr fragments which were separated on polyacrylamide gels in the presence of sodium dodecyl sulfate. Phosphorylation was present in both serine and threonine residues. Incubation of 32P-labeled C-protein with the catalytic subunit of protein phosphatase 1 or 2A rapidly removed 30-40% of the [32P]phosphate. The major site(s) dephosphorylated by either one of the phosphatases was a phosphothreonine residue(s) apparently located on the same tryptic peptide and on the same CNBr fragment. CNBr fragmentation also revealed a minor phosphorylation site which was dephosphorylated by either of the phosphatases. Increasing the incubation period or the phosphatase concentration did not result in any further dephosphorylation of C-protein by phosphatase 1, but phosphatase 2A at high concentrations could completely dephosphorylate C-protein. These results demonstrate that C-protein phosphorylated with cAMP-dependent protein kinase can be dephosphorylated by protein phosphatases 1 and 2A. It is suggested that the enzyme responsible for dephosphorylation of C-protein in vivo is phosphatase 2A.     

Phosphorylation of protein phosphatase inhibitor-1 by protein kinase C

Inhibitor-1 becomes a potent inhibitor of protein phosphatase 1 when phosphorylated by cAMP-dependent protein kinase at Thr(35). Moreover, Ser(67) of inhibitor-1 serves as a substrate for cyclin-dependent kinase 5 in the brain. Here, we report that dephosphoinhibitor-1 but not phospho-Ser(67) inhibitor-1 was efficiently phosphorylated by protein kinase C at Ser(65) in vitro. In contrast, Ser(67) phosphorylation by cyclin-dependent kinase 5 was unaffected by phospho-Ser(65). Protein kinase C activation in striatal tissue resulted in the concomitant phosphorylation of inhibitor-1 at Ser(65) and Ser(67), but not Ser(65) alone. Selective pharmacological inhibition of protein phosphatase activity suggested that phospho-Ser(65) inhibitor-1 is dephosphorylated by protein phosphatase 1 in the striatum. In vitro studies confirmed these findings and suggested that phospho-Ser(67) protects phospho-Ser(65) inhibitor-1 from dephosphorylation by protein phosphatase 1 in vivo. Activation of group I metabotropic glutamate receptors resulted in the up-regulation of diphospho-Ser(65)/Ser(67) inhibitor-1 in this tissue. In contrast, the activation of N-methyl-d-aspartate-type ionotropic glutamate receptors opposed increases in striatal diphospho-Ser(65)/Ser(67) inhibitor-1 levels. Phosphomimetic mutation of Ser(65) and/or Ser(67) did not convert inhibitor-1 into a protein phosphatase 1 inhibitor. On the other hand, in vitro and in vivo studies suggested that diphospho-Ser(65)/Ser(67) inhibitor-1 is a poor substrate for cAMP-dependent protein kinase. These observations extend earlier studies regarding the function of phospho-Ser(67) and underscore the possibility that phosphorylation in this region of inhibitor-1 by multiple protein kinases may serve as an integrative signaling mechanism that governs the responsiveness of inhibitor-1 to cAMP-dependent protein kinase activation.