The beta4 integrin interactor p27(BBP/eIF6) is an essential nuclear matrix protein involved in 60S ribosomal subunit assembly

p27(BBP/eIF6) is an evolutionarily conserved protein that was originally identified as p27(BBP), an interactor of the cytoplasmic domain of integrin beta4 and, independently, as the putative translation initiation factor eIF6. To establish the in vivo function of p27(BBP/eIF6), its topographical distribution was investigated in mammalian cells and the effects of disrupting the corresponding gene was studied in the budding yeast, Saccharomyces cerevisiae. In epithelial cells containing beta4 integrin, p27(BBP/eIF6) is present in the cytoplasm and enriched at hemidesmosomes with a pattern similar to that of beta4 integrin. Surprisingly, in the absence and in the presence of the beta4 integrin subunit, p27(BBP/eIF6) is in the nucleolus and associated with the nuclear matrix. Deletion of the IIH S. cerevisiae gene, encoding the yeast p27(BBP/eIF6) homologue, is lethal, and depletion of the corresponding gene product is associated with a dramatic decrease of the level of free ribosomal 60S subunit. Furthermore, human p27(BBP/eIF6) can rescue the lethal effect of the iihDelta yeast mutation. The data obtained in vivo suggest an evolutionarily conserved function of p27(BBP/eIF6) in ribosome biogenesis or assembly rather than in translation. A further function related to the beta4 integrin subunit may have evolved specifically in higher eukaryotic cells.     

Cloning of Dictyostelium eIF6 (p27BBP) and mapping its nucle(ol)ar localization subdomains

Eukaryotic translation initiation factor 6 (eIF6), also termed p27BBP, is an evolutionary conserved regulator of ribosomal function. The protein is involved in maturation and/or export from the nucleus of the 60S ribosomal subunit. Regulated binding to and release from the 60S subunit also regulates formation of 80S ribosomes, and thus translation. The protein is also found in hemidesmosomes of epithelial cells expressing beta4 integrin and is assumed to regulate cross-talk between beta4 integrin, intermediate filaments and ribosomes. In the present study we show that the Dictyostelium eIF6 (also called p27BBP) gene is expressed during growth, down-regulated during the first hours of starvation, and up-regulated again at the end of aggregation. Phagocytosis, and to a lesser extent pinocytic uptake of axenic medium, stimulate gene expression in starving cells. The eIF6 gene is present in single copy and its ablation is lethal. We utilized the green fluorescent protein (GFT) as fusion protein marker to investigate sequences responsible for eIF6 subcellular localization. The protein is found both in cytoplasm and nucleus, and is enriched in nucleoli. Deletion sequence analysis shows that nucle(ol)ar localization sequences are located within the N- and C-terminal subdomains of the protein.     

Formation of nuclear matrix filaments by p27(BBP)/eIF6

p27(BBP)/eIF6 is an evolutionarily conserved protein necessary for ribosome biogenesis which was cloned in mammals for its ability to bind the cytodomain of beta 4 integrin. In cultured cells, a conspicuous fraction of p27(BBP)/eIF6 is associated with the intermediate filaments/nuclear matrix (IF/NM) cytoskeleton. The mechanism of this association is not known. Here we show that in epidermis p27(BBP)/eIF6 is naturally associated with IF/NM. To analyze the intrinsic capability of p27(BBP)/eIF6 to generate cytoskeletal networks, the properties of the pure, recombinant, untagged protein were studied. Recombinant p27(BBP)/eIF6 binds beta 4 integrin. Upon dialysis against IF buffer, p27(BBP)/eIF6 forms polymers which, strikingly, have a morphology identical to NM filaments. Cross-linking experiments suggested that polymerization is favored by the formation of disulphide bridges. These data suggest that p27(BBP)/eIF6 is associated with the cytoskeleton, and contributes to formation of NM filaments. These findings help to settle the controversy on nuclear matrix.     

Evidence for alternate splicing within the mRNA transcript encoding the DNA damage response kinase ATR

The ITGB4BP gene encodes for a highly conserved protein, named p27BBP (also known as eIF6), originally identified in mammals as a cytoplasmic interactor of beta4 integrin. In vitro and in vivo studies demonstrated that p27BBP is essential for cell viability and has a primary function in the biogenesis of the 60S ribosomal subunit. Here we report the genomic organization of the human ITGB4BP gene and show that its gene product is expressed with features of a housekeeping element in vitro, but is regulated in a cell specific fashion in vivo. The human gene spans 10 kb and comprises seven exons and six introns. The 5' flanking region shows a TATA-less promoter, canonical CpG islands, and binding sites for serum responsive elements. In cultured cells, p27BBP mRNA and protein are constitutively expressed and stable. A gradual loss of p27BBP mRNA can be observed only after prolonged serum starvation, and heat shock treatment. In contrast, p27BBP mRNA and protein levels in vivo are variable among different organs. More strikingly, immunohistochemical analysis shows that the p27BBP protein is present in a cell specific fashion, even within the same tissue. Taken together, these data show that ITGB4BP gene expression is highly regulated in vivo, possibly by the combination of tissue specific factors and protein synthesis pathways.     

Phosphorylation of eukaryotic translation initiation factor 4B (EIF4B) by open reading frame 45/p90 ribosomal S6 kinase (ORF45/RSK) signaling axis facilitates protein translation during Kaposi sarcoma-associated herpesvirus (KSHV) lytic replication

Open reading frame 45 (ORF45) of Kaposi sarcoma-associated herpesvirus (KSHV) causes sustained activation of p90 ribosomal S6 kinase (RSK), which is crucial for KSHV lytic replication, but the exact functional roles remain to be determined. To characterize the biological consequence of persistent RSK activation by ORF45, we screened known cellular substrates of RSK. We found that ORF45 induced phosphorylation of eukaryotic translation initiation factor 4B (eIF4B), increased its assembly into translation initiation complex, and subsequently facilitated protein translation. The ORF45/RSK-mediated eIF4B phosphorylation was distinguishable from that caused by the canonical AKT/mammalian target of rapamycin/ribosomal S6 kinase and MEK/ERK/RSK pathways because it was resistant to both rapamycin (an mammalian target of rapamycin inhibitor) and U1026 (an MEK inhibitor). The rapamycin and U1026 doubly insensitive eIF4B phosphorylation was induced during KSHV reactivation but was abolished if either ORF45 or RSK1/2 were ablated by siRNA, a pattern that is correlated with reduced lytic gene expression as we observed previously. Ectopic expression of eIF4B but not its phosphorylation-deficient mutant form increased KSHV lytic gene expression and production of progeny viruses. Together, these results indicated that ORF45/RSK axis-induced eIF4B phosphorylation is involved in translational regulation and is required for optimal KSHV lytic replication.     

Pancreastatin,a chromogranin A-derived peptide,activates protein synthesis signaling cascade in rat adipocytes

Pancreastatin (PST), a chromogranin A-derived peptide, has been found to modulate glucose, lipid, and protein metabolism in rat adipocytes. PST has an overall counterregulatory effect on insulin action by activating a specific receptor-effector system (Galpha(q/11) protein-PLC-beta-PKC(classical)). However, PST stimulates both basal and insulin-mediated protein synthesis in rat adipocytes. In order to further investigate the mechanisms underlying the effect of PST stimulating protein synthesis, we sought to study the regulation of different components of the core translational machinery by the signaling triggered by PST. Thus, we studied ribosomal p70 S6 kinase, phosphorylation of the cap-binding protein (initiation factor) eIF4E, and phosphorylation of the eIF4E-binding protein 4E-BP1 (PHAS-I). We have found that PST stimulates the S6 kinase activity, as assessed by kinase assay using specific immunoprecipitates and substrate. This effect was checked by Western blot with specific antibodies against the phosphorylated S6 kinase. Thus, PST dose-dependently stimulates Thr421/Ser424 phosphorylation of S6 kinase. Moreover, PST promotes phosphorylation of regulatory sites in 4E-BP1 (PHAS-I) (Thr37, Thr46). The initiation factor eIF4E itself, whose activity is also increased upon phosphorylation, is phosphorylated in Ser209 by PST stimulation. Finally, we have found that these effects of PST on S6 kinase and the translation machinery can be blocked by preventing the activation of PKC. These results indicate that PST stimulates protein synthesis machinery by activating PKC and provides some evidence of the molecular mechanisms involved, i.e., the activation of S6K and the phosphorylation of 4E-BP1 (PHAS-I) and the initiation factor eIF4E.     

Arachidonic acid activation of translation initiation signaling in vascular smooth muscle cells

To understand the role of arachidonic acid (AA) in regulating vascular smooth muscle cell (VSMC) growth, its effects on phosphorylation of Akt, S6K1, ribosomal protein S6, 4EBP1, and eIF4E were studied. Arachidonic acid stimulated phosphorylation of Akt, S6K1, ribosomal protein S6, 4EBP1, and eIF4E in a time-dependent manner in VSMC. Arachidonic acid stimulation of phosphorylation of the above signaling molecules is specific, as these events were not affected by other unsaturated or saturated fatty acids. Metabolic conversion of AA via the LOX/MOX and/or COX pathways, to some extent, was required for its effects on the phosphorylation of Akt, S6K1, ribosomal protein S6, 4EBP1, and eIF4E. In addition, AA increased PI3K activity in a time-dependent manner in VSMC. LY294002, an inhibitor of PI3K, completely blocked AA-induced phosphorylation of Akt, S6K1, ribosomal protein S6, 4EBP1, and eIF4E, suggesting a role for PI3K in these effects. Consistent with its effects on translation initiation signaling events, AA induced global protein synthesis in VSMC and this response was dependent, to some extent, on its metabolism via the LOX/MOX and/or COX pathways, and mediated by the PI3K/Akt/mTOR pathway. Thus, the above observations provide the first biochemical evidence for the role of AA in the activation of translation initiation signaling in VSMC.     

The mTOR/PI3K and MAPK pathways converge on eIF4B to control its phosphorylation and activity

The eukaryotic translation initiation factor 4B (eIF4B) plays a critical role in recruiting the 40S ribosomal subunit to the mRNA. In response to insulin, eIF4B is phosphorylated on Ser422 by S6K in a rapamycin-sensitive manner. Here we demonstrate that the p90 ribosomal protein S6 kinase (RSK) phosphorylates eIF4B on the same residue. The relative contribution of the RSK and S6K modules to the phosphorylation of eIF4B is growth factor-dependent, and the two phosphorylation events exhibit very different kinetics. The S6K and RSK proteins are members of the AGC protein kinase family, and require PDK1 phosphorylation for activation. Consistent with this requirement, phosphorylation of eIF4B Ser422 is abrogated in PDK1 null embryonic stem cells. Phosphorylation of eIF4B on Ser422 by RSK and S6K is physiologically significant, as it increases the interaction of eIF4B with the eukaryotic translation initiation factor 3.     

Impaired myocardial protein synthesis induced by acute alcohol intoxication is associated with changes in eIF4F

The purpose of the present study was to examine potential mechanisms for the known inhibitory effect of acute alcohol exposure on myocardial protein synthesis. Rats were injected intraperitoneally with either ethanol (75 mmol/kg) or saline, and protein synthesis was measured in vivo 2.5 h thereafter by use of the flooding-dose L-[(3)H]phenylalanine technique. Rates of myocardial protein synthesis and translational efficiency in alcohol-treated rats were decreased compared with control values. Free (nonpolysome bound) 40S and 60S ribosomal subunits were increased 50% after alcohol treatment, indicating an impaired peptide-chain initiation. To identify mechanisms responsible for this impairment, several eukaryotic initiation factors (eIF) were analyzed. Acute alcohol intoxication did not significantly alter the myocardial content of eIF2 alpha or eIF2B epsilon, the extent of eIF2 alpha phosphorylation, or the activity of eIF2B. Acute alcohol exposure increased the binding of 4E-binding protein 1 (4E-BP1) to eIF4E (55%), diminished the amount of eIF4E bound to eIF4G (70%), reduced the amount of 4E-BP1 in the phosphorylated gamma-form (40%), and decreased the phosphorylation of p70S6 kinase and the ribosomal protein S6. There was no significant difference in either the plasma insulin-like growth factor (IGF) I concentration (total or free) or expression of IGF-I or IGF-II mRNA in heart between the two groups. These data suggest that the acute alcohol-induced impairment in myocardial protein synthesis results, in part, from an inhibition in peptide-chain initiation, which is associated with marked changes in eIF4E availability and p70S6 kinase phosphorylation but is independent of changes in the eIF2/2B system and IGFs.     

Leucine regulates translation of specific mRNAs in L6 myoblasts through mTOR-mediated changes in availability of eIF4E and phosphorylation of ribosomal protein S6

Regulation of translation of mRNAs coding for specific proteins plays an important role in controlling cell growth, differentiation, and transformation. Two proteins have been implicated in the regulation of specific mRNA translation: eukaryotic initiation factor eIF4E and ribosomal protein S6. Increased phosphorylation of eIF4E as well as its overexpression are associated with stimulation of translation of mRNAs with highly structured 5'-untranslated regions. Similarly, phosphorylation of S6 results in preferential translation of mRNAs containing an oligopyrimidine tract at the 5'-end of the message. In the present study, leucine stimulated phosphorylation of the eIF4E-binding protein, 4E-BP1, in L6 myoblasts, resulting in dissociation of eIF4E from the inactive eIF4E.4E-BP1 complex. The increased availability of eIF4E was associated with a 1.6-fold elevation in ornithine decarboxylase relative to global protein synthesis. Leucine also stimulated phosphorylation of the ribosomal protein S6 kinase, p70(S6k), resulting in increased phosphorylation of S6. Hyperphosphorylation of S6 was associated with a 4-fold increase in synthesis of elongation factor eEF1A. Rapamycin, an inhibitor of the protein kinase mTOR, prevented all of the leucine-induced effects. Thus, leucine acting through an mTOR-dependent pathway stimulates the translation of specific mRNAs both by increasing the availability of eIF4E and by stimulating phosphorylation of S6.     

A potential role for extracellular signal-regulated kinases in prostaglandin F2alpha-induced protein synthesis in smooth muscle cells

To understand the mechanisms of prostaglandin F2alpha (PGF2alpha)-induced protein synthesis in vascular smooth muscle cells (VSMC), we have studied its effect on two major signal transduction pathways: mitogen-activated protein kinases and phosphatidylinositol 3-kinase (PI3-kinase) and their downstream targets ribosomal protein S6 kinase (p70(S6k)) and eukaryotic initiation factor eIF4E and its regulator 4E-BP1. PGF2alpha induced the activities of extracellular signal-regulated kinase 2 (ERK2) and Jun N-terminal kinase 1 (JNK1) groups of mitogen-activated protein kinases, PI3-kinase, and p70(S6k) in a time-dependent manner in growth-arrested VSMC. PGF2alpha also induced eIF4E and 4E-BP1 phosphorylation, global protein synthesis, and basic fibroblast growth factor-2 (bFGF-2) expression in VSMC. Whereas inhibition of PI3-kinase by wortmannin completely blocked the p70(S6k) activation, it only partially decreased the ERK2 activity, and had no significant effect on global protein synthesis and bFGF-2 expression induced by PGF2alpha. Rapamycin, a potent inhibitor of p70(S6k), also failed to prevent PGF2alpha-induced global protein synthesis and bFGF-2 expression, although it partially decreased ERK2 activity. In contrast, inhibition of ERK2 activity by PD 098059 led to a significant loss of PGF2alpha-induced eIF4E and 4E-BP1 phosphorylation, global protein synthesis, and bFGF-2 expression. PGF2alpha-induced phosphorylation of eIF4E and 4E-BP1 was also found to be sensitive to inhibition by both wortmannin and rapamycin. These findings demonstrate that 1) PI3-kinase-dependent and independent mechanisms appear to be involved in PGF2alpha-induced activation of ERK2; 2) PGF2alpha-induced eIF4E and 4E-BP1 phosphorylation appear to be mediated by both ERK-dependent and PI3-kinase-dependent rapamycin-sensitive mechanisms; and 3) ERK-dependent eIF4E phosphorylation but not PI3-kinase-dependent p70(S6k) activation correlates with PGF2alpha-induced global protein synthesis and bFGF-2 expression in VSMC.     

Soy peptide-induced stem cell proliferation: involvement of ERK and TGF-β1

This study was conducted to investigate the proliferative effect of vegetable soy peptides on adult stem cells (ASCs) in the absence of serum and their possible mechanisms of action. The proliferation of human adipose tissue-derived mesenchymal stem cells (ADSCs) and cord blood-derived mesenchymal stem cells (CB-MSCs) treated with soy peptides was found to increase significantly upon 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and Click-iT 5-ethynyl-2'-deoxyuridine flow cytometry assay. In addition, soy peptides led to stepwise phosphorylation of the p44/42 MAPK (ERK), mammalian target of rapamycin (mTOR), p70 S6 kinase, S6 ribosomal protein (S6RP) and eukaryotic initiation factor 4E (eIF4E) in ADSCs. Furthermore, quantitative analysis of the cytokines revealed that the production of transforming growth factor-beta1 (TGF-β1), vascular endothelial growth factor and interleukin-6 increased significantly in response to treatment with soy peptides in both ADSCs and CB-MSCs. Similarly, soy peptide-induced phosphorylation of the ERK/mTOR/S6RP/eIF4E pathway was blocked in response to pretreatment with PD98059, a specific ERK inhibitor. Moreover, inhibition of TGF-β1 through PD98059 pretreatment and a consecutive decrease in ADSC proliferation revealed that TGF-β1 induces the phosphorylation of mTOR/S6RP/eIF4E. Collectively, the results of this study indicate that ERK-dependent production of TGF-β1 plays a crucial role in the soy peptide-induced proliferation of ADSCs under serum-free conditions.     

Angiotensin IV enhances phosphorylation of 4EBP1 by multiple signaling events in lung endothelial cells

Angiotensin IV (Ang IV)-stimulated cell proliferation is regulated through activation of multiple signaling modules in lung endothelial cells (EC). Because eukaryotic intitiation factor 4E (eIF4E) binding protein 1 (4EBP1) plays a critical role in the RNA translation and the regulation of cell growth, we examined whether Ang IV modulates expression and/or phosphorylation of eIF4E and 4EBP1 as well as the role of multiple signaling events associated with 4EBP1 phosphorylation in EC. Ang IV stimulation increased phosphorylation but not expression of eIF4E and 4EBP1 proteins. Ang IV stimulation selectively phosphorylated Thr46 > Thr70 > Ser65 but not Thr37 residues in 4EBP1. Pretreatment of cells with PD-98059 and rapamycin, inhibitors of mitogen-activated protein kinase (ERK1/2) and mammalian target for rapamycin (mTOR), respectively, partially blocked Ang IV-mediated phosphorylation of 4EBP1. In contrast, overexpression of p70 ribosomal S6 kinase (p70S6K) and protein kinase B (Akt) enhanced phosphorylation of 4EBP1 and eIF4E binding affinity to the cap region of mRNA. These results support critical roles of multiple signaling and phosphorylation of 4EBP1 by Ang IV in translation process and protein synthesis.     

c-Raf/MEK/ERK pathway controls protein kinase C-mediated p70S6K activation in adult cardiac muscle cells

p70S6 kinase (S6K1) plays a pivotal role in hypertrophic cardiac growth via ribosomal biogenesis. In pressure-overloaded myocardium, we show S6K1 activation accompanied by activation of protein kinase C (PKC), c-Raf, and mitogen-activated protein kinases (MAPKs). To explore the importance of the c-Raf/MAPK kinase (MEK)/MAPK pathway, we stimulated adult feline cardiomyocytes with 12-O-tetradecanoylphorbol-13-acetate (TPA), insulin, or forskolin to activate PKC, phosphatidylinositol-3-OH kinase, or protein kinase A (PKA), respectively. These treatments resulted in S6K1 activation with Thr-389 phosphorylation as well as mammalian target of rapamycin (mTOR) and S6 protein phosphorylation. Thr-421/Ser-424 phosphorylation of S6K1 was observed predominantly in TPA-treated cells. Dominant negative c-Raf expression or a MEK1/2 inhibitor (U0126) treatment showed a profound blocking effect only on the TPA-stimulated phosphorylation of S6K1 and mTOR. Whereas p38 MAPK inhibitors exhibited only partial effect, MAPK-phosphatase-3 expression significantly blocked the TPA-stimulated S6K1 and mTOR phosphorylation. Inhibition of mTOR with rapamycin blocked the Thr-389 but not the Thr-421/Ser-424 phosphorylation of S6K1. Therefore, during PKC activation, the c-Raf/MEK/extracellular signal-regulated kinase-1/2 (ERK1/2) pathway mediates both the Thr-421/Ser-424 and the Thr-389 phosphorylation in an mTOR-independent and -dependent manner, respectively. Together, our in vivo and in vitro studies indicate that the PKC/c-Raf/MEK/ERK pathway plays a major role in the S6K1 activation in hypertrophic cardiac growth.     

Cloning of Dictyostelium eIF6 (p27) and mapping its nucle(ol)ar localization subdomains

Eukaryotic translation initiation factor 6 (eIF6), also termed p27^BBP, is an evolutionary conserved regulator of ribosomal function. The protein is involved in maturation and/or export from the nucleus of the 60S ribosomal subunit. Regulated binding to and release from the 60S subunit also regulates formation of 80S ribosomes, and thus translation. The protein is also found in hemidesmosomes of epithelial cells expressing β4 integrin and is assumed to regulate cross-talk between β4 integrin, intermediate filaments and ribosomes. In the present study we show that the Dictyostelium eIF6 (also called p27^BBP) gene is expressed during growth, down-regulated during the first hours of starvation, and up-regulated again at the end of aggregation. Phagocytosis, and to a lesser extent pinocytic uptake of axenic medium, stimulate gene expression in starving cells. The eIF6 gene is present in single copy and its ablation is lethal. We utilized the green fluorescent protein (GFT) as fusion protein marker to investigate sequences responsible for eIF6 subcellular localization. The protein is found both in cytoplasm and nucleus, and is enriched in nucleoli. Deletion sequence analysis shows that nucle(ol)ar localization sequences are located within the N- and C-terminal subdomains of the protein.     

Photosynthetic Control of Arabidopsis Leaf Cytoplasmic Translation Initiation by Protein Phosphorylation

Photosynthetic CO₂ assimilation is the carbon source for plant anabolism, including amino acid production and protein synthesis. The biosynthesis of leaf proteins is known for decades to correlate with photosynthetic activity but the mechanisms controlling this effect are not documented. The cornerstone of the regulation of protein synthesis is believed to be translation initiation, which involves multiple phosphorylation events in Eukaryotes. We took advantage of phosphoproteomic methods applied to Arabidopsis thaliana rosettes harvested under controlled photosynthetic gas-exchange conditions to characterize the phosphorylation pattern of ribosomal proteins (RPs) and eukaryotic initiation factors (eIFs). The analyses detected 14 and 11 new RP and eIF phosphorylation sites, respectively, revealed significant CO₂-dependent and/or light/dark phosphorylation patterns and showed concerted changes in 13 eIF phosphorylation sites and 9 ribosomal phosphorylation sites. In addition to the well-recognized role of the ribosomal small subunit protein RPS6, our data indicate the involvement of eIF3, eIF4A, eIF4B, eIF4G and eIF5 phosphorylation in controlling translation initiation when photosynthesis varies. The response of protein biosynthesis to the photosynthetic input thus appears to be the result of a complex regulation network involving both stimulating (e.g. RPS6, eIF4B phosphorylation) and inhibiting (e.g. eIF4G phosphorylation) molecular events.     

Signaling pathways initiated by beta-hydroxy-beta-methylbutyrate to attenuate the depression of protein synthesis in skeletal muscle in response to cachectic stimuli

To investigate the mechanism by which beta-hydroxy-beta-methylbutyrate (HMB) attenuates the depression of protein synthesis in the skeletal muscle of cachectic mice, a study has been carried out in murine myotubes in the presence of proteolysis-inducing factor (PIF). PIF inhibited protein synthesis by 50% within 4 h, and this was effectively attenuated by HMB (25-50 muM). HMB (50 muM) alone stimulated protein synthesis, and this was attenuated by rapamycin (27 nM), an inhibitor of mammalian target of rapamycin (mTOR). Further evidence for an involvement of this pathway was shown by an increased phosphorylation of mTOR, the 70-kDa ribosomal S6 kinase (p70(S6k)), and initiation factor 4E-binding protein (4E-BP1) and an increased association of eukaryotic initiation factor 2 (eIF4E) with eIF4G. PIF alone induced a transient (1-2 h) stimulation of phosphorylation of mTOR and p70(S6k). However, in the presence of HMB, phosphorylation of mTOR, p70(S6k), and 4E-BP1 was increased, and inactive 4E-BP1-eIF4E complex was reduced, whereas the active eIF4G.eIF4E complex was increased, suggesting continual stimulation of protein synthesis. HMB alone reduced phosphorylation of elongation factor 2, but this effect was not seen in the presence of PIF. PIF induced autophosphorylation of the double-strand RNA-dependent protein kinase (PKR), leading to phosphorylation of eIF2 on the alpha-subunit, which would inhibit protein synthesis. However, in the presence of HMB, phosphorylation of PKR and eIF2alpha was attenuated, and this was also observed in skeletal muscle of cachectic mice administered HMB (0.25 g/kg). These results suggest that HMB attenuates the depression of protein synthesis by PIF in myotubes through multiple mechanisms.     

Nucleotide sequence and 40 S subunit assembly of Xenopus laevis ribosomal protein S22

We have isolated and determined the nucleotide sequence of a cDNA encoding Xenopus laevis ribosomal protein S22. A synthetic S22 mRNA derived from this cDNA directs the synthesis of an in vitro translation product that is indistinguishable from S22 purified from Xenopus ovarian ribosomes. In vitro translated S22 is assembled into 40 S subunits when microinjected into the cytoplasm of oocytes. Analysis of the derived amino acid sequence indicates that Xenopus S22 is homologous to Escherichia coli ribosomal protein S10.     

Initiation factor eIF2B not p70 S6 kinase is involved in the activation of the PI-3K signalling pathway induced by the v-src oncogene

Our data show that in hamster fibroblasts transformed by Rous sarcoma virus (RSV), the phosphoinositide 3'-kinase (PI-3K)/Akt/glycogen synthase kinase 3 antiapoptotic pathway is upregulated and involved in increased protein synthesis through activation of initiation factor eIF2B. Upon inhibition of PI-3K by wortmannin, phosphorylation of 70-kDa ribosomal protein S6 kinase (p70 S6k) and its physiological substrate, ribosomal protein S6, decreased in the non-transformed cells but not in RSV-transformed cells. Thus PI-3K, which is thought to be involved in regulation of p70 S6k, signals to p70 S6k in normal fibroblasts, but it does not appear to be an upstream effector of p70 S6k in fibroblasts transformed by v-src oncogene, suggesting that changes in the PI-3K signalling pathway upstream of p70 S6k are induced by RSV transformation.