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Biomedical subjects

C G Proud

Publications and source records attributed to C G Proud.

At least 19 recordsLinked to original sources
PubMed1999

Phosphorylated seryl and threonyl, but not tyrosyl, residues are efficient specificity determinants for GSK-3beta and Shaggy.

Glycogen synthase kinase-3 is involved in diverse functions including insulin signalling and development. In a number of substrates, phosphorylation by glycogen synthase kinase-3 is known to require prior phosphorylation at a Ser in the +4 position relative to its own phosphorylation site. Here we have used synthetic peptides derived from a putative glycogen synthase kinase-3 site in the Drosophila translation initiation factor eIF2B epsilon to investigate the efficacy of residues other than Ser(P) as priming residues for glycogen synthase kinase-3beta and its Drosophila homologue Shaggy. Glycogen synthase kinase-3beta phosphorylated peptides with Ser(P) and Thr(P) in the priming position, but peptides with Tyr(P), Thr, Glu or Asp were not phosphorylated. The Vmax for the Thr(P) peptide was three times higher than that of the Ser(P) peptide. These data suggest that glycogen synthase kinase-3 is unique among phosphate-directed kinases. The priming site specificity of Shaggy is similar to that of mammalian glycogen synthase kinase-3beta. This unpredicted efficacy of Thr(P) in the priming position suggests that there may be other unidentified substrates for these kinases.

Amino Acid Sequence↗
PubMed1999

cAMP inhibits translation by inducing Ca2+/calmodulin-independent elongation factor 2 kinase activity in IPC-81 cells.

Treatment of IPC-81 cells led to inhibition of protein synthesis, which was accompanied by an increase in the average size of polysomes and a decreased rate of elongation, indicating that it involved inhibition of peptide chain elongation. This inhibition was also associated with increased phosphorylation of elongation factor eEF2 (which inhibits its activity) and enhanced Ca2+/calmodulin-independent activity of eEF2 kinase. Previous work has shown that phosphorylation of eEF2 kinase by cAMP-dependent protein kinase (cAPK) in vitro induces such activator-independent activity, and the present data show that such a mechanism can occur in intact cells to link physiological levels of cAPK activation with inhibition of protein synthesis.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1998

Amino acid availability regulates p70 S6 kinase and multiple translation factors.

Incubation of Chinese hamster ovary cells without amino acids for up to 60 min caused a rapid marked decrease in p70 S6 kinase activity and increased binding of initiation factor eIF4E to its inhibitory regulator protein 4E-BP1. This was associated with dephosphorylation of 4E-BP1 and eIF4E and dissociation of eIF4E from eIF4G. All these effects were rapidly reversed by resupplying a mixture of amino acids and this was blocked by rapamycin and by inhibitors of phosphatidylinositol 3-kinase, implying a role for phosphatidylinositol 3-kinase in the signalling pathway linking amino acids with the control of p70 S6 kinase activity and the phosphorylation of these translation factors. Amino acid withdrawal also led to changes in the phosphorylation of other translation factors; phosphorylation of eIF4E decreased whereas elongation factor eEF2 became more heavily phosphorylated, each of these changes being associated with decreased activity of the factor in question. Earlier studies have suggested that protein kinase B (PKB) may act upstream of p70 S6 kinase. However, amino acids did not affect the activity of PKB, indicating that amino acids activate p70 S6 kinase through a pathway independent of this enzyme. Studies with individual amino acids suggested that the effects on p70 S6 kinase activity and translation-factor phosphorylation were independent of cell swelling. The data show that amino acid supply regulates multiple translation factors in mammalian cells.

Amino Acids↗
PubMed1998

The phosphorylation of eukaryotic initiation factor eIF4E in response to phorbol esters, cell stresses, and cytokines is mediated by distinct MAP kinase pathways.

Initiation factor eIF4E binds to the 5'-cap of eukaryotic mRNAs and plays a key role in the mechanism and regulation of translation. It may be regulated through its own phosphorylation and through inhibitory binding proteins (4E-BPs), which modulate its availability for initiation complex assembly. eIF4E phosphorylation is enhanced by phorbol esters. We show, using specific inhibitors, that this involves both the p38 mitogen-activated protein (MAP) kinase and Erk signaling pathways. Cell stresses such as arsenite and anisomycin and the cytokines tumor necrosis factor-alpha and interleukin-1beta also cause increased phosphorylation of eIF4E, which is abolished by the specific p38 MAP kinase inhibitor, SB203580. These changes in eIF4E phosphorylation parallel the activity of the eIF4E kinase, Mnk1. However other stresses such as heat shock, sorbitol, and H2O2, which also stimulate p38 MAP kinase and increase Mnk1 activity, do not increase phosphorylation of eIF4E. The latter stresses increase the binding of eIF4E to 4E-BP1, and we show that this blocks the phosphorylation of eIF4E by Mnk1 in vitro, which may explain the absence of an increase in eIF4E phosphorylation under these conditions.

Animals↗
PubMed1998

Nerve and epidermal growth factor induce protein synthesis and eIF2B activation in PC12 cells.

The regulation of protein synthesis and of eukaryotic initiation factor eIF2B was studied in PC12 cells. An increase in protein synthesis was observed after nerve growth factor (NGF) and epidermal growth factor (EGF) treatment of PC12 cells, and this increase coincided with activation of eIF2B. Growth factor addition in the presence of the phosphatidylinositol-3'-OH kinase inhibitor wortmannin showed that both NGF- and EGF-induced protein synthesis and eIF2B activation were phosphatidylinositol-3'-OH kinase dependent. The EGF-induced stimulation of protein synthesis and activation of eIF2B was dependent upon FK506-binding protein-rapamycin-associated protein, as shown with the immunosuppressant rapamycin, whereas NGF induction was partially dependent upon FK506-binding protein-rapamycin-associated protein. The activities of two kinases that act on eIF2B, glycogen synthase kinase-3 and casein kinase II, were measured to assess their potential roles in the activation of eIF2B in PC12 cells. Inactivation of glycogen synthase kinase-3 was seen in response to both NGF and EGF and this coincided with activation of eIF2B. However, inactivation of glycogen synthase kinase-3 was not rapamycin sensitive, in contrast to the activation of eIF2B. This indicates the involvement of another protein kinase or regulatory mechanism in the eIF2B activation. Both growth factors activated casein kinase II. However, the time course of its activation and its insensitivity to wortmannin and rapamycin suggest that casein kinase II does not play a major regulatory role in eIF2B activation under these conditions.

Androstadienes↗
PubMed1998

Regulation of eukaryotic initiation factor eIF2B: glycogen synthase kinase-3 phosphorylates a conserved serine which undergoes dephosphorylation in response to insulin.

Eukaryotic initiation factor eIF2B catalyses a key regulatory step in mRNA translation. eIF2B and total protein synthesis are acutely activated by insulin, and this requires phosphatidylinositol 3-kinase (PI 3-kinase). The epsilon-subunit of eIF2B is phosphorylated by glycogen synthase kinase-3 (GSK-3), which is inactivated by insulin in a PI 3-kinase-dependent manner. Here we identify the phosphorylation site in eIF2Bepsilon as Ser540 and show that treatment of eIF2B with GSK-3 inhibits its activity. Ser540 is phosphorylated in intact cells and undergoes dephosphorylation in response to insulin. This is blocked by PI 3-kinase inhibitors. Insulin-induced dephosphorylation of this inhibitory site in eIF2B seems likely to be important in the overall activation of translation by this hormone.

Amino Acid Sequence↗
PubMed1998

Activation of microtubule-associated protein kinase (Erk) and p70 S6 kinase by D2 dopamine receptors.

The ability of human and rat D2(short) and D2(long) dopamine receptors to activate microtubule-associated protein (MAP) kinase (Erk1/2) and p70 S6 kinase has been investigated in recombinant cells expressing these receptors. In cells expressing the D2(short) receptor, dopamine activated both enzymes in a transient manner but with very different time courses, with activation of Erk being much quicker. Activation of both enzymes by dopamine was dose-dependent and could be prevented by a range of selective dopamine antagonists. Excellent correlations were observed between the potencies of the antagonists for blocking enzyme activation and their affinities for the D2 dopamine receptor. Activation of Erk and of p70 S6 kinase via the D2 dopamine receptors was prevented by pretreatment of the cells with pertussis toxin, indicating the involvement of G proteins of the Gi or Go family. Inhibitors of phosphatidylinositol 3-kinase (PI 3-kinase) were found to block substantially, but not completely, activation of p70 S6 kinase by dopamine, suggesting the involvement of PI 3-kinase-dependent and -independent signalling pathways in its control by dopamine. p70 S6 kinase activation was completely blocked by rapamycin. In the case of Erk, activation was partially blocked by wortmannin or LY294002, indicating a possible link with PI 3-kinase.

Animals↗
PubMed1997

Heat shock increases the association of binding protein-1 with initiation factor 4E.

The effects of heat shock on the regulation of the cap-binding initiation factor 4E (eIF4E) and its inhibitory binding protein, 4E-BP1, have been examined in Chinese hamster ovary cells and in cardiac myocytes. Heat shock increased the association between eIF4E and 4E-BP1, and this was associated with a dephosphorylation of 4E-BP1. These effects did not appear to be due wholly to decreased activity of the p70 S6 kinase pathway, which is implicated in the control of 4E-BP1, and they were not mediated by the stress-activated p38 microtubule-associated protein kinase pathway. Increased binding of 4E-BP1 to eIF4E correlated with a decrease in the amount of eIF4G which co-purified with the latter. This could account for the previously observed impairment of eIF4F function during heat shock, and, since heat shock protein mRNAs are believed to be relatively cap-independent, could provide a mechanism for the selective up-regulation of the synthesis of heat shock proteins and other stress proteins during heat shock.

Animals↗
PubMed1997

Molecular mechanisms for the control of translation by insulin.

Insulin acutely stimulates protein synthesis in mammalian cells, and this involves activation of the process of mRNA translation. mRNA translation is a complex multi-step process mediated by proteins termed translation factors. Several translation factors are regulated in response to insulin, often as a consequence of changes in their states of phosphorylation. The initiation factor eIF4E binds to the cap structure at the 5'-end of the mRNA and mediates assembly of an initiation-factor complex termed eIF4F. Assembly of this complex can be regulated by eIF4E-binding proteins (4E-BPs), which inhibit eIF4F complex assembly. Insulin induces phosphorylation of the 4E-BPs, resulting in alleviation of the inhibition. This regulatory mechanism is likely to be especially important for the control of the translation of specific mRNAs whose 5'-untranslated regions (5'-UTRs) are rich in secondary structure. Translation of another class of mRNAs, those with 5'-UTRs containing polypyrimidine tracts is also activated by insulin and this, like phosphorylation of the 4E-BPs, appears to involve the rapamycin-sensitive signalling pathway which leads to activation of the 70 kDa ribosomal protein S6 kinase (p70 S6 kinase) and the phosphorylation of the ribosomal protein S6. Overall stimulation of translation may involve activation of initiation factor eIF2B, which is required for all initiation events. This effect is dependent upon phosphatidylinositol 3-kinase and may involve the inactivation of glycogen synthase kinase-3 and consequent dephosphorylation of eIF2B, leading to its activation. Peptide-chain elongation can also be activated by insulin, and this is associated with the dephosphorylation and activation of elongation factor eEF2, probably as a consequence of the insulin-induced reduction in eEF2 kinase activity. Thus multiple signalling pathways acting on different steps in translation are involved in the activation of this process by insulin and lead both to general activation of translation and to the selective regulation of specific mRNAs.

Gene Expression Regulation↗
PubMed1997

p70 S6 kinase is activated by sodium arsenite in adult rat cardiomyocytes: roles for phosphatidylinositol 3-kinase and p38 MAP kinase.

p70 S6 kinase (p70 S6k) is important in regulating a variety of cellular functions including mRNA translation and cell cycle progression and is activated by mitogens and hormones. Unexpectedly, we have found that, in adult rat cardiomyocytes, arsenite, which generally induces stress responses, markedly and rapidly activates p70 S6k. This activation of p70 S6k is completely blocked by rapamycin but only partially prevented by inhibitors of phosphatidylinositol 3-kinase. In trying to delineate the mechanism underlying this effect, we found that arsenite did not activate protein kinase B, JNK or MAP kinase, but did activate p38 MAP kinase in cardiac myocytes. A specific inhibitor of p38 MAP kinase (SB203580) partially attenuated the stimulation of p70 S6k by arsenite. These data indicate that the activation of p70 S6k by arsenite involves p38 MAP kinase and phosphatidylinositol 3-kinase but not PKB.

Animals↗
PubMed1997

Activation of translation initiation factor eIF2B by insulin requires phosphatidyl inositol 3-kinase.

Eukaryotic initiation factor eIF2B mediates a key regulatory step in peptide-chain initiation and is acutely activated by insulin, although, it is not clear how. Inhibitors of phosphatidylinositide 3-kinase blocked activation of eIF2B, although rapamycin, which inhibits the p70 S6 kinase pathway, did not. Furthermore, a dominant negative mutant of PI 3-kinase also prevented activation of eIF2B, while a Sos-mutant, which blocks MAP kinase activation, did not. The data demonstrate that a pathway distinct from MAP and p70 S6 kinases regulates eIF2B. Glycogen synthase kinase-3 (GSK-3) phosphorylates and inactivates eIF2B. In all cases, eIF2B and GSK-3 were regulated reciprocally. Dominant negative PI 3-kinase abolished the insulin-induced inhibition of GSK-3. These data strongly support the hypothesis that insulin activates eIF2B through a signalling pathway involving PI 3-kinase and inhibition of GSK-3.

Androstadienes↗
PubMed1997

Mitogen-activated protein kinases activate the serine/threonine kinases Mnk1 and Mnk2.

Mitogen-activated protein (MAP) kinases bind tightly to many of their physiologically relevant substrates. We have identified a new subfamily of murine serine/threonine kinases, whose members, MAP kinase-interacting kinase 1 (Mnk1) and Mnk2, bind tightly to the growth factor-regulated MAP kinases, Erk1 and Erk2. MNK1, but not Mnk2, also binds strongly to the stress-activated kinase, p38. MNK1 complexes more strongly with inactive than active Erk, implying that Mnk and Erk may dissociate after mitogen stimulation. Erk and p38 phosphorylate MNK1 and Mnk2, which stimulates their in vitro kinase activity toward a substrate, eukaryotic initiation factor-4E (eIF-4E). Initiation factor eIF-4E is a regulatory phosphoprotein whose phosphorylation is increased by insulin in an Erk-dependent manner. In vitro, MNK1 rapidly phosphorylates eIF-4E at the physiologically relevant site, Ser209. In cells, Mnk1 is post-translationally modified and enzymatically activated in response to treatment with either peptide growth factors, phorbol esters, anisomycin or UV. Mitogen- and stress-mediated MNK1 activation is blocked by inhibitors of MAP kinase kinase 1 (Mkk1) and p38, demonstrating that Mnk1 is downstream of multiple MAP kinases. MNK1 may define a convergence point between the growth factor-activated and one of the stress-activated protein kinase cascades and is a candidate to phosphorylate eIF-4E in cells.

3T3 Cells↗
PubMed1997

Regulation of protein kinase B and glycogen synthase kinase-3 by insulin and beta-adrenergic agonists in rat epididymal fat cells. Activation of protein kinase B by wortmannin-sensitive and -insensitive mechanisms.

Previous studies using L6 myotubes have suggested that glycogen synthase kinase-3 (GSK-3) is phosphorylated and inactivated in response to insulin by protein kinase B (PKB, also known as Akt or RAC) (Cross, D. A. E., Alessi, D. R., Cohen, P., Andjelkovic, M., and Hemmings, B. A. (1995) Nature 378, 785-789). In the present study, marked increases in the activity of PKB have been shown to occur in insulin-treated rat epididymal fat cells with a time course compatible with the observed decrease in GSK-3 activity. Isoproterenol, acting primarily through beta3-adrenoreceptors, was found to decrease GSK-3 activity to a similar extent (approximately 50%) to insulin. However, unlike the effect of insulin, the inhibition of GSK by isoproterenol was not found to be sensitive to inhibition by the phosphatidylinositol 3'-kinase inhibitors, wortmannin or LY 294002. The change in GSK-3 activity brought about by isoproterenol could not be mimicked by the addition of permeant cyclic AMP analogues or forskolin to the cells, although at the concentrations used, these agents were able to stimulate lipolysis. Isoproterenol, but again not the cyclic AMP analogues, was found to increase the activity of PKB, although to a lesser extent than insulin. While wortmannin abolished the stimulation of PKB activity by insulin, it was without effect on the activation seen in response to isoproterenol. The activation of PKB by isoproterenol was not accompanied by any detectable change in the electrophoretic mobility of the protein on SDS-polyacrylamide gel electrophoresis. It would therefore appear that distinct mechanisms exist for the stimulation of PKB by insulin and isoproterenol in rat fat cells.

Adipocytes↗
PubMed1997

Peptide substrates suitable for assaying glycogen synthase kinase-3 in crude cell extracts.

In this study we describe the characterization and use of new peptide substrates for assaying glycogen synthase kinase-3 (GSK-3) which are based on the sequence around the single GSK-3 phosphorylation site in the translation factor eIF2B. The new peptides offer important advantages over previous substrates, which were based on the sequence around the multiple GSK-3 phosphorylation sites in glycogen synthase (GS), for the assay of GSK-3 in cell extracts. In particular, decreases in GSK-3 activity following, e.g., insulin treatment, are partially or completely masked when the GS-based peptides are used but are readily measured using the new, eIF2B-based, peptides. The new peptides, unlike those based on GS, are therefore suitable for the assay of changes in GSK-3 activity in cell extracts without the need for prior immunoprecipitation or ion-exchange chromatography.

3T3 Cells↗
PubMed1997

Eukaryotic initiation factor 2B (eIF2B).

Eukaryotic initiation factor 2B (eIF2B) is a guanine nucleotide-exchange factor which mediates the exchange of GDP (bound to initiation factor eIF2) for GTP, thus regenerating the active [eIF2.GTP] complex that is required for peptide-chain initiation. The activity of eIF2B is a key control point for eukaryotic protein synthesis and is altered in response to viral infection, hormones, nutrients, growth factors and certain stresses. It may be regulated directly by its own phosphorylation, phosphorylation of its substrate, eIF2 and by allosteric effectors. Understanding its regulation will lead to a better knowledge of insulin action, regulation of the immune system and of insulin production in the pancreas.

Allosteric Regulation↗
PubMed1996

Cloning of cDNA for the gamma-subunit of mammalian translation initiation factor 2B, the guanine nucleotide-exchange factor for eukaryotic initiation factor 2.

Peptide sequence data were obtained from rabbit protein synthesis initiation factor subunit eIF2B gamma. Searching the database of expressed sequence tags (dbEST) revealed nucleotide sequences potentially encoding human eIF2B gamma that contained peptides corresponding to those from the rabbit subunit. PCR primers were derived from these sequences and used to generate a probe. This was used to screen a rat skeletal muscle cDNA library, and a clone encoding rat eIF2B gamma was isolated. This cDNA gave a product in coupled transcription/translation that co-migrated with the gamma-subunit of purified eIF2B under SDS/PAGE. The sequence of this rat eIF2B gamma cDNA is reported. The protein sequence shows homology with that of yeast eIF2B gamma (the GCD1 gene product). We have also identified an open reading frame from the Caenorhabditis elegans genome project that probably encodes the gamma-subunit of C. elegans eIF2B. All these sequences show similarity to nucleotidyl- and acyltransferases, as previously reported for GCD1 [Koonin (1995) Protein Sci. 4, 1608-1617], and contain conserved motifs potentially involved in nucleotide binding. They also contain "I-patch' motifs: isoleucine-rich hexamer repeats that have been associated with the binding of acyl groups in bacterial acyltransferases. The roles of these motifs are discussed in relation to the known properties of eIF2B.

Amino Acid Sequence↗