Biomedical subjects

C G Proud

Publications and source records attributed to C G Proud.

At least 37 records · Page 2Linked to original sources

PubMed2001

Staurosporine inhibits phosphorylation of translational regulators linked to mTOR.

Treatment of Swiss 3T3 cells with staurosporine resulted in dephosphorylation of two proteins which play key roles in regulating mRNA translation. This occurred before the execution of apoptosis, assessed by caspase-3 activity. These translation regulators are p70 S6 kinase, which phosphorylates ribosomal protein S6, and eukaryotic initiation factor (eIF) 4E binding protein 1 (4E-BP1), which both lie downstream of the mammalian target of rapamycin (mTOR). This resulted in decreased p70 S6 kinase activity, dephosphorylation of ribosomal protein S6, increased binding of 4E-BP1 to eIF4E and a concomitant decrease in eIF4F complexes. Our data show that staurosporine impairs mTOR signalling in vivo but that this not due to direct inhibition of mTOR or to inhibition of protein kinase C. It is becoming clear that agents which cause apoptosis inactivate mTOR signalling as a common early response prior to the execution of apoptosis, i.e., before caspase activation.

3T3 Cells↗

PubMed2001

Regulation of mRNA translation.

The control of mRNA translation plays an important role in regulating gene expression in diverse situations. Nutrients, especially amino acids, regulate translation to control the expression of specific proteins including transcriptional activators and ribosomal proteins. Sequence elements in the 5'- or 3'-untranslated regions of mRNAs can control their translation. Hormones such as insulin activate protein synthesis by switching on translation factors required for overall mRNA translation. Viruses employ a range of stratagems to ensure efficient translation of their mRNAs and in some cases to inhibit host cell translation. During development, control of mRNA translation regulates gene expression both spatially and temporally.

Animals↗

PubMed2001

Interplay between insulin and nutrients in the regulation of translation factors.

Protein synthesis in mammalian cells is regulated through alterations in the states of phosphorylation of eukaryotic initiation factors and elongation factors (eIFs and eEFs respectively) and of other regulatory proteins. This modulates their activities or their abilities to interact with one another. Insulin activates several of these proteins including the following: the guanine-nucleotide exchange factor eIF2B; the eIF4F complex, which (through eIF4E) interacts with the cap of the mRNA; p70 S6 kinase; and elongation factor eEF2, which mediates the translocation step of elongation. Control of the last three of these is linked to mTOR (mammalian target of rapamycin). In Chinese hamster ovary cells, regulation of all these proteins by insulin is modulated by the presence of amino acids and/or glucose in the medium. For example, p70 S6 kinase activity declines in the absence of amino acids and cannot be stimulated by insulin under this condition. The readdition of amino acids, especially leucine, restores activity and sensitivity to insulin. With eIF2B and eEF2, both amino acids and glucose must be provided for insulin to regulate their activities. In contrast, insulin-stimulation of the formation of eIF4F complexes requires glucose but not amino acids. Glucose metabolism is required for this permissive effect. Our recent studies have also identified the mechanism by which mTOR signalling regulates the phosphorylation of eEF2. eEF2 kinase is phosphorylated by p70 S6 kinase at Ser-366; this results in the inactivation of eEF2 kinase, especially at low (micromolar) Ca concentrations.

Adaptor Proteins, Signal Transducing↗

PubMed2001

Changes in the phosphorylation of initiation factor eIF-2alpha, elongation factor eEF-2 and p70 S6 kinase after transient focal cerebral ischaemia in mice.

Mice were subjected to 60 min occlusion of the left middle cerebral artery (MCA) followed by 1-6 h of reperfusion. Tissue samples were taken from the MCA territory of both hemispheres to analyse ischaemia-induced changes in the phosphorylation of the initiation factor eIF-2alpha, the elongation factor eEF-2 and p70 S6 kinase by western blot analysis. Tissue sections from additional animals were taken to evaluate ischaemia-induced changes in global protein synthesis by autoradiography and changes in eIF-2alpha phosphorylation by immunohistochemistry. Transient MCA occlusion induced a persistent suppression of protein synthesis. Phosphorylation of eIF-2alpha was slightly increased during ischaemia, it was markedly up-regulated after 1 h of reperfusion and it normalized after 6 h of recirculation despite ongoing suppression of protein synthesis. Similar changes in eIF-2alpha phosphorylation were induced in primary neuronal cell cultures by blocking of endoplasmic reticulum (ER) calcium pump, suggesting that disturbances of ER calcium homeostasis may play a role in ischaemia-induced changes in eIF-2alpha phosphorylation. Dephosphorylation of eIF-2alpha was not paralleled by a rise in levels of p67, a glycoprotein that protects eIF-2alpha from phosphorylation, even in the presence of active eIF-2alpha kinase. Phosphorylation of eEF-2 rose moderately during ischaemia, but returned to control levels after 1 h of reperfusion and declined markedly below control levels after 3 and 6 h of recirculation. In contrast to the only short-lasting phosphorylation of eIF-2a and eEF-2, transient focal ischaemia induced a long-lasting dephosphorylation of p70 S6 kinase. The results suggest that blocking of elongation does not play a major role in suppression of protein synthesis induced by transient focal cerebral ischaemia. Investigating the factors involved in ischaemia-induced suppression of the initiation step of protein synthesis and identifying the underlying mechanisms may help to further elucidate those disturbances directly related to the pathological process triggered by transient cerebral ischaemia and leading to neuronal cell injury.

Animals↗

PubMed2001

The mitogen-activated protein kinase signal-integrating kinase Mnk2 is a eukaryotic initiation factor 4E kinase with high levels of basal activity in mammalian cells.

The cap-binding translation initiation factor eukaryotic initiation factor 4E (eIF4E) is phosphorylated in vivo at Ser209 in response to a variety of stimuli. In this paper, we show that the mitogen-activated protein kinase (MAPK) signal-integrating kinase Mnk2 phosphorylates eIF4E at this residue. Mnk2 binds to the scaffolding protein eIF4G, and overexpression of Mnk2 results in increased phosphorylation of endogenous eIF4E, showing that it can act as an eIF4E kinase in vivo. We have identified eight phosphorylation sites in Mnk2, of which at least three potential MAPK sites are likely to be essential for Mnk2 activity. In contrast to that of Mnk1, the activity of overexpressed Mnk2 is high under control conditions and could only be reduced substantially by a combination of PD98059 and SB203580, while the activity of endogenous Mnk2 in Swiss 3T3 cells was hardly affected upon treatment with these inhibitors. These compounds did not abolish phosphorylation of eIF4E, implying that Mnk2 may mediate phosphorylation of eIF4E in Swiss 3T3 cells. In vitro phosphorylation studies show that Mnk2 is a significantly better substrate than Mnk1 for extracellular signal-regulated kinase 2 (ERK2), p38MAPKalpha, and p38MAPKbeta. Therefore, the high levels of activity of Mnk2 under several conditions may be explained by efficient activation of Mnk2 by low levels of activity of the upstream kinases. Interestingly, we found that the association of both Mnk1 and Mnk2 with eIF4G increased upon inhibition of the MAPK pathways while activation of ERK resulted in decreased binding to eIF4G. This might reflect a mechanism to ensure rapid, but transient, phosphorylation of eIF4E upon stimulation of the MAPK pathways.

3T3 Cells↗

PubMed2000

ABC50 interacts with eukaryotic initiation factor 2 and associates with the ribosome in an ATP-dependent manner.

Eukaryotic initiation factor 2 (eIF2) plays a key role in the process of translation initiation and in its control. Here we demonstrate that highly purified mammalian eIF2 contains an additional polypeptide of apparent molecular mass of 110 kDa. This polypeptide co-purified with eIF2 through five different chromatography procedures. A cDNA clone encoding the polypeptide was isolated, and its sequence closely matched that of a protein previously termed ABC50, a member of the ATP-binding cassette (ABC) family of proteins. Antibodies to ABC50 co-immunoprecipitated eIF2 and vice versa, indicating that the two proteins interact. The presence of ABC50 had no effect upon the ability of eIF2 to bind GDP but markedly enhanced the association of methionyl-tRNA with the factor. Unlike the majority of ABC proteins, which are membrane-associated transporters, ABC50 associates with the ribosome and co-sediments in sucrose gradients with the 40 and 60 S ribosomal subunits. The association of ABC50 with ribosomal subunits was increased by ATP and decreased by ADP. ABC50 is related to GCN20 and eEF3, two yeast ABC proteins that are not membrane-associated transporters and are instead implicated in mRNA translation and/or its control. Thus, these data identify ABC50 as a third ABC protein with a likely function in mRNA translation, which associates with eIF2 and with ribosomes.

ATP-Binding Cassette Transporters↗

PubMed2000

Characterization of the initiation factor eIF2B and its regulation in Drosophila melanogaster.

Eukaryotic initiation factor (eIF) 2B catalyzes a key regulatory step in the initiation of mRNA translation. eIF2B is well characterized in mammals and in yeast, although little is known about it in other eukaryotes. eIF2B is a hetropentamer which mediates the exchange of GDP for GTP on eIF2. In mammals and yeast, its activity is regulated by phosphorylation of eIF2alpha. Here we have cloned Drosophila melanogaster cDNAs encoding polypeptides showing substantial similarity to eIF2B subunits from yeast and mammals. They also exhibit the other conserved features of these proteins. D. melanogaster eIF2Balpha confers regulation of eIF2B function in yeast, while eIF2Bepsilon shows guanine nucleotide exchange activity. In common with mammalian eIF2Bepsilon, D. melanogaster eIF2Bepsilon is phosphorylated by glycogen synthase kinase-3 and casein kinase II. Phosphorylation of partially purified D. melanogaster eIF2B by glycogen synthase kinase-3 inhibits its activity. Extracts of D. melanogaster S2 Schneider cells display eIF2B activity, which is inhibited by phosphorylation of eIF2alpha, showing the insect factor is regulated similarly to eIF2B from other species. In S2 cells, serum starvation increases eIF2alpha phosphorylation, which correlates with inhibition of eIF2B, and both effects are reversed by serum treatment. This shows that eIF2alpha phosphorylation and eIF2B activity are under dynamic regulation by serum. eIF2alpha phosphorylation is also increased by endoplasmic reticulum stress in S2 cells. These are the first data concerning the structure, function or control of eIF2B from D. melanogaster.

Animals↗

PubMed2000

The activation of eukaryotic initiation factor (eIF)2B by growth factors in PC12 cells requires MEK/ERK signalling.

Epidermal and nerve growth factors (EGF and NGF) activate protein synthesis and initiation factor eIF2B in rat phaeochromocytoma (PC12) cells. The activation of protein synthesis by EGF or NGF depends upon extracellular regulated kinase kinase (MEK)/extracellular regulated kinase signalling. Here we show that PD98059, an inhibitor of MEK activation, blocks the activation of eIF2B by EGF or NGF. It is known that eIF2B activity can be inhibited by phosphorylation at Ser535 in its epsilon-subunit by glycogen synthase kinase (GSK)-3. We find that inactivation of GSK-3 by EGF or NGF is blocked by PD98059. However, neither EGF nor NGF caused a detectable change in phosphorylation of Ser535 of eIF2Bepsilon. Thus, the EGF- and NGF-induced activation of eIF2B in PC12 cells involves regulatory mechanisms distinct from dephosphorylation of the GSK-3 site.

Animals↗

PubMed2000

Rapid induction of apoptosis mediated by peptides that bind initiation factor eIF4E.

Overexpression of the translation initiation factor eIF4E leads to cell transformation and occurs in a number of human cancers [1]. mRNA translation and cell growth can be regulated through the availability of eIF4E to form initiation complexes by binding to eIF4G. The availability of eIF4E is blocked through the binding of members of a family of eIF4E-binding proteins (4E-BPs) [2] [3]. Indeed, cell transformation caused by the overexpression of eIF4E can be reversed by the overexpression of 4E-BPs [4] [5] [6] [7] [8]. To study the role of eIF4E in cell transformation, we developed a series of peptides based on the conserved eIF4E-binding motifs in 4E-BPs and eIF4G [9] linked to the penetratin peptide-carrier sequence, which mediates the rapid transport of peptides across cell membranes. Surprisingly, introduction of these eIF4E-binding peptides into MRC5 cells led to rapid, dose-dependent cell death, with characteristics of apoptosis. Single alanine substitutions at key positions in the peptides impair their binding to eIF4E and markedly reduce their ability to induce apoptosis. A triple alanine substitution, which abolishes binding to eIF4E, renders the peptide unable to induce apoptosis. Our data provide strong evidence that the peptides induce apoptosis through binding to eIF4E. They do not induce apoptosis through inhibition of protein synthesis, as chemical inhibitors of translation did not induce apoptosis or affect peptide-induced cell death. Thus these new data indicate that eIF4E has a direct role in controlling cell survival that is not linked to its known role in mRNA translation.

Adaptor Proteins, Signal Transducing↗

PubMed2000

DNA-damaging agents cause inactivation of translational regulators linked to mTOR signalling.

Treatment of cells with DNA-damaging agents, such as etoposide, can cause growth arrest or apoptosis. Treatment of Swiss 3T3 or RAT-1 cells with etoposide led to the dephosphorylation of both p70 S6 kinase and eukaryotic initiation factor (eIF) 4E-binding protein 1 (4E-BP1), resulting in decreased p70 S6 kinase activity and an increase in 4E-BP1 binding to eIF4E. These effects were not prevented by the general caspase inhibitor, Z-VAD.FMK. These findings indicate caspase-independent inhibition of signalling pathways that involve the mammalian target of rapamycin (mTOR). Similar effects were observed in response to two other DNA-damaging agents, cisplatin and mitomycin-C. These events preceded apoptosis, which was assessed by caspase-3 activity assays and FACS analysis. This shows that inhibition of mTOR signalling is not a consequence of apoptosis, although it may play a role in the events that precede cell death. 4E-BP1 was cleaved during apoptosis yielding a fragment that retained the ability to bind eIF4E. Cleavage of 4E-BP1 was inhibited by treatment of the cells with Z-VAD.FMK, indicating it is caspase-dependent. Insulin elicited full activation of p70 S6 kinase and phosphorylation of 4E-PB1 in etoposide-treated cells prior to the onset of apoptosis, but not during cell death. This suggests that mTOR signalling becomes irreversibly inhibited only after entry into apoptosis. Oncogene (2000).

3T3 Cells↗

PubMed2000

Glucose and amino acids modulate translation factor activation by growth factors in PC12 cells.

In PC12 phaeochromocytoma cells, protein synthesis is activated by epidermal and nerve growth factors (EGF and NGF). EGF and NGF also regulate a number of components of the translational machinery in these cells. Here we show that the ability of EGF and NGF to induce the phosphorylation of the 70 kDa ribosomal protein, S6 kinase, and the eukaryotic initiation factor (eIF), 4E-binding protein 1, is dependent upon the presence of amino acids (but not glucose) in the medium. This resembles the regulation of these proteins by insulin, which also requires amino acids. Glucose, but not amino acids, is required for the activation of eIF2B by EGF and NGF. In contrast, EGF and NGF can still activate protein synthesis in the absence of nutrients, suggesting that other regulatory events are important in this. In nutrient-deprived cells, an increase in the phosphorylation of eIF4E, and the assembly of the eIF4F complex by EGF and NGF, coincided with the activation of protein synthesis. In serum-starved cells, activation of protein synthesis, phosphorylation of eIF4E, and formation of the eIF4F complex, were blocked by inhibition of MEK, a component of the extracellular regulated kinase (ERK) signalling pathway. Thus the ERK pathway plays a key role in the regulation of protein synthesis in PC12 cells.

Amino Acids↗

PubMed2000

Distinct signalling pathways mediate insulin and phorbol ester-stimulated eukaryotic initiation factor 4F assembly and protein synthesis in HEK 293 cells.

Stimulation of serum-starved human embryonic kidney (HEK) 293 cells with either the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), or insulin resulted in increases in the phosphorylation of 4E-BP1 and p70 S6 kinase, eIF4F assembly, and protein synthesis. All these effects were blocked by rapamycin, a specific inhibitor of mTOR. Phosphatidylinositol 3-kinase and protein kinase B were activated by insulin but not by TPA. Therefore TPA can induce eIF4F assembly, protein synthesis, and the phosphorylation of p70 S6 kinase and 4E-BP1 independently of both phosphatidylinositol 3-kinase and protein kinase B. Using two structurally unrelated inhibitors of MEK (PD098059 and U0126), we provide evidence that Erk activation is important in TPA stimulation of eIF4F assembly and the phosphorylation of p70 S6 kinase and 4E-BP1 and that basal MEK activity is important for basal, insulin, and TPA-stimulated protein synthesis. Transient transfection of constitutively active mitogen-activated protein kinase interacting kinase 1 (the eIF4E kinase) indicated that inhibition of protein synthesis and eIF4F assembly by PD098059 is not through inhibition of eIF4E phosphorylation but of other signals emanating from MEK. This report also provides evidence that increased eIF4E phosphorylation alone does not affect the assembly of the eIF4F complex or general protein synthesis.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Detailed analysis of the phosphorylation of the human La (SS-B) autoantigen. (De)phosphorylation does not affect its subcellular distribution.

The La (SS-B) autoantigen is an evolutionarily conserved phosphoprotein which plays an important role, most likely as an RNA chaperone, in various processes, such as the biosynthesis and maturation of RNA polymerase III transcripts in the cell nucleus and (internal) initiation of translation in the cytoplasm. In this study, the phosphorylation state of this protein from human HeLa and HEp-2 cells was characterized by high-resolution two-dimensional IEF/SDS-PAGE analysis, and phosphorylation sites were mapped by nanoelectrospray mass spectrometry. Furthermore, the effect of phosphorylation at the sites identified on the subcellular distribution of the protein was studied by site-directed mutagenesis. At least 14 isoelectric isoforms were discerned on 2-D gels with La protein from both types of cells. Metabolic labeling in combination with alkaline phosphatase treatment revealed that only a limited number of these isoforms could be attributed to phosphorylation. Four phosphorylation sites, Thr-302, Ser-325, Thr-362, and Ser-366, were mapped by mass spectrometric analysis of the isolated La protein from HeLa cells or the carboxy-terminal half of this protein. The analysis of mutants of La, in which the respective phosphorylated residues were replaced by either a neutral (alanine) or an acidic (aspartate) residue, by microinjection into Xenopus laevis oocytes on the one hand and transfection of HEp-2 cells on the other hand revealed that the subcellular distribution of this protein was not affected by these amino acid substitutions. These results strongly suggest that the signals that determine the subcellular distribution of this protein are not regulated by (de)phosphorylation of the target residues examined.

Amino Acid Sequence↗

PubMed2000

Activation of mRNA translation in rat cardiac myocytes by insulin involves multiple rapamycin-sensitive steps.

Insulin acutely activates protein synthesis in ventricular cardiomyocytes from adult rats. In this study, we have established the methodology for studying the regulation of the signaling pathways and translation factors that may be involved in this response and have examined the effects of acute insulin treatment on them. Insulin rapidly activated the 70-kDa ribosomal S6 kinase (p70 S6k), and this effect was inhibited both by rapamycin and by inhibitors of phosphatidylinositol 3-kinase. The activation of p70 S6k is mediated by a signaling pathway involving the mammalian target of rapamycin (mTOR), which also modulates other translation factors. These include the eukaryotic initiation factor (eIF) 4E binding proteins (4E-BPs) and eukaryotic elongation factor 2 (eEF2). Insulin caused phosphorylation of 4E-BP1 and induced its dissociation from eIF4E, and these effects were also blocked by rapamycin. Concomitant with this, insulin increased the binding of eIF4E to eIF4G. Insulin also activated protein kinase B (PKB), which may lie upstream of p70 S6k and 4E-BP1, with the activation of the different isoforms being in the order alpha>beta>gamma. Insulin also caused inhibition of glycogen synthase kinase 3, which lies downstream of PKB, and of eEF2 kinase. The phosphorylation of eEF2 itself was also decreased by insulin, and this effect and the inactivation of eEF2 kinase were attenuated by rapamycin. The activation of overall protein synthesis by insulin in cardiomyocytes was substantially inhibited by rapamycin (but not by inhibitors of other specific signaling pathways, e.g., mitogen-activated protein kinase), showing that signaling events linked to mTOR play a major role in the control of translation by insulin in this cell type.

Age Factors↗

PubMed1999

Nutrients differentially regulate multiple translation factors and their control by insulin.

Eukaryotic initiation factor eIF2B and eukaryotic elongation factor eEF2 each mediate regulatory steps important for the overall regulation of mRNA translation in mammalian cells and are activated by insulin. Here, we demonstrate that their activation by insulin requires the presence, in the medium in which the cells are maintained, of both amino acids and glucose: insulin only induced activation of eIF2B and the dephosphorylation of eEF2 when cells were exposed to both types of nutrient. Other translational regulators, e.g. the 70 kDa ribosomal protein S6 kinase (p70 S6 kinase) and the eIF4E binding protein 1, 4E-BP1, are also regulated by insulin but their control does not require glucose, only amino acids. The effects of nutrients on the activation of eIF2B do not reflect changes in the phosphorylation of eIF2 (and, by inference, operation of a kinase analogous to yeast Gcn2p), or a requirement for nutrients for inactivation of glycogen synthase kinase-3 or dephosphorylation of eIF2B. Nutrients did not affect the ability of insulin to activate protein kinase B. These data show that activation by insulin of p70 S6 kinase, which modulates the translation of specific mRNAs, depends on the availability of amino acids whereas regulation of factors involved in overall activation of translation (eIF2B, eEF2) requires both amino acids and glucose. These results add substantially to the emerging evidence that nutrients themselves modulate functions of mammalian cells and indicate that (i) nutrients modulate the activation of eIF2B and eEF2 through as-yet unidentified mechanisms and (ii) regulation of p70 S6 kinase and 4E-BP1 by insulin requires other inputs in addition to protein kinase B.

Amino Acids↗

PubMed1999

Rapamycin-resistant phosphorylation of the initiation factor-4E-binding protein (4E-BP1) in v-SRC-transformed hamster fibroblasts.

Increased phosphorylation of the translational repressor protein 4E-BP1 was found in the cell line derived from the tumor induced in Syrian hamster by Rous sarcoma virus (RSV). This was accompanied by its dissociation from the complex with initiation factor eIF4E. The ribosomal S6 protein kinase p70S6k is supposed to be regulated by the same or a closely related rapamycin-sensitive signalling pathway to that which modulates 4E-BP1. Phosphorylation and activity of p70S6k were found to be also increased in RSV-transformed H19 cells that express significantly higher amounts of the Src protein (p60src) relative to the non-transformed hamster fibroblasts NIL-2. The increased activity and phosphorylation of p70S6k were blocked by rapamycin, indicating that the rapamycin-sensitive pathway is involved in its regulation in v-src-transformed hamster fibroblasts. In agreement with this, rapamycin reduced the expression of elongation factor eEF1alpha (whose translation is regulated by a rapamycin-sensitive mechanism thought to involve p70S6k) and did not affect the production of a housekeeping protein, alpha-tubulin, in these cells. Synthesis of Src protein was also inhibited in cells treated with rapamycin. However, treatment of cells with a concentration of rapamycin sufficient to completely inhibit the activity and phosphorylation of p70S6k resulted in only partial de-phosphorylation of 4E-BP1 and its re-association with eIF4E in the transformed cells, indicating that additional rapamycin-insensitive mechanisms/pathways are implicated in the control of 4E-BP1 phosphorylation in RSV-transformed hamster fibroblasts. Over-expression of eIF4E favours cell proliferation and can lead to a transformed phenotype, while over-expression of 4E-BP1 has the opposite effect. The altered signalling to the phosphorylation of 4E-BP1 in RSV-transformed cells, which leads to its dissociation from eIF4E and thus relief of inhibition of eIF4E function, may therefore represent an important regulatory mechanism in malignant cell growth.

Animals↗

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↗