scholarly journals Glycogen synthase kinase-3 is rapidly inactivated in response to insulin and phosphorylates eukaryotic initiation factor eIF-2B

1993 ◽  
Vol 294 (3) ◽  
pp. 625-629 ◽  
Author(s):  
G I Welsh ◽  
C G Proud
Keyword(s):  
Kinase Activity ◽  
Glycogen Synthase ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Glycogen Synthase Kinase ◽  
Initiation Factor ◽  
Glycogen Synthase Kinase 3 ◽  
Eukaryotic Initiation Factor ◽  
Nucleotide Exchange ◽  
Cell Extracts

We have studied the control of insulin-regulated protein kinases in Chinese hamster ovary cells transfected with the human insulin receptor (CHO.T cells). Among these enzymes is one that is obtained after chromatography of cell extracts on Mono-S, whose activity is decreased (7.3 +/- 1.9-fold) within 10 min of insulin treatment. This enzyme phosphorylates glycogen synthase and the largest subunit of protein synthesis eukaryotic initiation factor (eIF)-2B (the guanine nucleotide exchange factor). The kinase appears to be glycogen synthase kinase-3 (GSK-3), on the basis of: (1) its ability to phosphorylate a peptide based on the phosphorylation sites for GSK-3 in glycogen synthase, and (2) the finding that the fractions possessing this activity contain immunoreactive GSK-3, whose peak is coincident with that of kinase activity, as judged by immunoblotting using antibodies specific for the alpha- and beta-isoforms of GSK-3. The decrease in kinase activity induced by insulin was reversed by treatment of the column fractions with protein phosphatase-2A. These data indicate that insulin rapidly causes inactivation of GSK-3 and that this is due to phosphorylation of GSK-3. The implications of these findings for the control of glycogen and protein metabolism are discussed.

scholarly journals Evidence that the dephosphorylation of Ser535 in the ∊-subunit of eukaryotic initiation factor (eIF) 2B is insufficient for the activation of eIF2B by insulin

2002 ◽  
Vol 367 (2) ◽  
pp. 475-481 ◽  
Author(s):  
Xuemin WANG ◽  
Maarten JANMAAT ◽  
Anne BEUGNET ◽  
Fiona E.M. PAULIN ◽  
Christopher G. PROUD
Keyword(s):  
Protein Synthesis ◽  
Glycogen Synthase ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Initiation Factor ◽  
Eukaryotic Initiation Factor ◽  
Nucleotide Exchange ◽  
Ovary Cells

Eukaryotic initiation factor (eIF) 2B is a guanine-nucleotide exchange factor that plays a key role in the regulation of protein synthesis. It is activated by insulin, serum and other agents that stimulate general protein synthesis. The largest (∊) subunit of eIF2B is a substrate for glycogen synthase kinase (GSK)-3 in vitro, and phosphorylation by GSK3 inhibits the activity of eIF2B. The site of phosphorylation has previously been identified as Ser535. GSK3 is inactivated by phosphorylation in response to insulin or serum. In Chinese-hamster ovary cells, insulin and serum bring about the dephosphorylation of Ser535in vivo, concomitantly with the phosphorylation of GSK3, and these effects are mediated through signalling via phosphoinositide 3-kinase. We have made use of inhibitors of GSK3 to determine whether GSK3 is responsible for phosphorylation of Ser535in vivo and to explore the role of phosphorylation of Ser535 in the regulation of eIF2B. Treatment of cells with LiCl or with either of two recently developed GSK3 inhibitors, SB-415286 and SB-216763, brought about the dephosphorylation of Ser535, which strongly indicates that this site is indeed a target for GSK3 in vivo. However, these compounds did not elicit significant activation of eIF2B, indicating, consistent with conclusions from one of our previous studies, that additional inputs are required for the activation of eIF2B. Our results also show that each of the inhibitors used affects overall protein synthesis and have additional effects on translation factors or signalling pathways apparently unrelated to their effects on GSK3, indicating that caution must be exercised when interpreting data obtained using these compounds.

scholarly journals The kinase DYRK phosphorylates protein-synthesis initiation factor eIF2Bɛ at Ser539 and the microtubule-associated protein tau at Thr212: potential role for DYRK as a glycogen synthase kinase 3-priming kinase

2001 ◽  
Vol 355 (3) ◽  
pp. 609-615 ◽  
Author(s):  
Yvonne L. WOODS ◽  
Philip COHEN ◽  
Walter BECKER ◽  
Ross JAKES ◽  
Michel GOEDERT ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Protein Kinases ◽  
Glycogen Synthase ◽  
Glycogen Synthase Kinase ◽  
Initiation Factor ◽  
Glycogen Synthase Kinase 3 ◽  
General Role ◽  
Protein Tau ◽  
Microtubule Associated Protein Tau ◽  
Protein Synthesis Initiation

The substrate specificity of glycogen synthase kinase 3 (GSK3) is unusual in that efficient phosphorylation only occurs if another phosphoserine or phosphothreonine residue is already present four residues C-terminal to the site of GSK3 phosphorylation. One such substrate is the ε-subunit of rat eukaryotic protein-synthesis initiation factor 2B (eIF2Bε), which is inhibited by the GSK3-catalysed phosphorylation of Ser535. There is evidence that GSK3 is only able to phosphorylate eIF2Bε at Ser535 if Ser539 is already phosphorylated by another protein kinase. However, no protein kinases capable of phosphorylating Ser539 have so far been identified. Here we show that Ser539 of eIF2Bε, which is followed by proline, is phosphorylated specifically by two isoforms of dual-specificity tyrosine phosphorylated and regulated kinase (DYRK2 and DYRK1A), but only weakly or not at all by other ‘proline-directed’ protein kinases tested. We also establish that phosphorylation of Ser539 permits GSK3 to phosphorylate Ser535in vitro and that eIF2Bε is highly phosphorylated at Ser539in vivo. The DYRK isoforms also phosphorylate human microtubule-associated protein tau at Thr212in vitro, a residue that is phosphorylated in foetal tau and hyperphosphorylated in filamentous tau from Alzheimer's-disease brain. Phosphorylation of Thr212 primes tau for phosphorylation by GSK3 at Ser208in vitro, suggesting a more general role for DYRK isoforms in priming phosphorylation of GSK3 substrates.

scholarly journals Inactivation of eukaryotic initiation factor 2B in vitro by heat shock

1998 ◽  
Vol 334 (2) ◽  
pp. 463-467 ◽  
Author(s):  
Gert C. SCHEPER ◽  
Adri A. M. THOMAS ◽  
van Roel WIJK
Keyword(s):  
Heat Shock ◽  
Thermal Inactivation ◽  
Ribosomal Subunit ◽  
Initiation Factor ◽  
Eukaryotic Initiation Factor ◽  
Nucleotide Exchange ◽  
Mild Heat ◽  
Eif2α Phosphorylation ◽  
Cell Extracts

Protein synthesis in rat H35 Reuber hepatoma cells is rapidly inhibited on heat shock. At mild heat-shock temperatures the main cause for inhibition is the inactivation of the guanine nucleotide exchange factor eukaryotic initiation factor 2B (eIF2B); under more severe heat-shock conditions the activity of several initiation factors is compromised. eIF2B is required for GDP/GTP exchange on eIF2, which delivers methionyl-tRNA to the 40 S ribosomal subunit. We have tried to elucidate the mechanism underlying the inactivation of eIF2B by assays in vitro. Incubation of cell extracts at 41 °C or higher led to the inactivation of eIF2B. In agreement with observations in cells exposed to mild heat shocks, the thermal inactivation of eIF2B could be ascribed to neither eIF2α phosphorylation nor the induction of another inhibitor. With the use of glycerol gradients we show that eIF2B forms aggregates in heat-treated extracts. Furthermore eIF2B activity is protected against heat shock in thermotolerant cells. Taken together, these results suggest a role for chaperones in the control of eIF2B activity.

scholarly journals Wortmannin inhibits the effects of insulin and serum on the activities of glycogen synthase kinase-3 and mitogen-activated protein kinase

1994 ◽  
Vol 303 (1) ◽  
pp. 15-20 ◽  
Author(s):  
G I Welsh ◽  
E J Foulstone ◽  
S W Young ◽  
J M Tavaré ◽  
C G Proud
Keyword(s):  
Map Kinase ◽  
Phorbol Ester ◽  
Glycogen Synthase ◽  
Chinese Hamster Ovary Cells ◽  
Glycogen Synthase Kinase ◽  
Glycogen Synthase Kinase 3 ◽  
Parental Cell Line ◽  
S6 Kinase ◽  
Mitogen Activated Protein ◽  
Kinase Pathway

We have previously shown that insulin causes inactivation of glycogen synthase kinase-3 (GSK-3) in Chinese hamster ovary cells over-expressing the human insulin receptor (CHO.T cells). We now show that serum and phorbol ester also cause rapid inactivation of GSK-3, both in CHO.T cells and in the nontransfected parental cell line, CHO.K1 cells. Rapamycin was without effect on the inactivation of GSK-3 by insulin, serum or phorbol ester, indicating that the p70 S6 kinase pathway is not involved. In contrast, wortmannin, a potent inhibitor of phosphatidylinositol 3-kinase, blocked the effects of both insulin and serum on GSK-3 activity, and also substantially reduced the activation of both p90 S6 kinase (by insulin) and mitogen-activated protein (MAP) kinase (by insulin and serum). These findings imply (i) that GSK-3 activity is regulated by a cascade involving MAP kinase and p90 S6 kinase and (ii) that wortmannin affects an early step in the MAP kinase pathway. One can infer from this that GSK-3 may be an important regulatory enzyme for the control of several biosynthetic pathways, key enzymes in which are regulated by GSK-3-mediated phosphorylation. Wortmannin had a smaller effect on the activation of MAP kinase by phorbol ester, indicating that phorbol esters may stimulate MAP kinase by a different or additional mechanism to that employed by insulin or serum. Wortmannin had very little effect on the inactivation of GSK-3 by phorbol ester: possible reasons for this are discussed.

scholarly journals Insulin-Induced Cell Cycle Progression Is Impaired in Chinese Hamster Ovary Cells Overexpressing Insulin Receptor Substrate-3

Endocrinology ◽  
2004 ◽  
Vol 145 (12) ◽  
pp. 5862-5874 ◽  
Author(s):  
Yasushi Kaburagi ◽  
Ryo Yamashita ◽  
Yuzuru Ito ◽  
Hitoshi Okochi ◽  
Ritsuko Yamamoto-Honda ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Insulin Receptor ◽  
Cyclin D1 ◽  
Cell Cycle Progression ◽  
Glycogen Synthase ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Glycogen Synthase Kinase 3 ◽  
Cycle Progression ◽  
Ovary Cells

Abstract To analyze the roles of insulin receptor substrate (IRS) proteins in insulin-stimulated cell cycle progression, we examined the functions of rat IRS-1 and IRS-3 in Chinese hamster ovary cells overexpressing the human insulin receptor. In this type of cell overexpressing IRS-1 or IRS-3, we showed that: 1) overexpression of IRS-3, but not IRS-1, suppressed the G1/S transition induced by insulin; 2) IRS-3 was more preferentially localized to the nucleus than IRS-1; 3) phosphorylation of glycogen synthase kinase 3 and MAPK/ERK was unaffected by IRS-3 overexpression, whereas that of protein kinase B was enhanced by either IRS; 4) overexpressed IRS-3 suppressed cyclin D1 expression in response to insulin; 5) among the signaling molecules regulating cyclin D1 expression, activation of the small G protein Ral was unchanged, whereas insulin-induced gene expression of c-myc, a critical component for growth control and cell cycle progression, was suppressed by overexpressed IRS-3; and 6) insulin-induced expression of p21, a cyclin-dependent kinase inhibitor, was decreased by overexpressed IRS-3. These findings imply that: 1) IRS-3 may play a unique role in mitogenesis by inhibiting insulin-stimulated cell cycle progression via a decrease in cyclin D1 and p21 expressions as well as suppression of c-myc mRNA induction in a manner independent of the activation of MAPK, protein kinase B, glycogen synthase kinase 3 and Ral; and 2) the interaction of IRS-3 with nuclear proteins may be involved in this process.

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