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 Mitogen inactivation of glycogen synthase kinase-3β in intact cells via serine 9 phosphorylation

1994 ◽  
Vol 303 (3) ◽  
pp. 701-704 ◽  
Author(s):  
V Stambolic ◽  
J R Woodgett
Keyword(s):  
Cell Fate ◽  
Glycogen Synthase ◽  
Regulatory Proteins ◽  
Glycogen Synthase Kinase ◽  
Glycogen Synthase Kinase 3 ◽  
Serine Kinase ◽  
Intact Cells ◽  
S6 Kinase ◽  
Mitogen Activated Protein

Glycogen synthase kinase-3 (GSK-3), a protein-serine kinase implicated in cell-fate determination and differentiation, phosphorylates several regulatory proteins that are activated by dephosphorylation in response to hormones or growth factors. GSK-3 beta is phosphorylated in vitro at serine 9 by p70 S6 kinase and p90rsk-1, resulting in its inhibition [Sutherland, Leighton, and Cohen (1993) Biochem. J. 296, 15-19]. Using HeLa cells expressing GSK-3 beta or a mutant containing alanine at residue 9, we demonstrate that serine 9 is modified in intact cells and is targeted specifically by p90rsk-1, and that phosphorylation leads to loss of activity. Since p90rsk-1 is directly activated by mitogen-activated protein kinases, agonists of this pathway, such as insulin, repress GSK-3 function.

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 The inhibition of glycogen synthase kinase-3 by insulin or insulin-like growth factor 1 in the rat skeletal muscle cell line L6 is blocked by wortmannin, but not by rapamycin: evidence that wortmannin blocks activation of the mitogen-activated protein kinase pathway in L6 cells between Ras and Raf

1994 ◽  
Vol 303 (1) ◽  
pp. 21-26 ◽  
Author(s):  
D A E Cross ◽  
D R Alessi ◽  
J R Vandenheede ◽  
H E McDowell ◽  
H S Hundal ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Map Kinase ◽  
Protein Phosphatase ◽  
Glycogen Synthase ◽  
Glycogen Synthase Kinase 3 ◽  
Rat Skeletal Muscle ◽  
S6 Kinase ◽  
P70 S6 Kinase ◽  
Skeletal Muscle Cell Line ◽  
Stimulation Of

Glycogen synthase kinase-3 (GSK3) is inactivated in vitro by p70 S6 kinase or MAP kinase-activated protein kinase-1 beta (MAPKAP kinase-1 beta; also known as Rsk-2). Here we show that GSK3 isoforms are inhibited by 40% within minutes after stimulation of the rat skeletal-muscle cell line L6 with insulin-like growth factor-1 (IGF-1) or insulin. GSK3 was similarly inhibited in rabbit skeletal muscle after an intravenous injection of insulin. Inhibition resulted from increased phosphorylation of GSK3, probably at a serine/threonine residue(s), because it was reversed by incubation with protein phosphatase-2A. Rapamycin blocked the activation of p70 S6 kinase by IGF-1 in L6 cells, but had no effect on the inhibition of GSK3 or the activation of MAPKAP kinase-1 beta. In contrast, wortmannin, a potent inhibitor of PtdIns 3-kinase, prevented the inactivation of GSK3 and the activation of MAPKAP kinase-1 beta and p70 S6 kinase by IGF-1 or insulin. Wortmannin also blocked the activation of p74raf-1. MAP kinase kinase and p42 MAP kinase, but not the formation of GTP-Ras by IGF-1. The results suggest that the stimulation of glycogen synthase by insulin/IGF-1 in skeletal muscle involves the MAP-KAP kinase-1-catalysed inhibition of GSK3, as well as the previously described activation of the glycogen-associated form of protein phosphatase-1.

scholarly journals The mechanism by which epidermal growth factor inhibits glycogen synthase kinase 3 in A431 cells

1994 ◽  
Vol 303 (1) ◽  
pp. 27-31 ◽  
Author(s):  
Y Saito ◽  
J R Vandenheede ◽  
P Cohen
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Glycogen Synthase ◽  
Glycogen Synthase Kinase ◽  
Glycogen Synthase Kinase 3 ◽  
A431 Cells ◽  
S6 Kinase ◽  
P70 S6 Kinase ◽  
Epidermal Growth

Glycogen synthase kinase 3 (GSK3) was inhibited by 50% within 5 min when A431 cells were stimulated with epidermal growth factor (EGF). The inhibition was unaffected by rapamycin at concentrations which blocked the activation of p70 S6 kinase, and reversed by incubation with protein phosphatase-1. EGF stimulation of A431 cells inhibited GSK3 alpha and GSK3 beta to a similar extent, and inhibition was accompanied by phosphorylation of the tryptic peptides containing the serine residues phosphorylated in vitro by p70 S6 kinase or MAP kinase-activated protein (MAPKAP) kinase-1 beta (also termed Rsk-2). These results demonstrate that EGF inhibits GSK3 by inducing phosphorylation of a serine residue and that GSK3 is not phosphorylated in vivo by either p70 S6 kinase or protein kinase C.

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