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.

scholarly journals Functional Effects of PTPN11 (SHP2) Mutations Causing LEOPARD Syndrome on Epidermal Growth Factor-Induced Phosphoinositide 3-Kinase/AKT/Glycogen Synthase Kinase 3β Signaling

2010 ◽  
Vol 30 (10) ◽  
pp. 2498-2507 ◽  
Author(s):  
Thomas Edouard ◽  
Jean-Philippe Combier ◽  
Audrey Nédélec ◽  
Sophie Bel-Vialar ◽  
Mélanie Métrich ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Glycogen Synthase ◽  
Glycogen Synthase Kinase ◽  
Hek293 Cells ◽  
Glycogen Synthase Kinase 3Β ◽  
Leopard Syndrome ◽  
Epidermal Growth ◽  
Gsk 3Β ◽  
Phosphoinositide 3 Kinase

ABSTRACT LEOPARD syndrome (LS), a disorder with multiple developmental abnormalities, is mainly due to mutations that impair the activity of the tyrosine phosphatase SHP2 (PTPN11). How these alterations cause the disease remains unknown. We report here that fibroblasts isolated from LS patients displayed stronger epidermal growth factor (EGF)-induced phosphorylation of both AKT and glycogen synthase kinase 3β (GSK-3β) than fibroblasts from control patients. Similar results were obtained in HEK293 cells expressing LS mutants of SHP2. We found that the GAB1/phosphoinositide 3-kinase (PI3K) complex was more abundant in fibroblasts from LS than control subjects and that both AKT and GSK-3β hyperphosphorylation were prevented by reducing GAB1 expression or by overexpressing a GAB1 mutant unable to bind to PI3K. Consistently, purified recombinant LS mutants failed to dephosphorylate GAB1 PI3K-binding sites. These mutants induced PI3K-dependent increase in cell size in a model of chicken embryo cardiac explants and in transcriptional activity of the atrial natriuretic factor (ANF) gene in neonate rat cardiomyocytes. In conclusion, SHP2 mutations causing LS facilitate EGF-induced PI3K/AKT/GSK-3β stimulation through impaired GAB1 dephosphorylation, resulting in deregulation of a novel signaling pathway that could be involved in LS pathology.

scholarly journals Recruitment of epidermal growth factor and transferrin receptors into coated pits in vitro: differing biochemical requirements.

1993 ◽  
Vol 4 (7) ◽  
pp. 715-727 ◽  
Author(s):  
C Lamaze ◽  
T Baba ◽  
T E Redelmeier ◽  
S L Schmid
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Kinase Inhibitor ◽  
Protein Complexes ◽  
Egf Receptors ◽  
A431 Cells ◽  
Coated Pits ◽  
Morphological Studies ◽  
Epidermal Growth

The biochemical requirements for epidermal growth factor (EGF) and transferrin receptor-mediated endocytosis were compared using perforated human A431 cells. Morphological studies showed that horseradish peroxidase (HRP)-conjugated EGF and gold-labeled antitransferrin (Tfn) receptor antibodies were colocalized during endocytosis in vitro. The sequestration of both ligands into deeply invaginated coated pits required ATP hydrolysis and cytosolic factors and was inhibited by GTP gamma S, indicating mechanistic similarities. Importantly, several differences in the biochemical requirements for sequestration of EGF and Tfn were also detected. These included differing requirements for soluble AP (clathrin assembly protein) complexes, differing cytosolic requirements, and differing sensitivities to the tyrosine kinase inhibitor, genistein. The biochemical differences detected between EGF and Tfn sequestration most likely reflect specific requirements for the recruitment of EGF-receptors (R) into coated pits. This assay provides a novel means to identify the molecular bases for these biochemical distinctions and to elucidate the mechanisms involved in ligand-induced recruitment of EGF-R into coated pits.

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 Inactivation of glycogen synthase kinase-3β by phosphorylation: new kinase connections in insulin and growth-factor signalling

1993 ◽  
Vol 296 (1) ◽  
pp. 15-19 ◽  
Author(s):  
C Sutherland ◽  
I A Leighton ◽  
P Cohen
Keyword(s):  
Growth Factor ◽  
Protein Kinase ◽  
Glycogen Synthase ◽  
Inhibitory Effect ◽  
Glycogen Synthase Kinase ◽  
Glycogen Synthase Kinase 3 ◽  
Beta Activity ◽  
S6 Kinase ◽  
Growth Factor Signalling ◽  
Phosphatase 2A

The beta-isoform of glycogen synthase kinase-3 (GSK3 beta) isolated from rabbit skeletal muscle was inactivated 90-95% following incubation with MgATP and either MAP kinase-activated protein kinase-1 (MAPKAP kinase-1, also termed RSK-2) or p70 S6 kinase (p70S6K), and re-activated with protein phosphatase 2A. MAPKAP kinase-1 and p70S6K phosphorylated the same tryptic peptide on GSK3 beta, and the site of phosphorylation was identified as the serine located nine residues from the N-terminus of the protein. The inhibitory effect of Ser-9 phosphorylation on GSK3 beta activity was observed with three substrates, (inhibitor-2, c-jun and a synthetic peptide), and also with glycogen synthase provided that 0.15 M KCl was added to the assays. The results suggest that Ser-9 phosphorylation underlies the reported inhibition of GSK3 beta by insulin and that GSK3 may represent a point of convergence of two major growth-factor-stimulated protein kinase cascades.

Processing of epidermal growth factor by suckling and adult rat intestinal cells

1986 ◽  
Vol 250 (6) ◽  
pp. G850-G855 ◽  
Author(s):  
R. K. Rao ◽  
W. Thornburg ◽  
M. Korc ◽  
L. M. Matrisian ◽  
B. E. Magun ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Oral Feeding ◽  
Intestinal Cells ◽  
A431 Cells ◽  
Adult Rats ◽  
Suckling Rats ◽  
Epidermal Growth

Preparations of intestinal villus and crypt cells were isolated from jejunal segments of suckling (14-day-old) and adult (6- to 7-wk-old) rats. These cell preparations were incubated with 125I-labeled epidermal growth factor (EGF) at 37 degrees C to determine the extent of cellular processing of 125I-EGF in vitro. 125I-EGF bound specifically to both crypt and villus cells of suckling rats and was internalized and degraded to similar extents in both cell preparations. Analysis of the 125I radioactivity in the medium and cellular extract by gel filtration on Sephadex G-25 columns demonstrated the presence of [125I]iodotyrosine (24–31%) following 30 min of incubation. This degradation of EGF was accompanied by a loss in the capacity to bind to anti-EGF antibodies (34–52%) and A431 cells (28–48%). Binding, internalization, and processing of 125I-EGF by crypt cell preparations of adult rats was similar to that of suckling rats. In contrast, little degradation of 125I-EGF to iodotyrosine and loss of cell binding capability occurred following incubation with adult villus cells. However, a considerable loss in binding to anti-EGF antibody was detected (48%). The results indicate that isolated intestinal cells are capable of degrading 125I-EGF in vitro in a manner similar to that seen after oral feeding in vivo. They also indicate differences in the processing of 125I-EGF by isolated villus cells of adult compared with suckling rat.

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