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J. Biol. Chem., Vol. 278, Issue 20, 18177-18183, May 16, 2003
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From the Departments of The role of elongation factor (EF)-2
phosphorylation in the regulation of pancreatic
Glucose Regulates EF-2 Phosphorylation and Protein
Translation by a Protein Phosphatase-2A-dependent Mechanism
in INS-1-derived 832/13 Cells*
,
Medicine and
¶ Neurobiology, Pharmacology and Physiology, University of
Chicago, Chicago, Illinois 60637 and § Department of
Psychiatry, Yale University School of Medicine, New Haven,
Connecticut 06508
-cell protein
synthesis by glucose was investigated in the INS-1-derived cell line
832/13. Incubation of cells in media containing 1 mM
glucose resulted in a progressive increase in EF-2 phosphorylation that
was maximal by 1-2 h. Readdition of 10 mM glucose promoted
a rapid dephosphorylation of EF-2 that was complete in 10 min and
maintained over the ensuing 2 h. Similar results were obtained
using primary rat islets or Min-6 insulinoma cells. The glucose effect
in 832/13 cells was replicated by addition of pyruvate or
-ketocaproate, but not 2-deoxyglucose, suggesting that mitochondrial
metabolism was required. Accordingly, glucose-mediated dephosphorylation of EF-2 was completely blocked by the mitochondrial respiratory antagonists antimycin A and oligomycin. The hyperglycemic effect was not mimicked by incubation of cells in 100 nM
insulin, 30 mM potassium chloride, or 0.25 mM
diazoxide, indicating that insulin secretion and/or depolarization of
cells was not required. The locus of the high glucose effect
appeared to be protein phosphatase-2A, the principal phosphatase acting
on EF-2. Protein phosphatase-2A activity was stimulated by glucose
addition to 832/13 cells, but neither protein phosphatase-1 nor
calmodulin kinase III (EF-2 kinase) activity was affected under these
conditions. The slower rephosphorylation of EF-2 during the transition
from high to low glucose may involve effects on EF-2 kinase activity.
Addition of 5-aminoimidazole-4-carboxamide
1--D-ribofuranoside in high glucose led to a marked
stimulation of EF-2 phosphorylation, consistent with the possibility
that increased AMP kinase activity in low glucose stimulates EF-2
kinase. In parallel with the effects on EF-2 dephosphorylation,
addition of high glucose to 832/13 cells markedly increased the
incorporation of [35S]methionine into total protein.
Taken together, these results suggest that modulation of
extracellular glucose impacts protein translation rate in cells at
least in part through regulation of the elongation step, via
phosphorylation/dephosphorylation of EF-2.
*
This work was supported by a Career Development Award (to
M. J. B.) and a Research Award (to H. C. P.) from the American
Diabetes Association.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Medicine,
University of Chicago, MC1027, 5841 S. Maryland Ave., Chicago, IL
60637. Tel.: 773-702-2346; Fax: 773-834-0486; E-mail:
mbrady@medicine.bsd.uchicago.edu.
Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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