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J. Biol. Chem., Vol. 277, Issue 37, 34168-34175, September 13, 2002
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From the Glucokinase (GK) activity is essential for the
physiological regulation of insulin secretion by glucose. Because the
enzyme exerts nearly total control over glucose metabolism in the
A Functional Link between Glucokinase Binding to Insulin Granules
and Conformational Alterations in Response to Glucose and
Insulin*
,,¶
Department of Molecular Physiology and
Biophysics, Vanderbilt University Medical Center, Nashville,
Tennessee 37232 and the § Department of Cell Biology and
Physiology, University of Pittsburgh School of Medicine,
Pittsburgh, Pennsylvania 15261
-cell, even small changes in GK activity exert effects on
glucose-stimulated insulin secretion and, consequently, the blood
glucose concentration. Using quantitative imaging of multicolor
fluorescent proteins fused to GK, we found that the association of GK
with insulin granules is regulated by glucose in the -cell. Glucose
stimulation increased the rate of fluorescence recovery after
photobleaching of GK to insulin granules, indicating that GK is
released into the cytoplasm after glucose stimulation. Changes in
fluorescence resonance energy transfer between two different
fluorescent protein variants inserted on opposing ends of GK were
observed after glucose stimulation and correlated with increased enzyme
activity. Furthermore, glucose-stimulated changes in GK regulation were
blocked by two inhibitors of insulin secretion. Insulin treatment
restored GK regulation in inhibited cells and stimulated GK
translocation and activation by itself. Together, these data support a
model for post-translational regulation of GK whereby insulin regulates both the association of GK with secretory granules and the
activity of the enzyme within the pancreatic -cell.
*
This work was supported by National Institutes of Health
Grants DK60275 (to M. A. R.), DK42612 and DK42502 (both to
M. A. M.), and DK53434 and CA86283 (both to D. W. P.), and by National Science Foundation Grant BBI-9871063 (to D. W. P.).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.
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