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J. Biol. Chem., Vol. 276, Issue 34, 31919-31928, August 24, 2001
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From the Barbara Davis Center for Childhood Diabetes, University of
Colorado Health Sciences Center, Denver, Colorado 80262
Phogrin, a 60/64-kDa integral membrane protein of
dense-core granules in neuroendocrine cells, is phosphorylated in a
Ca2+-sensitive manner in response to secretagogue
stimulation of pancreatic
Secretagogue-dependent Phosphorylation of
the Insulin Granule Membrane Protein Phogrin Is Mediated by
cAMP-dependent Protein Kinase*
and
-cells. Phosphorylation of the phogrin
cytosolic domain by -cell homogenates was
Ca2+-independent but stimulated by cAMP. Recombinant
protein kinase A (PKA) could phosphorylate phogrin directly. High
performance liquid chromatography analysis of tryptic phosphopeptides,
combined with site-directed mutagenesis of candidate sites, revealed
the presence of two phosphorylation sites at Ser-680 and Thr-699, located in the juxtamembrane region between the transmembrane span and
the protein-tyrosine phosphatase homology domain of phogrin. Full-length wild-type phogrin, as well as mutant versions where Ser-680
and Thr-699 had been replaced either by alanines or by aspartic acid
residues, were targeted to secretory granules in transfected AtT20
neuroendocrine cells. Stimulation of these cells with a range of
secretagogues, including K+, BaCl2, and
forskolin, demonstrated that the in vivo phosphorylation sites are the same as those identified in vitro. In MIN6
-cells, the PKA inhibitor H-89 prevented
Ca2+-dependent phogrin phosphorylation in
response to glucose, suggesting that Ca2+ exerts its
effect on phogrin phosphorylation through regulating the
activity of PKA.
*
This work was supported in part by National Institutes of
Health Grants DK 55597-02 (RO1), DK 57516-02 (P30) and DK 52068-02 (RO1).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.
Recipient of a Juvenile Diabetes Foundation International
postdoctoral fellowship.
§
To whom correspondence should be addressed: Barbara Davis Center
for Childhood Diabetes, University of Colorado Health Sciences Center,
4200 E. 9th Ave., Denver, CO 80262. Tel.: 303-315-8197; Fax:
303-315-4892; E-mail: john.hutton@uchsc.edu.
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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