Secretagogue-dependent phosphorylation of the insulin granule membrane protein phogrin is mediated by cAMP-dependent protein kinase.
Phogrin, a 60/64-kDa integral membrane protein of dense-core granules in neuroendocrine cells, is phosphorylated in a Ca(2+)-sensitive manner in response to secretagogue stimulation of pancreatic beta-cells. Phosphorylation of the phogrin cytosolic domain by beta-cell homogenates was Ca(2+)-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(+), BaCl(2), and forskolin, demonstrated that the in vivo phosphorylation sites are the same as those identified in vitro. In MIN6 beta-cells, the PKA inhibitor H-89 prevented Ca(2+)-dependent phogrin phosphorylation in response to glucose, suggesting that Ca(2+) exerts its effect on phogrin phosphorylation through regulating the activity of PKA.
Wasmeier
C
C
Barbara Davis Center for Childhood Diabetes, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
Hutton
J C
JC
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D007328:Insulin / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D010766:Phosphorylation; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8; D011994:Recombinant Proteins / Q000378:metabolism; D022142:Secretory Vesicles / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D007328:Insulin; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; D017868:Cyclic AMP-Dependent Protein Kinases; C102889:PTPRN2 protein, human; D017027:Protein Tyrosine Phosphatases; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8; D014357:Trypsin