Regulated phosphorylation of secretory granule membrane proteins of the rat parotid gland.
An antiserum raised against purified rat parotid secretory granule membrane proteins has been used to identify organelle-specific protein phosphorylation events following stimulation of intact cells from the rat parotid gland. After lobules were prelabeled with [32P]orthophosphate and exposed to secretagogues, phosphoproteins were immunoprecipitated with the granule membrane protein antiserum, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and visualized by autoradiography. Parallel studies of stimulated amylase release were performed. Isoproterenol treatment of parotid lobules resulted in an increase in the phosphate content of immunoprecipitable 60- and 72-kDa proteins that correlated with amylase release in a time-dependent manner. Forskolin addition mimicked these effects, but only the isoproterenol effects were reversed by propranolol treatment. To confirm the specificity of the antiserum to the secretory granule membrane fraction, subcellular isolation techniques were employed following in situ phosphorylation. The 60- and 72-kDa phosphoproteins were immunoprecipitated from both a particulate fraction and a purified secretory granule fraction. Furthermore, the extraction properties of both species suggest that they are integral membrane proteins. These findings support the possibility that stimulus-regulated secretion may involve phosphorylation of integral membrane proteins of the exocrine secretory granule.
Marino
C R
CR
Department of Medicine, Yale University School of Medicine, New Haven, Connecticut 06510.
Castle
J D
JD
Gorelick
F S
FS
D000681:Amylases / Q000378:metabolism; D000818:Animals; D001345:Autoradiography; D005576:Colforsin / Q000494:pharmacology; D003594:Cytoplasmic Granules / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008297:Male; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008854:Microscopy, Electron; D008970:Molecular Weight; D010306:Parotid Gland / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D010710:Phosphates / Q000378:metabolism; D010750:Phosphoproteins / Q000302:isolation & purification; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D051381:Rats; D011919:Rats, Inbred Strains
D008565:Membrane Proteins; D010710:Phosphates; D010750:Phosphoproteins; D010761:Phosphorus Radioisotopes; D005576:Colforsin; D000681:Amylases