title: Access of a membrane protein to secretory granules is facilitated by phosphorylation.
abstract: Peptidylglycine alpha-amidating monooxygenase (PAM), an integral membrane protein essential for the biosynthesis of amidated peptides, was used to assess the role of cytosolic acidic clusters in trafficking to regulated secretory granules. Casein kinase II phosphorylates Ser(949) and Thr(946) of PAM, generating a short, cytosolic acidic cluster. P-CIP2, a protein kinase identified by its ability to interact with several juxtamembrane determinants in the PAM cytosolic domain, also phosphorylates Ser(949). Antibody specific for phospho-Ser(949)-PAM-CD demonstrates that a small fraction of the PAM-1 localized to the perinuclear region bears this modification. Pituitary cell lines expressing PAM-1 mutants that mimic (TS/DD) or prevent (TS/AA) phosphorylation at these sites were studied. PAM-1 TS/AA yields a lumenal monooxygenase domain that enters secretory granules inefficiently and is rapidly degraded. In contrast, PAM-1 TS/DD is routed to regulated secretory granules more efficiently than wild-type PAM-1 and monooxygenase release is more responsive to secretagogue. Furthermore, this acidic cluster affects exit of internalized PAM-antibody complexes from late endosomes; internalized PAM-1 TS/DD accumulates in a late endocytic compartment instead of the trans-Golgi network. The increased ability of solubilized PAM-1 TS/DD to aggregate at neutral pH may play an important role in its altered trafficking.
authors.affiliation: Department of Neuroscience, University of Connecticut Health Center, 263 Farmington Ave., Farmington, CT 06030, USA.
authors.forename: T C
authors.initials: TC
authors.lastname: Steveson
authors.forename: G C
authors.initials: GC
authors.lastname: Zhao
authors.forename: H T
authors.initials: HT
authors.lastname: Keutmann
authors.forename: R E
authors.initials: RE
authors.lastname: Mains
authors.forename: B A
authors.initials: BA
authors.lastname: Eipper
mesh_terms: D047390:Casein Kinase II; D004004:Dichlororibofuranosylbenzimidazole / Q000494:pharmacology; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D021382:Protein Sorting Signals; D021381:Protein Transport; D011994:Recombinant Proteins / Q000378:metabolism; D022142:Secretory Vesicles / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D013912:Threonine / Q000378:metabolism; D021601:trans-Golgi Network / Q000378:metabolism
chemical_list: D008565:Membrane Proteins; D009097:Multienzyme Complexes; D010446:Peptide Fragments; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D013912:Threonine; D012694:Serine; D004004:Dichlororibofuranosylbenzimidazole; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases