Access of a membrane protein to secretory granules is facilitated by phosphorylation

Peptidylglycine a-amidating monooxygenase (PAM), an integral membrane prote in 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, ge nerating a short, cytosolic acidic cluster. P-CIP2, a protein kinase identi fied by its ability to interact with several juxtamembrane determinants in the PAM cytosolic domain, also phosphorylates Ser(949). Antibody specific f or 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) phosp horylation at these sites were studied. PAM-1 TS/AA yields a lumenal monoox ygenase domain that enters secretory granules inefficiently and is rapidly degraded. In contrast, PAM-1 TS/DD is routed to regulated secretory granule s more efficiently than wild-type PAM-1 and monooxygenase release is more r esponsive 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-Go lgi network. The increased ability of solubilized PAM-1 TS/DD to aggregate at neutral pH may play an important role in its altered trafficking.