Phosphorylation of cytosolic domain Ser(937) affects both biosynthetic and endocytic trafficking of peptidylglycine alpha-amidating monooxygenase.
Peptidylglycine alpha-amidating monooxygenase (PAM), a bifunctional enzyme, catalyzes the COOH-terminal amidation of bioactive peptides. In test tube assays, PAM is phosphorylated by protein kinase C at Ser(937). The roles of phosphorylation and dephosphorylation of Ser(937) in the biosynthetic and endocytic trafficking of integral membrane PAM were examined using an antiserum specific for the phosphorylation of Ser(937) and using AtT-20 cells expressing membrane PAM in which Ser(937) was mutated to Ala or Asp. Although phosphorylation at Ser(937) can occur while PAM is in the endoplasmic reticulum, early steps in the biosynthetic trafficking of membrane PAM were not affected by Ser(937) phosphorylation. The inability to phosphorylate PAM/S937A increased its intracellular degradation and decreased secretion of the soluble monooxygenase portion of PAM. In contrast, the biosynthetic trafficking of PAM/S937D was indistinguishable from wild-type PAM. Despite the fact that Ser(937) is adjacent to the only Tyr-based internalization motif in PAM, internalization and trafficking through early endosomes were unaffected by phosphorylation. However, PAM antibody internalized by wild-type PAM acquired a perinuclear localization, while antibody internalized by PAM/S937A was routed to lysosomes, and antibody bound to PAM/S937D maintained a dispersed, punctate pattern. In cells stimulated with phorbol ester, phosphorylation of Ser(937) increased and phosphorylated PAM accumulated in large vesicular structures. Therefore, phosphorylation of PAM-1 at Ser(937) directs newly synthesized and internalized protein away from lysosomes, while dephosphorylation is needed for a different step in the late endocytic pathway.
Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
T C
TC
Steveson
H T
HT
Keutmann
R E
RE
Mains
B A
BA
Eipper
D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D004705:Endocytosis; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D009097:Multienzyme Complexes; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D014176:Protein Biosynthesis; D012694:Serine / Q000378:metabolism; D015398:Signal Transduction
D009097:Multienzyme Complexes; D012694:Serine; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase