Phosphorylation of cytosolic domain Ser(937) affects both biosynthetic andendocytic trafficking of peptidylglycine alpha-amidating monooxygenase

Peptidylglycine alpha-amidating monooxygenase (PARI), 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 o f phosphorylation and dephosphorylation of Ser(937) in the biosynthetic and endocytic trafficking of integral membrane PAM were examined using an anti serum specific for the phosphorylation of Ser(937) and using AtT-20 cells e xpressing membrane PAM in which Ser(937) was mutated to Ala or Asp. Althoug h phosphorylation at Ser(937) can occur while PAM is in the endoplasmic ret iculum,, early steps in the biosynthetic trafficking of membrane PAM were n ot affected by Ser(937) phosphorylation. The inability to phosphorylate PAM /S937A increased its intracellular degradation and decreased secretion of t he soluble monooxygenase portion of PAM. In contrast, the biosynthetic traf ficking of PAM/S937A was indistinguishable from wild-type PAM. Despite the fact that Ser(937) is adjacent to the only Tyr-based internalization motif in PARZ internalization and trafficking through early endosomes were unaffe cted by phosphorylation, However, PAM antibody internalized by wild-type PA M acquired a perinuclear localization, while antibody internalized by PAM/S 937A was routed to lysosomes, and antibody bound to PAM/S937D maintained a dispersed, punctate pattern. In cells stimulated with phorbol ester, phosph orylation of Ser(937) increased and phosphorylated PAM accumulated in large vesicular structures. Therefore, phosphorylation of PAM-1 at Ser(937) dire cts newly synthesized and internalized protein away from lysosomes, while d ephosphorylation is needed for a different step in the late endocytic pathw ay.