Phosphorylation of Cytosolic Domain Ser937 Affects Both Biosynthetic and Endocytic Trafficking of Peptidylglycine α-Amidating Monooxygenase*
- From the ‡Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 and the §Endocrine Unit, Massachusetts General Hospital, Boston, Massachusetts 02114
Abstract
Peptidylglycine α-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 Ser937. The roles of phosphorylation and dephosphorylation of Ser937 in the biosynthetic and endocytic trafficking of integral membrane PAM were examined using an antiserum specific for the phosphorylation of Ser937 and using AtT-20 cells expressing membrane PAM in which Ser937 was mutated to Ala or Asp. Although phosphorylation at Ser937 can occur while PAM is in the endoplasmic reticulum, early steps in the biosynthetic trafficking of membrane PAM were not affected by Ser937 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 Ser937 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 Ser937increased and phosphorylated PAM accumulated in large vesicular structures. Therefore, phosphorylation of PAM-1 at Ser937directs newly synthesized and internalized protein away from lysosomes, while dephosphorylation is needed for a different step in the late endocytic pathway.
Footnotes
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↵* This work was supported by National Institutes of Health Grants DK-32949 (to B. A. E.) and DK-09520 (to T. C. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵¶ To whom correspondence should be addressed: Dept. of Neuroscience, WBSB 907, The Johns Hopkins University School of Medicine, 725 N. Wolfe St., Baltimore, MD, 21205. Tel.: 410-955-6937; Fax: 410-955-0681; E-mail: beipper@jhmi.edu.
- Abbreviations:
- TGN
-
trans-Golgi network
- PAM
-
peptidylglycine α-amidating monooxygenase
- PHM
-
peptidylglycine α-hydroxylating monooxygenase
- PAL
-
peptidyl-α-hydroxyglycine α-amidating lyase
- CD
-
cytosolic COOH-terminal domain
- LAMP-1
-
lysosome-associated membrane protein 1
- CI-MPR
-
cation-independent mannose 6-phosphate receptor
- PMA
-
phorbol 12-myristate 13-acetate
- ER
-
endoplasmic reticulum
- TES
-
2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid
- Ab
-
antibody
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- Received December 30, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
