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J. Biol. Chem., Vol. 276, Issue 43, 40326-40337, October 26, 2001
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From the Peptidylglycine
Access of a Membrane Protein to Secretory Granules Is Facilitated
by Phosphorylation*
,, and
Department of Neuroscience, University of
Connecticut Health Center, Farmington, Connecticut 06030, the
§ Georgetown Medical Center, Georgetown University,
Washington, D. C. 20007, and the ¶ Endocrine Unit,
Massachusetts General Hospital, Boston, Massachusetts 02114.
-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 Ser949 and Thr946 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 Ser949. Antibody specific for
phospho-Ser949-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.
*
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.
To whom correspondence should be addressed: Dept. of
Neuroscience, MC 3401, University of Connecticut Health Center, 263 Farmington Ave., Farmington, CT 06030. Tel.: 860-679-8898; Fax:
860-679-7958; E-mail: eipper@uchc.edu.
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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