Phosphorylation of the Cytosolic Domain of Peptidylglycine -Amidating Monooxygenase

doi:10.1074/jbc.270.50.30075

Phosphorylation of the Cytosolic Domain of Peptidylglycine -Amidating Monooxygenase (*)

From the Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 and the
(1)Endocrine Unit, Massachusetts General Hospital, Boston, Massachusetts 02114

** To whom correspondence should be addressed:
Dept. of Neuroscience, Wood Basic Science Bldg., Rm. 907, The Johns Hopkins University School of Medicine, 725 North Wolfe St., Baltimore, MD 21205.
Tel.: 410-955-6921; Fax: 410-955-0681; betty_eipper{at}qmail.bs.jhu.edu.

↵^§ Present address: Dept. of Neurology, The Johns Hopkins University School of Medicine, Pathology 2-210, 600 N. Wolfe St., Baltimore, MD 21287.
↵^¶ Present address: Dept. of Physiology, University of North Carolina, Chapel Hill, NC 27599.

Abstract

Peptidylglycine α-amidating monooxygenase (PAM) is a bifunctional enzyme that catalyzes the COOH-terminal α-amidation of neural and endocrine peptides through a two-step reaction carried out sequentially by its monooxygenase and lyase domains. PAM occurs in soluble and integral membrane forms. Metabolic labeling of stably transfected hEK-293 and AtT-20 cells showed that [P]PO⁴ was efficiently incorporated into Ser and Thr residues of membrane PAM but not into soluble PAM. Truncation of integral membrane PAM proteins (which terminate with Ser) at Tyr eliminated their phosphorylation, suggesting that the COOH-terminal region of the protein was the site of phosphorylation. Recombinant PAM COOH-terminal domain was phosphorylated on Ser and Ser by protein kinase C (PKC). PAM-1 protein recovered from different subcellular fractions of stably transfected AtT-20 cells was differentially susceptible to calcium-dependent, staurosporine-inhibitable phosphorylation catalyzed by endogenous cytosolic protein kinase(s). Although phorbol ester treatment of hEK-293 cells expressing PAM-1 stimulated the cleavage/release of a bifunctional 105-kDa PAM protein, the effect was an indirect one since it was also observed in hEK-293 cells expressing a truncated PAM-1 protein that was not phosphorylated. AtT-20 cells expressing PAM-1 lacking one of the PKC sites (PAM-1/Ser Ala) exhibited an altered pattern of PAM•PAM antibody internalization, with the mutant protein targeted to lysosomes upon internalization. Thus, phosphorylation of Ser in the COOH-terminal cytosolic domain of membrane PAM plays a role in a specific step in the targeting of this protein.

Footnotes

↵* This work was supported by United States Public Health Service Grant DK-32949. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

PAM

peptidylglycine α-amidating monooxygenase

rPAM

rat PAM

PHM

peptidylglycine α-hydroxylating monooxygenase

PAL

peptidyl-α-hydroxyglycine α-amidating lyase

TES

N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid

PAGE

polyacrylamide gel electrophoresis

FITC

fluorescein isothiocyanate

PMA

phorbol 12-myristate 13-acetate

PKC

protein kinase C

HPLC

high performance liquid chromatography

TGF

transforming growth factor.
- Received May 9, 1995.
- Revision received October 12, 1995.