Menkes protein (ATP7A) is a P-type ATPase involved in copper uptake and homeostasis. Disturbed copper homeostasis occurs in patients with Menkes disease, an X-linked disorder characterized by mental retardation, neurodegeneration, connective tissue disorders, and early childhood death. Mutations in ATP7A result in malfunction of copper-requiring enzymes, such as tyrosinase and copper/zinc superoxide dismutase. The first step of the two-step amidation reaction carried out by peptidylglycine alpha-amidating monooxygenase (PAM) also requires copper. We used tissue from wild-type rats and mice and an ATP7A-specific antibody to determine that ATP7A is expressed at high levels in tissues expressing high levels of PAM. ATP7A is largely localized to the trans Golgi network in pituitary endocrine cells. The Atp7a mouse, bearing a mutation in the Atp7a gene, is an excellent model system for examining the consequences of ATP7A malfunction. Despite normal levels of PAM protein, levels of several amidated peptides were reduced in pituitary and brain extracts of Atp7a mice, demonstrating that PAM function is compromised when ATP7A is inactive. Based on these results, we conclude that a reduction in the ability of PAM to produce bioactive end-products involved in neuronal growth and development could contribute to many of the biological effects associated with Menkes disease.
Peptidylglycine ␣-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 Ser 949 and Thr 946 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 Ser 949 . Antibody specific for phospho-Ser 949 -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.
The luminal domains of membrane peptidylglycine ␣-amidating monooxygenase (PAM) are essential for peptide ␣-amidation, and the cytosolic domain (CD) is essential for trafficking. Overexpression of membrane PAM in corticotrope tumor cells reorganizes the actin cytoskeleton, shifts endogenous adrenocorticotropic hormone (ACTH) from mature granules localized at the tips of processes to the TGN region, and blocks regulated secretion. PAM-CD interactor proteins include a protein kinase that phosphorylates PAM (P-CIP2) and Kalirin, a Rho family GDP/GTP exchange factor. We engineered a PAM protein unable to interact with either P-CIP2 or Kalirin (PAM-1/K919R), along with PAM proteins able to interact with Kalirin but not with P-CIP2. AtT-20 cells expressing PAM-1/K919R produce fully active membrane enzyme but still exhibit regulated secretion, with ACTH-containing granules localized to process tips. Immunoelectron microscopy demonstrates accumulation of PAM and ACTH in tubular structures at the trans side of the Golgi in AtT-20 cells expressing PAM-1 but not in AtT-20 cells expressing PAM-1/K919R. The ability of PAM to interact with P-CIP2 is critical to its ability to block exit from the Golgi and affect regulated secretion. Consistent with this, mutation of its P-CIP2 phosphorylation site alters the ability of PAM to affect regulated secretion. INTRODUCTIONAtT-20 corticotrope tumor cells have long served as a reliable model system for studying the biosynthesis, storage, and regulated secretion of pituitary peptide hormones (Mains and Eipper, 1978;Moore and Kelly, 1986;Tooze and Tooze, 1986). Cleavage of endogenous pro-opiomelanocortin (POMC) by PC1 (PC1) and carboxypeptidase E yields both adrenocorticotropic hormone (ACTH) and -lipotropin (Fricker and Devi, 1993;Zhou et al., 1993). Production of other POMC peptides requires the action of peptidylglycine ␣-amidating monooxygenase (PAM) (Eipper et al., 1986). Although levels of PAM in AtT-20 cells are 20-fold lower than in the anterior pituitary, complete amidation of POMCderived products occurs (Eipper et al., 1986).PAM is one of the few peptide-processing enzymes that spans the secretory granule membrane. The fact that its luminal, catalytic domains are pH sensitive and are further activated on cleavage from the membrane (Husten and Eipper, 1991;Husten et al., 1993) raised the possibility that PAM might play a role in signaling luminal conditions to the cytosolic machinery involved in secretory granule formation. This type of signaling is essential in communicating information about events occurring in the lumen of the endoplasmic reticulum to cytosolic proteins (Pahl and Baeuerle, 1997;Shamu, 1997;Brown and Goldstein, 1998), as well as in cargo selection during vesicle budding (Kuehn and Herrmann, 1998).We were surprised to find that expression of exogenous PAM in AtT-20 cells at levels equivalent to those in the anterior pituitary blocked the regulated secretion of ACTH, Abbreviations used: ACTH, adrenocorticotropic hormone; DC, COOH-terminal domain of PAM; CHO, C...
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