Signaling Mediated by the Cytosolic Domain of Peptidylglycine α-Amidating Monooxygenase

Abstract

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...

Retrieval and Reuse of Pituitary Secretory GranuleProteins

“…D, cell lysates were immunoprecipitated with a VAMP2 antibody; one of two experiments is shown. The VAMP2 signals from fluorography were desensitized and plotted as in C. (10,11,29,30). In this study, we set out to establish the fate of PAM molecules that visit the cell surface using surface biotinylation.…”

The U2AF homology motif kinase 1 (UHMK1) is upregulated upon hematopoietic cell differentiation

“…Not surprisingly, most of UHMK1-related functions have been addressed in the context of adult nervous system [6,8,[10][11][12][13][14][15][16][17][18].…”

A Signal Sequence Is Sufficient for Green Fluorescent Protein to Be Routed to Regulated Secretory Granules**This work was supported by NIH Grants DK-32948, DA-00266 (to R.E.M.), and NS-35891 (to R.B.E.).

“…Evidence from our own studies indicate that PAM and ACTH are present together in some trans cisternae of the Golgi, and both are absent from other stacks, implying sorting before reaching the trans Golgi stack, perhaps within the lumen of cis or medial Golgi stacks (63). There is little evidence for sorting as early as the ER.…”