Signaling mediated by the cytosolic domain of peptidylglycine alpha-amidating monooxygenase.
The luminal domains of membrane peptidylglycine alpha-amidating monooxygenase (PAM) are essential for peptide alpha-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.
Department of Neuroscience, University of Connecticut Health Center, Farmington, Connecticut 06030-3401, USA.
M R
MR
Alam
T C
TC
Steveson
R C
RC
Johnson
N
N
Bäck
B
B
Abraham
R E
RE
Mains
B A
BA
Eipper
D000324:Adrenocorticotropic Hormone / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases; D003593:Cytoplasm / Q000201:enzymology; D017931:DNA Primers / Q000235:genetics; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D016253:Microscopy, Immunoelectron; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D009097:Multienzyme Complexes; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D017346:Protein Serine-Threonine Kinases; D021382:Protein Sorting Signals / Q000235:genetics; D017434:Protein Structure, Tertiary
D002352:Carrier Proteins; D017931:DNA Primers; D020662:Guanine Nucleotide Exchange Factors; D047908:Intracellular Signaling Peptides and Proteins; C102365:Kalrn protein, rat; D009097:Multienzyme Complexes; D021382:Protein Sorting Signals; C102364:Uhmk1 protein, rat; D000324:Adrenocorticotropic Hormone; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases