Secretory granule to the nucleus: role of a multiply phosphorylated intrinsically unstructured domain.
Intrinsically unstructured domains occur in one-third of all proteins and are characterized by conformational flexibility, protease sensitivity, and the occurrence of multiple phosphorylation. They provide large interfaces for diverse protein-protein interactions. Peptidylglycine alpha-amidating monooxygenase (PAM), an enzyme essential for neuropeptide biosynthesis, is a secretory granule membrane protein. As one of the few proteins spanning the granule membrane, PAM is a candidate to relay information about the status of the granule pool and conditions in the granule lumen. Here, we show that the PAM cytosolic domain is unstructured. Mass spectroscopy and two-dimensional gel electrophoresis demonstrated phosphorylation at 10-12 sites in the cytosolic domain. Stimulation of exocytosis resulted in coupled phosphorylation and dephosphorylation of specific sites and in the endoproteolytic release of a soluble, proteasome-sensitive cytosolic domain fragment. Analysis of granule-rich tissues, such as pituitary and heart, showed that a similar fragment was generated endogenously and translocated to the nucleus. This multiply phosphorylated unstructured domain may act as a signaling molecule that relays information from secretory granules to both cytosol and nucleus.
Rajagopal
Chitra
C
Department of Molecular, Microbial, and Structural Biology, University of Connecticut Health Center, Farmington, Connecticut 06030, USA.
Stone
Kathryn L
KL
Francone
Victor P
VP
Mains
Richard E
RE
Eipper
Betty A
BA
D021581:Active Transport, Cell Nucleus / Q000502:physiology; D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002467:Cell Nucleus / Q000201:enzymology* / Q000235:genetics; D005089:Exocytosis / Q000502:physiology; D006801:Humans; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D022142:Secretory Vesicles / Q000737:chemistry* / Q000201:enzymology* / Q000235:genetics; D044767:Ubiquitin-Protein Ligases
D048868:Adaptor Proteins, Signal Transducing; D006899:Mixed Function Oxygenases; C114022:MYCBP2 protein, human; D044767:Ubiquitin-Protein Ligases