Nerve growth factor‐induced phosphorylation of amphiphysin‐1 by casein kinase 2 regulates clathrin–amphiphysin interactions

Amphiphysins interact directly with clathrin and have a function in clathrin‐mediated synaptic vesicle recycling and clathrin‐mediated endocytosis. The neuronal isoform amphiphysin‐1 is a serine/threonine phosphoprotein that is dephosphorylated upon stimulation of synaptic vesicle endocytosis. Repho...

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Bibliographic Details
Published in:Journal of neurochemistry 2006-09, Vol.98 (6), p.2013-2022
Main Authors: Döring, Markus, Loos, Anneke, Schrader, Nina, Pfander, Boris, Bauerfeind, Rudolf
Format: Article
Language:English
Subjects:
amphiphysin
Animals
Binding sites
Biological and medical sciences
Casein Kinase II - metabolism
casein kinase 2
Cell physiology
clathrin
Clathrin - metabolism
Endocytosis
Fundamental and applied biological sciences. Psychology
HeLa Cells
Humans
Inhibitor drugs
Molecular and cellular biology
Mutation
nerve growth factor
Nerve Growth Factor - pharmacology
Nerve Growth Factor - physiology
Nerve Tissue Proteins - genetics
Nerve Tissue Proteins - metabolism
Nerve Tissue Proteins - pharmacology
Nervous system
PC12 Cells
Peripheral nervous system. Autonomic nervous system. Neuromuscular transmission. Ganglionic transmission. Electric organ
Pharmacology
Phosphorylation - drug effects
Protein Kinase Inhibitors - pharmacology
Proteins
Rats
Responses to growth factors, tumor promotors, other factors
Tetradecanoylphorbol Acetate - pharmacology
Transfection
Vertebrates: nervous system and sense organs
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Summary:Amphiphysins interact directly with clathrin and have a function in clathrin‐mediated synaptic vesicle recycling and clathrin‐mediated endocytosis. The neuronal isoform amphiphysin‐1 is a serine/threonine phosphoprotein that is dephosphorylated upon stimulation of synaptic vesicle endocytosis. Rephosphorylation was stimulated by nerve growth factor. We analysed the regulation of amphiphysin–clathrin interactions by phosphorylation. The N‐terminal domain of clathrin bound to unphosphorylated amphiphysin‐1, but not to the phosphorylated protein. A search for possible phosphorylation sites revealed two casein kinase 2 consensus motifs in close proximity to the clathrin binding sites in amphiphysin‐1 and ‐2. We mutagenized these residues (T350 and T387) to glutamate, mimicking a constitutive phosphorylation. The double mutant showed a strong reduction in clathrin binding. The assumption that casein kinase 2 phosphorylates amphiphysin‐1 at T350 and T387 was corroborated by experiments showing that: (i) casein kinase 2 phosphorylated these residues directly in vitro, (ii) when expressed in HeLa cells, the glutamate mutant showed reduced phosphorylation, and (iii) casein kinase 2 inhibitors blocked nerve growth factor‐induced phosphorylation of endogenous amphiphysin‐1 in PC12 cells. These observations are consistent with the hypothesis that, upon activation by nerve growth factor, casein kinase 2 phosphorylates amphiphysin‐1 and thereby regulates the endocytosis of clathrin‐coated vesicles via the interaction between clathrin and amphiphysin.
ISSN:0022-3042
1471-4159
DOI:10.1111/j.1471-4159.2006.04037.x