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. 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.
Döring
Markus
M
Department of Cell Biology, Hannover Medical School, Hannover, Germany.
Loos
Anneke
A
Schrader
Nina
N
Pfander
Boris
B
Bauerfeind
Rudolf
R
D000818:Animals; D047390:Casein Kinase II / Q000378:metabolism*; D002966:Clathrin / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D009154:Mutation; D020932:Nerve Growth Factor / Q000494:pharmacology / Q000502:physiology*; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D016716:PC12 Cells; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection
D002966:Clathrin; D009419:Nerve Tissue Proteins; D047428:Protein Kinase Inhibitors; C075637:amphiphysin; D020932:Nerve Growth Factor; D047390:Casein Kinase II; D013755:Tetradecanoylphorbol Acetate