Protein phosphorylation is required for endocytosis in nerve terminals: potential role for the dephosphins dynamin I and synaptojanin, but not AP180 or amphiphysin.
Dynamin I and at least five other nerve terminal proteins, amphiphysins I and II, synaptojanin, epsin and eps15 (collectively called dephosphins), are coordinately dephosphorylated by calcineurin during endocytosis of synaptic vesicles. Here we have identified a new dephosphin, the essential endocytic protein AP180. Blocking dephosphorylation of the dephosphins is known to inhibit endocytosis, but the role of phosphorylation has not been determined. We show that the protein kinase C (PKC) antagonists Ro 31-8220 and Go 7874 block the rephosphorylation of dynamin I and synaptojanin that occurs during recovery from an initial depolarizing stimulus (S1). The rephosphorylation of AP180 and amphiphysins 1 and 2, however, were unaffected by Ro 31-8220. Although these dephosphins share a single phosphatase, different protein kinases phosphorylated them after nerve terminal stimulation. The inhibitors were used to selectively examine the role of dynamin I and/or synaptojanin phosphorylation in endocytosis. Ro 31-8220 and Go 7874 did not block the initial S1 cycle of endocytosis, but strongly inhibited endocytosis following a second stimulus (S2). Therefore, phosphorylation of a subset of dephosphins, which includes dynamin I and synaptojanin, is required for the next round of stimulated synaptic vesicle retrieval.
Cousin
M A
MA
Cell Signalling Unit, Children's Medical Research Institute, Sydney, New South Wales, Australia.
Tan
T C
TC
Robinson
P J
PJ
D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D019703:Calcineurin / Q000378:metabolism; D016572:Cyclosporine / Q000494:pharmacology; D034284:Dynamin I; D034281:Dynamins; D004591:Electrophoresis, Polyacrylamide Gel; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D020558:GTP Phosphohydrolases / Q000378:metabolism; D007211:Indoles / Q000494:pharmacology; D034041:Monomeric Clathrin Assembly Proteins; D009419:Nerve Tissue Proteins / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D017729:Presynaptic Terminals / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D013572:Synaptic Vesicles / Q000378:metabolism
D033942:Adaptor Proteins, Vesicular Transport; D004791:Enzyme Inhibitors; D007211:Indoles; D034041:Monomeric Clathrin Assembly Proteins; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; D011506:Proteins; C439023:clathrin assembly protein AP180; C075637:amphiphysin; D016572:Cyclosporine; D011493:Protein Kinase C; C097702:synaptojanin; D019703:Calcineurin; D010744:Phosphoric Monoester Hydrolases; D034284:Dynamin I; D020558:GTP Phosphohydrolases; D034281:Dynamins; C064758:Ro 31-8220