The AP-3 complex required for endosomal synaptic vesicle biogenesis is associated with a casein kinase Ialpha-like isoform.
The formation of small vesicles is mediated by cytoplasmic coats the assembly of which is regulated by the activity of GTPases, kinases, and phosphatases. A heterotetrameric AP-3 adaptor complex has been implicated in the formation of synaptic vesicles from PC12 endosomes (). When the small GTPase ARF1 is prevented from hydrolyzing GTP, we can reconstitute AP-3 recruitment to synaptic vesicle membranes in an assembly reaction that requires temperatures above 15 degrees C and the presence of ATP suggesting that an enzymatic step is involved in the coat assembly. We have now found an enzymatic reaction, the phosphorylation of the AP-3 adaptor complex, that is linked with synaptic vesicle coating. Phosphorylation occurs in the beta3 subunit of the complex by a kinase similar to casein kinase 1alpha. The kinase copurifies with neuronal-specific AP-3. In vitro, purified casein kinase I selectively phosphorylates the beta3A and beta3B subunit at its hinge domain. Inhibiting the kinase hinders the recruitment of AP-3 to synaptic vesicles. The same inhibitors that prevent coat assembly in vitro also inhibit the formation of synaptic vesicles in PC12 cells. The data suggest, therefore, that the mechanism of AP-3-mediated vesiculation from neuroendocrine endosomes requires the phosphorylation of the adaptor complex at a step during or after AP-3 recruitment to membranes.
Faundez
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Department of Biochemistry and Biophysics, Hormone Research Institute, University of California, San Francisco 94143-0534, USA.
Kelly
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D033965:Adaptor Protein Complex alpha Subunits; D033942:Adaptor Proteins, Vesicular Transport; D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D001921:Brain / Q000201:enzymology / Q000378:metabolism / Q000502:physiology; D047388:Casein Kinases; D004004:Dichlororibofuranosylbenzimidazole / Q000494:pharmacology; D011992:Endosomes / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007546:Isoquinolines / Q000494:pharmacology; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000737:chemistry / Q000187:drug effects / Q000502:physiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D008818:Mice, Neurologic Mutants; D034041:Monomeric Clathrin Assembly Proteins; D016716:PC12 Cells; D010766:Phosphorylation / Q000187:drug effects; D020033:Protein Isoforms / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D011494:Protein Kinases / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D051381:Rats; D013572:Synaptic Vesicles / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*
D033965:Adaptor Protein Complex alpha Subunits; D033942:Adaptor Proteins, Vesicular Transport; D004791:Enzyme Inhibitors; D007546:Isoquinolines; D046911:Macromolecular Substances; D008565:Membrane Proteins; D034041:Monomeric Clathrin Assembly Proteins; D020033:Protein Isoforms; C439023:clathrin assembly protein AP180; C058815:N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide; D004004:Dichlororibofuranosylbenzimidazole; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D047388:Casein Kinases