Casein kinase II phosphorylates the synaptic vesicle protein p65.
p65 (synaptotagmin), an abundant synaptic vesicle protein, has been implicated in the processes of vesicle docking and fusion. To characterize further the properties of this important neuronal protein, we have investigated its phosphorylation in vitro. Immunoprecipitation of p65 results in coprecipitation of a protein kinase that phosphorylates p65 as well as syntaxin, a plasma membrane protein that interacts with p65. p65 is phosphorylated on a threonine residue (Thr-128) within the cytoplasmic domain near the transmembrane region. The coprecipitating protein kinase was identified as casein kinase II based on its catalytic properties, the sequence surrounding Thr-128, and Western blot analysis of the anti-p65 immunoprecipitates. Affinity chromatography utilizing bacterially expressed fragments of p65 demonstrated that casein kinase II interacts with a domain of p65 distinct from the phosphorylation site. In a synaptic vesicle fraction, the phosphorylation of p65 is stimulated by sphingosine and by detergent solubilization, suggesting that p65 phosphorylation may be subject to regulatory processes.
Bennett
M K
MK
Howard Hughes Medical Institute, Department of Molecular and Cellular Physiology, Beckman Center for Molecular and Genetic Medicine, Stanford University Medical Center, California 94305.
Miller
K G
KG
Scheller
R H
RH
D000595:Amino Acid Sequence; D000818:Animals; D001345:Autoradiography; D002135:Calcium-Binding Proteins; D047390:Casein Kinase II; D004591:Electrophoresis, Polyacrylamide Gel; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism* / Q000502:physiology; D050863:Synaptotagmin I; D050857:Synaptotagmins
D002135:Calcium-Binding Proteins; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D050863:Synaptotagmin I; D050857:Synaptotagmins; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases