Characterization of the phosphorylation site of PP59, a substrate for cyclic AMP-dependent protein kinase that is enriched in the synaptic membrane fraction of rat cerebellum.
We have identified previously a synaptic membrane-associated protein, PP59, that serves as a substrate for cyclic AMP-dependent protein kinase and is enriched in rat cerebellum. We show here that PP59 can be extracted from synaptic plasma membranes with a combination of 2% Triton X-100 plus 1 M KCl. A 290-fold purification of PP59 was achieved by selective solubilization, followed by continuous-elution preparative gel electrophoresis. To determine the amino acid sequence surrounding the cyclic AMP-dependent protein kinase phosphorylation site within PP59, the partially purified 32P-phosphorylated protein was digested with chymotrypsin, and radiolabeled peptides were purified by sequential reversed-phase HPLC in two different solvent systems. Automated Edman degradation revealed a single phosphorylation site contained within the sequence Ala-Arg-Glu-Arg-Ser-Asp-Ser(P)-Thr-Gly-Ser-Ser-Ser-Val-Tyr. No strong sequence homology to this peptide fragment with other known peptides or proteins in the SwissProt, PIR, or GenPept databases could be found. A synthetic peptide containing this unique 14-amino acid sequence was used to develop polyclonal anti-peptide antibodies that were affinity-purified and shown to recognize intact PP59 as determined by western blotting. These antibodies specifically inhibited the phosphorylation of PP59 by cyclic AMP-dependent protein kinase in an in vitro phosphorylation assay containing synaptic plasma membranes.
Rich
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School of Biological Sciences, University of California, Irvine 92697-4550, USA.
Aswad
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DW
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D001665:Binding Sites / Q000502:physiology; D015153:Blotting, Western; D002458:Cell Fractionation; D002531:Cerebellum / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008297:Male; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology / Q000302:isolation & purification; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D013379:Substrate Specificity; D013570:Synaptic Membranes / Q000737:chemistry* / Q000201:enzymology / Q000276:immunology
D010455:Peptides; D017868:Cyclic AMP-Dependent Protein Kinases