Autophosphorylation of

No abstract

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.