CHARACTERIZATION OF THE PHOSPHORYLATION SITE OF PP59, A SUBSTRATE FORCYCLIC-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 kina se and is enriched in rat cerebellum. We show here that PP59 can be ex tracted from synaptic plasma membranes with a combination of 2% Triton X-100 plus 1 M KCI. A 290-fold purification of PP59 was achieved by s elective solubilization, followed by continuous-elution preparative ge l electrophoresis. To determine the amino acid sequence surrounding th e cyclic AMP-dependent protein kinase phosphorylation site within PP59 , the partially purified P-32-phosphorylated protein was digested with chymotrypsin, and radiolabeled peptides were purified by sequential r eversed-phase HPLC in two different solvent systems. Automated Edman d egradation revealed a single phosphorylation site contained within the sequence lu-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 wa s used to develop polyclonal anti-peptide antibodies that were affinit y-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 phospho rylation assay containing synaptic plasma membranes.