Identification of a 59-kDa Substrate for cAMP-Dependent Protein Kinase That Is Enriched in Rat Cerebellar Membranes.

Following incubation of rat brain membranes with the catalytic subunit of cAMP-dependent protein kinase and [(32)P]ATP, a previously unreported phosphoprotein, pp59, was found to be enriched in cerebellar synaptic plasma membrane preparations, but not in those prepared from cerebral cortex, hippocampus, olfactory bulb, or striatum. This protein, which has an M(r) of 59,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is not phosphorylated by the cGMP-dependent protein kinase. While pp59 was consistently detected in cerebellar membranes from adult Sprague-Dawley rats, it was not detected in bovine or rabbit cerebellar membranes. Moreover, pp59 did not comigrate with any of the autophosphorylated subunits of the Ca(2+)/calmodulindependent protein kinase in rat cerebellar membranes. Extraction of pp59 from these membranes could be accomplished with 6 M urea, but not with 0.4 M NaCl or 0.5% (v/v) Triton X-100. The urea solubility suggests that pp59 is not an integral membrane protein. Acid hydrolysis of the protein phosphorylated in vitro yielded phosphoserine but no significant amount of phosphothreonine or phosphotyrosine. Further analysis of pp59 may provide new insights into the role of cAMP in modulation of synaptic function in the cerebellum.