IDENTIFICATION OF PHOSPHORYLATION SITES IN THE RECOMBINANT CATALYTIC SUBUNIT OF CAMP-DEPENDENT PROTEIN-KINASE

The catalytic subunit of cAMP-dependent protein kinase expressed in Es cherichia coli is a phosphoprotein. By in vivo labeling with [P-32i]or thophosphate, the sites of phosphorylation were identified as Ser-10, Ser-139, Thr-197, and Ser-338. Two of these sites, Thr-197 and Ser-338 , are found in the mammalian enzyme (Shoji, S., Titani, K., Demaille, J. G., and Fischer, E. H. (1979) J. Biol. Chem. 254, 6211-6214). The p redominant isoform is phosphorylated at Ser-10, Ser-338, and Thr-197. The isoforms cannot be readily interconverted by in vitro autophosphor ylation, suggesting that the phosphates are relatively stable once the mature protein is assembled. Unlike the mammalian enzyme, the recombi nant enzyme is not myristylated at its animo terminus. By coexpressing the catalytic subunit and N-myristyl transferase, the recombinant cat alytic subunit is myristylated, and, under these conditions, phosphory lation at Ser-10 is reduced. The fact that recombinant catalytic subun it mutants that are enzymatically impaired are not phosphorylated in v ivo indicates that the phosphorylation of the catalytic subunit observ ed in E. coli is due to autophosphorylation. Whether this process is i ntramolecular or intermolecular cannot be distinguished. Although auto phosphorylation accounts for the modification of the catalytic subunit when it is expressed in E. coli, there may be heterologous protein ki nases that are responsible for its in vivo phosphorylation when the en zyme is expressed in eukaryotic cells.