When the catalytic (rC) subunit of cAMP-dependent protein kinase (cAPK) is expressed in Escherichia coli, it is autophosphorylated at four sites, Ser10, Ser139, Ser338 and Thr197 (49). Three of these sites, Ser10, Ser338 and Thr197, are also found in the mammalian enzyme. To understand the functional importance of these phosphorylation sites, each was replaced with Ala, Glu or Asp. The expression, solubility and phosphorylation state of each mutant protein was characterized by immunoprecipitation following in vivo labeling with 32Pi. When possible, isoforms were resolved and kinetic properties were measured. The two stable phosphorylation sites in the mammalian enzyme, Ser338 and Thr197, were shown to play different roles. Ser338, which stabilizes a turn near the C-terminus, is important for stability. Both rC(S338A) and rC(S338E) were very labile; however, the kinetic properties of rC(S338E) were similar to the wild-type catalytic subunit (C-subunit). Ser338 most likely helps to anchor the C-terminus to the surface of the small lobe. Thr197 is in the activation loop near the cleft interface. Mutagenesis of T197 caused a significant loss of catalytic activity with increases in Kms for both peptide and MgATP, as well as a small decrease in k(cat) indicating that this phosphate is important for the correct orientation of catalytic residues at the active site. Replacement of Ser139, positioned at the beginning of the E-helix, with Ala had no effect on the kinetic parameters, stability or phosphorylation at the remaining sites. In contrast, mutation of Ser10, located at the beginning of the A-helix, produced mostly insoluble, inactive, unphosphorylated protein, suggesting that this region, though far removed from the active site, is structurally important at least for the expression of soluble phosphoprotein in E.coli. Since the mutation of active site residues as well as deletion mutants generate underphosphorylated proteins, these phosphorylations in E.coli all result from autophosphorylation.