Autophosphorylation of the catalytic subunit of cAMP-dependent protein kinase in Escherichia coli

When the catalytic (rC) subunit of cAMP-dependent protein kinase (cAPK) isexpressed in Escherichia coli, it is autophosphorylated at four sites,Ser10, Ser139, Ser338 and Thr197 (49). Three of these sites, Ser10, Ser338and Thr197, are also found in the mammalian enzyme. To understand thefunctional importance of these phosphorylation sites, each was replacedwith Ala, Glu or Asp. The expression, solubility and phosphorylation stateof each mutant protein was characterized by immunoprecipitation followingin vivo labeling with 32Pi. When possible, isoforms were resolved andkinetic properties were measured. The two stable phosphorylation sites inthe mammalian enzyme, Ser338 and Thr197, were shown to play differentroles. Ser338, which stabilizes a turn near the C-terminus, is importantfor stability. Both rC(S338A) and rC(S338E) were very labile; however, thekinetic properties of rC(S338E) were similar to the wild-type catalyticsubunit (C-subunit). Ser338 most likely helps to anchor the C-terminus tothe surface of the small lobe. Thr197 is in the activation loop near thecleft interface. Mutagenesis of T197 caused a significant loss of catalyticactivity with increases in Kms for both peptide and MgATP, as well as asmall decrease in k(cat) indicating that this phosphate is important forthe correct orientation of catalytic residues at the active site.Replacement of Ser139, positioned at the beginning of the E-helix, with Alahad no effect on the kinetic parameters, stability or phosphorylation atthe remaining sites. In contrast, mutation of Ser10, located at thebeginning of the A-helix, produced mostly insoluble, inactive,unphosphorylated protein, suggesting that this region, though far removedfrom the active site, is structurally important at least for the expressionof soluble phosphoprotein in E.coli. Since the mutation of active siteresidues as well as deletion mutants generate underphosphorylated proteins,these phosphorylations in E.coli all result from autophosphorylation.