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

Abstract

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.

Folding and activity of cAMP‐dependent protein kinase mutants

“…Similar results have been described for the single mutants PKA T197D and PKA T197E [11]. As reported previously PKA T197E was found to be nearly inactive whereas PKA T197D has an activity comparable to the wild-type [11]. By contrast, we have measured significant, albeit lower activity for both the PKA-4P(D) and the PKA-4P(E) mutants (57% and 39% activity, respectively) relative to the wild type.…”

“…Replacing the additional phosphorylation site T197 by either D or E yields poorly soluble protein. Similar results have been described for the single mutants PKA T197D and PKA T197E [11]. As reported previously PKA T197E was found to be nearly inactive whereas PKA T197D has an activity comparable to the wild-type [11].…”

“…Replacing S338 by alanine yields a more unstable, but fully active protein [13]. In contrast, changing S139 to alanine does not display adverse effects [11].…”

Identification of ChChd3 as a Novel Substrate of the cAMP-dependent Protein Kinase (PKA) Using an Analog-sensitive Catalytic Subunit

“…It is not uncommon for mutant PKA C-subunits to be insoluble because of defective phosphorylation of the protein. Typically when the wild type C-subunit is expressed in E. coli it is fully active and contains 2-4 phosphates due to autophosphorylation (16). Using an antibody generated against the activation loop of PKC that works well for PKA (17), we demonstrated that the His-tagged M120G mutant was not phosphorylated on either Thr 197 in the activation loop or on Ser 338 in the C-terminal tail (supplemental Fig.…”

Balanol Analogues Probe Specificity Determinants and the Conformational Malleability of the Cyclic 3‘,5‘-Adenosine Monophosphate-Dependent Protein Kinase Catalytic Subunit,

“…Recombinant murine PKA CR was purified as described previously (12). Briefly, the C subunit was expressed in BL21 E. coli and grown at 37°C for 6-8 h in LB media prior to induction of protein expression with IPTG.…”