Autoactivation of catalytic (C alpha) subunit of cyclic AMP-dependent protein kinase by phosphorylation of threonine 197.

Abstract

We recently found, using cultured mouse cell systems, that newly synthesized catalytic (C) subunits of cyclic AMP-dependent protein kinase undergo a posttranslational modification that reduces their electrophoretic mobilities in sodium dodecyl sulfate (SDS)-polyacrylamide gels and activates them for binding to a Sepharoseconjugated inhibitor peptide. Using an Escherichia coli expression system, we now show that recombinant murine Ca subunit undergoes a similar modification and that the modification results in a large increase in protein kinase activity. Threonine phosphorylation appears to be responsible for both the enzymatic activation and the electrophoretic mobility shift. The phosphothreonine-deficient form of C subunit had reduced affinities for the ATP analogs p-fluorosulfonyl-[14CJbenzoyl 5'-adenosine and adenosine 5'-O-(3-thiotriphosphate) as well as for the Sepharose-conjugated inhibitor peptide; it also had markedly elevated Kms for both ATP and peptide substrates. Autophosphorylation of C-subunit preparations enriched for this phosphothreonine-deficient form reproduced the changes in enzyme activity and SDS-gel mobility that occur in intact cells. A mutant form of the recombinant C subunit with Ala substituted for Thr-197 (the only C-subunit threonine residue known to be phosphorylated in mammalian cells) was similar in SDS-polyacrylamide gel electrophoresis mobility and activity to the phosphothreonine-deficient form of wild-type C subunit. In contrast to the wild-type subunit, however, the Ala-197 mutant form could not be shifted or activated by incubation with the phosphothreoninecontaining wild-type form. We conclude that posttranslational autophosphorylation of Thr-197 is a critical step in intracellular expression of active C subunit.It has been known for some time that catalytic (C) subunit of cyclic AMP (cAMP)-dependent protein kinase purified from animal tissues is phosphorylated at two sites, 22); these sites are also apparently phosphorylated in Escherichia coli (results cited in reference 32). A third site (Ser-10) can be phosphorylated by C subunit in vitro (29). Dephosphorylation of native C subunit has been difficult to achieve (22), and as a result, the importance of phosphorylation to C subunit function has remained unknown. Because of its specific labeling by a peptide-based affinity reagent, Thr-197 has been localized to a region in or near the active site of C subunit (17); recent crystallographic data substantiate this localization and suggest that the phosphate on Thr-197 might contribute to stabilization of the active conformation of C subunit (11,12

Phosphorylation of Ser-241 is essential for the activity of 3-phosphoinositide-dependent protein kinase-1: identification of five sites of phosphorylation in vivo

“…Ser-241 of PDK1 is similar in many respects to the stable phosphorylation site in the T-loop of PKA (Thr-197) [18]. This residue lies in an equivalent position in the kinase domain to Ser-241 of PDK1 (see Figure 6), and is resistant to dephosphorylation by protein phosphatases, because it is buried inside the protein [8,18].…”

“…This residue lies in an equivalent position in the kinase domain to Ser-241 of PDK1 (see Figure 6), and is resistant to dephosphorylation by protein phosphatases, because it is buried inside the protein [8,18]. Ser-241 of PDK1 is also not dephosphorylated following incubation with high concentrations of PP2A " , and PDK1 purified from rabbit skeletal muscle could not be inactivated by incubation with PP2A or protein phosphatase-1 [10].…”

Ligand-induced global transitions in the catalytic domain of protein kinase A

“…Three pairs of distance reporters (21) (ii) The His-87/P-Thr-197 pair monitors the distance between the C-helix and activation loop. The activation loop in PKA is stabilized by the phosphorylation of Thr-197, a posttranslational modification that is essential for optimal activity (37)(38)(39)(40)(41). The crystal structure of the closed form suggests that the hydrogen bond between His-87 and P-Thr-197 is a key element for this stabilization/activation.…”

Cotranslational cis -phosphorylation of the COOH-terminal tail is a key priming step in the maturation of cAMP-dependent protein kinase

“…The activation loop phosphate, which has been studied in many AGC kinases including PKA, has a global effect on structure and function (13,15). Recent results for PKA show that removing the activation loop phosphate abolishes the rapid phosphotransfer burst kinetics without affecting the steady state kcat (16).…”