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

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Volume 130, no. 1, p. 429, column 2, lines 5 to 7 of Materials and Methods: Should read ... (31) supplemented with 0.5% glucose, 10 ,ug of thiamine per ml, and 50 to ..

Phosphorylation of the catalytic subunit of cyclic AMP-dependent protein kinase, or protein kinase A, on Thr-197 is required for optimal enzyme activity, and enzyme isolated from either animal sources or bacterial expression strains is found phosphorylated at this site. Autophosphorylation of Thr-197 occurs in Escherichia coli and in vitro but is an inefficient intermolecular reaction catalyzed primarily by active, previously phosphorylated molecules. In contrast, the Catalytic (C) subunit of cyclic AMP (cAMP)-dependent protein kinase (protein kinase A [PKA]) requires phosphorylation at Thr-197 for expression of full activity, and this residue is found phosphorylated in both the enzyme isolated from animal tissues and in recombinant C subunit expressed in Escherichia coli (26,33,38). In addition to lowering the K m values for both ATP and peptide substrates, the Thr-197 phosphate causes a distinctive reduction in the mobility of the protein in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (33). Although C subunit is also phosphorylated at Ser-338 in both bacteria and mammalian cells and can be phosphorylated on additional Ser residues, these phosphorylations do not appear to affect C-subunit activity and have only minor effects on the SDS-PAGE mobility of the protein (6,26,33,38).Thr-197 falls in the activation loop region contained within subdomain VIII that also is associated with activating phosphorylation sites in many other protein kinases, including CDC2 kinase, the mitogen-activated protein (MAP) kinases, the MAP kinase kinases, and most protein tyrosine kinases (12,13,38). The sequence in this region is fully conserved in mammalian C subunits, including C␣, C␤, and C␥ isoforms (3, 27, 37). Activation of protein tyrosine kinases by phosphorylation in this region appears to be by autophosphorylation (13), while that of CDC2, MAP kinases, and MAP kinase kinases is by heterologous enzymes (8, 12). C-subunit phosphorylation in E. coli is apparently an intermolecular autophosphorylation reaction, and the purified recombinant protein is capable of autophosphorylation with concomitant activation (33,38). In the present report, we present evidence that the phosphorylation of C subunit in intact mammalian cells is catalyzed by a heterologous PKA kinase. Furthermore, we describe an activity from extracts of a PKA-deficient mutant of S49 mouse lymphoma cells that appears to phosphorylate C subunit specifically at Thr-197. MATERIALS AND METHODSExpression and radiolabeling of recombinant C subunits. Wild-type and mutant forms of recombinant murine C␣ subunit were expressed from the pET-8c expression vector in E. coli BL21(DE3) as described previously (33). Construction of the wild-type and Thr-1973Ala plasmids has been described elsewhere (33). The Lys-723Met mutation was introduced by replacement of an NcoIBstEII restriction fragment from sequences amplified from pMT-C␣K72M-EV (17), using an upstream PCR primer modified to introduce an NcoI restriction site overlapping the C-subunit initiati...