Physiological Phosphorylation of Protein Kinase A at Thr-197 Is by a Protein Kinase A Kinase

Abstract

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...

Shear stress stimulates phosphorylation of eNOS at Ser635by a protein kinase A-dependent mechanism

“…One mechanism is phosphorylation of Thr 197 in the activation loop of C subunit by PDK1 (6,9). Other mechanisms include interactions of PKA with caveolin, A-kinaseanchoring protein (AKAP110), and inhibitory nuclear factor B (I B) protein (16,39,42).…”

Phosphorylation of the Catalytic Subunit of Protein Kinase A

“…Whether PDK-1 is the in vivo PKA kinase or not remains to be resolved. However, it is clear from the work of Steinberg and co-workers (14) that the C-subunit is not autophosphorylated in mammalian cells; it is phosphorylated by a heterologous kinase.…”

“…What is not well understood is the in vivo mechanism for this phosphorylation. Although C-subunit expressed in Escherichia coli autophosphorylates on both Thr-197 and Ser-338, this most likely does not reflect the mechanism used in the eukaryotic cell (14).…”

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

“…Recent results for PKA show that removing the activation loop phosphate abolishes the rapid phosphotransfer burst kinetics without affecting the steady state kcat (16). In mammalian cells, the C-subunit is phosphorylated in trans at Thr 197 by either another C-subunit or phosphoinositide-dependent PK 1 (PDK1), a master kinase of the AGC subfamily (17,18). In Schizosaccharomyces pombe, the activation loop site is phosphorylated only by PDK1 (19).…”