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

Pompe's disease is caused by a deficiency of the lysosomal enzyme acid ␣-glucosidase (GAA). GAA is synthesized as a 110-kDa precursor containing N-linked carbohydrates modified with mannose 6-phosphate groups. Following trafficking to the lysosome, presumably via the mannose 6-phosphate receptor, the 110-kDa precursor undergoes a series of complex proteolytic and Nglycan processing events, yielding major species of 76 and 70 kDa. During a detailed characterization of human placental and recombinant human GAA, we found that the peptides released during proteolytic processing remained tightly associated with the major species. The 76-kDa form (amino acids (aa) 122-782) of GAA is associated with peptides of 3.9 kDa (aa 78 -113) and 19.4 kDa (aa 792-952). The 70-kDa form (aa 204 -782) contains the 3.9-and 19.4-kDa peptide species as well as a 10.3-kDa species (aa 122-199). A similar set of proteolytic fragments has been identified in hamster GAA, suggesting that the multicomponent character is a general phenomenon. Rabbit anti-peptide antibodies have been generated against sequences in the proteolytic fragments and used to demonstrate the time course of uptake and processing of the recombinant GAA precursor in Pompe's disease fibroblasts. The results indicate that the observed fragments are produced intracellularly in the lysosome and not as a result of nonspecific proteolysis during purification. These data demonstrate that the mature forms of GAA characterized by polypeptides of 76 or 70 kDa are in fact larger molecular mass multicomponent enzyme complexes.Lysosomal acid ␣-glucosidase (GAA 1 ; EC 3.2.1.3) is an exo-1,4-and -1,6-␣-glucosidase that hydrolyzes glycogen to glucose. The cDNA for GAA encodes a protein of 952 amino acids with a predicted molecular mass of 105 kDa (1). The newly synthesized precursor has an amino-terminal signal peptide for cotranslational transport into the lumen of the endoplasmic reticulum, where it is N-glycosylated at seven glycosylation sites, resulting in a glycosylated precursor with an apparent molecular mass of 110 kDa.The intracellular processing of GAA has been investigated previously (2, 3). It was proposed that, after transport through the Golgi complex and targeting to the endosome/lysosome, the 110-kDa precursor is proteolytically processed at the amino terminus, resulting in a 95-kDa intermediate with a sequence beginning at amino acid 122. Prior to this study, the 95-kDa intermediate was proposed to be proteolytically processed to a 76-kDa form, which was believed to occur between amino acids 816 and 881 (3). The 76-kDa form is then proteolytically processed at the amino terminus at amino acid 204 to give the 70-kDa mature form (3). The nomenclature used for the processed forms of GAA is based on apparent molecular mass as determined by SDS-PAGE.The identities of the proteases involved in the maturation of GAA have never been established. GAA has been purified from many different tissues such as bovine testis (4), rat liver (5), pig liver (6), human liver (7), rabbit mus...

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...

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