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 Thr-197 phosphorylation of newly synthesized protein kinase A in intact S49 mouse lymphoma cells is both efficient and insensitive to activators or inhibitors of intracellular protein kinase A. Using [35S]methionine-labeled, nonphosphorylated, recombinant catalytic subunit as the substrate in a gel mobility shift assay, we have identified an activity in extracts of protein kinase A-deficient S49 cells that phosphorylates catalytic subunit on Thr-197. The protein kinase A kinase activity partially purified by anion-exchange and hydroxylapatite chromatography is an efficient catalyst of protein kinase A phosphorylation in terms of both a low Km for ATP and a rapid time course. Phosphorylation of wild-type catalytic subunit by the kinase kinase activates the subunit for binding to a pseudosubstrate peptide inhibitor of protein kinase A. By both the gel shift assay and a [gamma-32P]ATP incorporation assay, the enzyme is active on wild-type catalytic subunit and on an inactive mutant with Met substituted for Lys-72 but inactive on a mutant with Ala substituted for Thr-197. Combined with the results from mutant subunits, phosphoamino acid analysis suggests that the enzyme is specific for phosphorylation of Thr-197.

FIG. 1

The Thr-197 phosphorylation of C subunits expressed in E. coli is limited by the intracellular activity of C subunit and inhibitable with H-89. E. coli BL21(DE3) containing both a wild-type C-subunit expression plasmid and the yeast N-myristoyltransferase expression plasmid pBB131 were induced in minimal medium at about 24°C for 45 (lanes a and b), 67 (lanes c and d), or 90 (lanes e and f) min before addition of rifampin and incubation an additional hour as described in Materials and Methods. Samples of 250 μl were then shifted to 37°C and incubated for 5 min without (lanes a, c, and e) or with (lanes b, d, and f) 100 μM H-89 before addition of 10 μCi of [³⁵S]methionine and labeling for 10 min at this temperature. Additional samples without H-89 were incubated in parallel but without the addition of radioactivity for determinations of C-subunit specific activity (see text). Cells were extracted by sonication, and about 5,000 cpm of soluble extract protein was subjected to SDS-PAGE. Shown are C-subunit patterns from a 4-day autoradiographic exposure of the resulting gel. Positions of the Thr-197-phosphorylated (C_P) and nonphosphorylated (C_N) forms of C subunit are indicated.

FIG. 2

Thr-197 phosphorylation of newly synthesized C subunit is rapid in cyclase-negative S49 cells whether or not they are stimulated with a cAMP analog. Cyclase-negative S49 cells were pulse-labeled for 10 min with [³⁵S]methionine in the absence (lanes a, c, and e) or presence (lanes b and d) of 100 μM CPT-cAMP and either harvested immediately (lanes a, b, and e) or chased for 30 min after dilution with conditioned medium without or with drug (lanes c and d). Samples for lanes a to d were extracted with HEB and immunoadsorbed in NaF-RIPA buffer, while that for lane e was extracted in EB and immunoadsorbed in RIPA buffer, as described in Materials and Methods. The radiolabeled C subunit species were resolved by SDS-PAGE and visualized by fluorography. Positions of the Thr-197-phosphorylated (Cα_P and Cβ_P) and nonphosphorylated (Cα_N and Cβ_N) forms of Cα and Cβ subunits are indicated.

FIG. 3

An extract of kinase-negative S49 cells can apparently phosphorylate recombinant C subunit. Soluble protein from kinase-negative S49 cells was mixed with [³⁵S]methionine-labeled recombinant C subunit to give about 3.0 (lanes a and f), 1.5 (lane b), 0.75 (lanes c and g), 0.38 (lane d), or 0.19 (lanes e and h) mg of extract protein per ml in 15 mM Tris-HCl (pH 7.5)–10 mM 2-mercaptoethanol–0.15 mM EDTA–11 mM magnesium sulfate–1 mM ATP–150 mM sodium fluoride–0.15 mg of bovine serum albumin per ml. Samples for lanes a to e were incubated for 1 h at 30°C before mixing with SDS-gel sample buffer, while those for lanes f to h were stopped immediately by addition of the SDS-containing buffer. Samples were analyzed as described in Materials and Methods, and positions of Thr-197-phosphorylated (C_P) and nonphosphorylated (C_N) forms of C subunit in the resulting autoradiographic patterns are indicated as for Fig. 1.

FIG. 4

Partial purification of PKA kinase activity by anion-exchange chromatography. An extract of kinase-negative cells was fractionated by chromatography on Accell Plus QMA, and fractions were assayed for absorbance at 280 nm (solid line), conductivity (dotted line), and PKA kinase activity (•) as described in Materials and Methods. PKA kinase activity is expressed as the percentage of total C-subunit radioactivity in the phosphorylated form after incubation under standard conditions (Materials and Methods).

FIG. 5

The PKA kinase activity shifts the SDS-PAGE mobility of wild-type and Lys-72→Met mutant C subunits but not that of a Thr-197→Ala mutant C subunit. [³⁵S]methionine-labeled wild-type (lanes a to c), Lys-72→Met (lanes d to f), or Thr-197→Ala (lanes g to h) C subunit were either incubated for 1 h at 30°C without PKA kinase (lanes a, d, and g) or mixed with a partially purified preparation of PKA kinase (first peak from Accell Plus QMA purified further by chromatography on ceramic hydroxylapatite and heparin-Sepharose) and incubated for 0 (lanes b, e, and h) or 1 (lanes c, f, and i) h at 30°C under standard conditions (Materials and Methods). Samples were analyzed by SDS-PAGE, and the positions of Thr-phosphorylated (C_P) and nonphosphorylated (C_N) forms of wild-type C subunit are indicated in autoradiographic patterns as in Fig. 1 and 3).

FIG. 6

The PKA kinase activity has an apparent K_m for ATP of about 12 μM. The ATP dependence of partially purified PKA kinase (first peak) was assessed under the standard assay conditions described in Materials and Methods but with 5 mM free magnesium sulfate and various concentrations of an equimolar mixture of ATP and magnesium sulfate. Data shown are the results from densitometric analysis of SDS-PAGE patterns and are expressed as the percentage of total C-subunit radioactivity in the phosphorylated form (C_P) as for Fig. 4.

FIG. 7

The initial phase of C-subunit phosphorylation by the PKA kinase is quite rapid. Partially purified PKA kinase (first peak) was mixed with [³⁵S]methionine-labeled C subunit in standard assay buffer (Materials and Methods) and incubated for various times at 30 (•) or 37°C (○) before analysis of C-subunit phosphorylation as for Fig. 6.

FIG. 8

PKA kinase can catalyze transfer of [³²P]phosphate from [γ-³²P]ATP to wild-type or Met-72 C subunit but not to Ala-197 C subunit. Bacterial extracts containing unlabeled, nonphosphorylated wild-type (lanes a to d), Met-72 (lanes e to h), or Ala-197 C (lanes i to k) subunit were incubated with [γ-³²P]ATP and active C subunit (lanes a, e, and i), no enzyme (lanes b, f, and j), PKA kinase (lanes c, g, and k), or PKA kinase and H-89 (lanes d and h) as described in Materials and Methods. Samples were subjected to SDS-PAGE and visualized by either Western immunoblot detection using an anti-C subunit antibody (A) or autoradiography (B). Only portions of gel patterns containing C subunits are shown, and positions of the Thr-197-phosphorylated (C_P) and nonphosphorylated (C_N) forms of wild-type C subunit are indicated as for Fig. 1, 3, and 5.

FIG. 9

PKA kinase-dependent phosphorylation of C subunit is specific for threonine. Wild-type (lanes a to c) or Met-72 (lanes d and e) C subunit was incubated with active C subunit (lane a) or with partially purified PKA kinase in the absence (lanes b and d) or presence (lanes c and e) of H-89 as for Fig. 8 but with 10 times the amounts of [γ-³²P]ATP (Materials and Methods). The C subunits were resolved by SDS-PAGE, excised from dried gels, and hydrolyzed with hydrochloric acid to allow analysis of their labeled phosphoamino acids by thin-layer electrophoresis. Ser-P and Thr-P indicate positions of unlabeled phosphoserine and phosphothreonine markers in the resulting autoradiographic patterns.

FIG. 10

PKA kinase-mediated phosphorylation of wild-type, but not of Met-72 mutant, C subunit activates the protein for binding to a pseudosubstrate inhibitor peptide. [³⁵S]methionine-labeled preparations of wild-type (lanes a to d) or Met-72 mutant (lanes e to h) C subunit were incubated for 1 h at 30°C without (lanes a, c, e, and g) or with (lanes b, d, f, and h) a partially purified preparation of PKA kinase under standard conditions but with only 100 μM ATP. Samples were either diluted immediately with SDS-gel sample buffer for SDS-PAGE analysis (lanes a, b, e, and f) or purified on columns of PKIP-Sepharose as described in Materials and Methods (lanes c, d, g, and h). For the samples taken before affinity column purification, equal amounts of protein radioactivity (∼10,000 cpm) were loaded onto the SDS-polyacrylamide gel. For the samples bound to—and subsequently eluted from—the affinity columns, equal proportions (∼23%) were subjected to gel analysis, although twice as much mutant protein radioactivity had been loaded onto the columns (Materials and Methods). Portions of autoradiographic patterns containing C subunits are shown, and positions of the Thr-197-phosphorylated (C_P) and nonphosphorylated (C_N) forms of wild-type C subunit are indicated as in Fig. 1, 3, 5, and 8).

FIG. 11

A number of protein kinases related to PKA have targets for Thr phosphorylation in the activation loop region in subdomain VIII that are followed by highly conserved sequences. Subdomain VIII sequences and the residues immediately upstream are shown for PKA, CGK, PKCβ, CAMK I and IV, and AMPK as described by Hanks et al. (13), using additional sequence data from references and . Thr-197 of PKA C subunit is indicated with an asterisk, and residues conserved among PKA, CGK, PKCβ, CAMK I, and CAMK IV are shown in boldface.