Regulation of cyclic nucleotide-dependent protein kinase activity by histones and poly(L-arginine).

G.M Walton,G.N Gill

doi:10.1016/s0021-9258(19)69861-3

Abstract

Interaction of cGMP-dependent protein kinase with histones H2A, H2B, H3, and H4, or poly(L-arginine) resulted in changes in enzyme conformation such that inactivation of cGMP binding and activation of basal catalytic activity (assayed without cGMP) occurred. Total kinase activity as determined by phosphorylation of exogenous substrates subsequently decreased, but autophosphorylation of the enzyme was enhanced. The reaction was specific for nucleosome core histones and poly(L-arginine); H1, troponin, and poly(L-lysine) had no effect. Inactivation of cyclic nucleotide binding sites followed pseudo-first order kinetics and, at various histone concentrations, exhibited saturation kinetics at low ionic strength (2 mM potassium phosphate, pH 6.8), but non-saturation kinetics at higher ionic strength (37.5 mM potassium phosphate, pH 6.8, 12.5 mM MgCl2). Saturation kinetics was observed with poly(L-arginine) at both low and high ionic strength. Kinetic parameters measured under saturation conditions were determined for each core histone and poly(L-arginine). Core histones and poly(L-arginine) were noncompetitive inhibitors of cGMP binding; core histones and poly(L-arginine) interacted competitively at an enzyme site designated as the poly(L-arginine) binding site. Regulatory subunits of cAMP-dependent protein kinase contain a similar poly(L-arginine) binding site. Modulator proteins bind to poly(L-arginine) or arginyl residues in histone to prevent interaction with the poly(L-arginine) binding site on the enzymes. Through this mechanism, modulator proteins maintain cyclic nucleotide dependency and full enzyme activity.

Highlights

Total kinase activity as determined by phosphorylation kinase activity is irreversible and, noevidence exists of exogenous substrates subsequently decreased, but autophosphorylation of the enzyme was enhanced
Saturation kinetics was observed with poly@-arginine) Arginyl residues are essential for the modifying activity of at both low and high ionic strength
Binding of Cyclic Nucleotides-Duplicate assays were performed in 0.5-ml plastic tubes in 0.05-ml reaction mixtures containing 30 mM potassium phosphate, 10 mMMgC12, 2 mM dithiothreitol, 10 mg/ml of bovine serum albumin, 0.4 PM [3H]cGMPor [3H]cAMPand varying amounts of cGMP-dependent protein kinase or RII.Equilibrium was achieved after incubation at 0°C for 30 min; protein-bound [3H]cGMP and [3H]cAMP wereisolated on Millipore filters [25, 26]

Summary

Purification Procedures

CGMP-dependent protein kinase was purified to homogeneity from bovine lung using 8-(2-aminoethyl)-amino-cAMP-Sepharosaes an affinity chromatography media [9, 10]. cGMP was removed by sedimenting the enzyme on glycerolgradients followed bytreatment with charcoal [5, 11]. Binding of Cyclic Nucleotides-Duplicate assays were performed in 0.5-ml plastic tubes in 0.05-ml reaction mixtures containing 30 mM potassium phosphate (pH 6.8), 10 mMMgC12, 2 mM dithiothreitol, 10 mg/ml of bovine serum albumin, 0.4 PM [3H]cGMPor [3H]cAMPand varying amounts of cGMP-dependent protein kinase or RII.Equilibrium was achieved after incubation at 0°C for 30 min; protein-bound [3H]cGMP and [3H]cAMP wereisolated on Millipore filters [25, 26]. Interaction of cGMP-dependentProteinKinase or Regulatory Subunits of CAMP-dependentProtein Kinase with Poly(L-arginine) or Histones-cGMP-dependent protein kinase (0.23 to 2.3 pg/ml) or RII(18units/ml) was incubated with varying concentrations of poly(Larginine) or histone at 30°C in a reaction mixture containing 37.5 mM potassium phosphate (pH 6.8), 12.5mMMgC12,2.5mM dithiothreitol, 12.5mg/ml of bovine serum albumin (high salt buffer).Alternatively, incubation was carried out in a buffer containing 2 mM potassium phosphate (pH 6.8), 2.5 mM dithiothreitol, 12.5 mg/ml of bovine serum albumin (lowsalt buffer). Results are plotted as ti,' (the time of50% inactivation of binding sites) uersus the reciprocal inhibitor concentration [27]

RESULTS

Modulator protein

EffectPs roonKteininase

DISCUSSION