Abstract
All mammalian cGMP-dependent protein kinases (PKGs) are dimeric. Dimerization of PKGs involves sequences located near the amino termini, which contain a conserved, extended leucine zipper motif. In PKG Ibeta this includes eight Leu/Ile heptad repeats, and in the present study, deletion and site-directed mutagenesis have been used to systematically delete these repeats or substitute individual Leu/Ile. The enzymatic properties and quaternary structures of these purified PKG mutants have been determined. All had specific enzyme activities comparable to wild type PKG. Simultaneous substitution of alanine at four or more of the Leu/Ile heptad repeats ((L3A/L10A/L17A/I24A), (L31A/I38A/L45A/I52A), (L17A/I24A/L31A/I38A/L45A/I52A), and (L3A/L10A/L45A/I52A)) of the motif produces a monomeric PKG Ibeta. Mutation of two Leu/Ile heptad repeats can produce either a dimeric (L3A/L10A) or monomeric (L17A/I24A and L31A/I38A) PKG. Point mutation of Leu-17 or Ile-24 (L17A or I24A) does not disrupt dimerization. These results suggest that all eight Leu/Ile heptad repeats are involved in dimerization of PKG Ibeta. Six of the eight repeats are sufficient to mediate dimerization, but substitutions at some positions (Leu-17, Ile-24, Leu-31, and Ile-38) appear to have greater impact than others on dimerization. The Ka of cGMP for activation of monomeric mutants (PKG Ibeta (delta1-52) and PKG Ibeta L17A/I24A/L31A/I38A/L45A/I52A) is 2- to 3-fold greater than that for wild type dimeric PKG Ibeta, and there is a corresponding 2- to 3-fold increase in cGMP-dissociation rate of the high affinity cGMP-binding site (site A) of these monomers. These results indicate that dimerization increases sensitivity for cGMP activation of the enzyme.
Highlights
Cyclic GMP-dependent protein kinase (PKG)1 is one of the major intracellular receptors for cGMP [1, 2]
Point mutation of Leu-17 or Ile-24 (L17A or I24A) does not disrupt dimerization. These results suggest that all eight Leu/Ile heptad repeats are involved in dimerization of PKG I
Lower eukaryotes have monomeric PKGs that lack the leucine zipper motif; PKG from Paramecium has been shown to be a monomer [31], but this kinase retains functional activities of PKG seen in PKGs of higher eukaryotes, careful studies of cGMP binding and activation of this PKG compared with the mammalian isoforms are lacking
Summary
PKG I Site-directed Mutagenesis—Human PKG I (hcGKI) clone encodes a full-length human placental PKG I. Determination of Stokes Radius—Purified PKG (ϳ7–10 g) was combined with two internal standards, crystalline catalase (3 mg), and ovalbumin (4 mg), in a volume of 200 l and loaded onto a Sephacryl S-200 gel filtration column (0.9 ϫ 35 cm) equilibrated in KPEM and 150 mM NaCl at 4 °C. Determination of Sedimentation Coefficient—Purified PKG (ϳ7–10 g) was combined with two internal standards, crystalline phosphorylase-b (3 mg), and hemoglobin (0.5 mg), in a volume of 200 l in KPEM/ 0.15 M NaCl with protease inhibitors and applied to a 13-ml linear 5–20% sucrose gradient containing KPEM/0.15 M NaCl. The gradients were centrifuged at 37,000 rpm in a Beckman SW 41 rotor for 30 – 44 h at 4 °C, and fractions (0.5 ml) were collected from the bottom of the tubes.
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