Abstract
Cyclic nucleotides are shown to stimulate the autophosphorylation of type II cGMP-dependent protein kinase (cGK) on multiple sites. Mass spectrometric based analyses, using a quadrupole time-of-flight-mass spectrometry instrument revealed that cGMP stimulated the in vitro phosphorylation of residues Ser110 and Ser114, and, at a slow rate, of Ser126 and Thr109 or Ser117, all located in the autoinhibitory region. In addition Ser445 was found to be phosphorylated in a cGMP-dependent manner, whereas Ser110 and Ser97 were already prephosphorylated to a large extent in Sf9 cells. cGMP-dependent phosphorylation of cGK II was also demonstrated in intact COS-1 cells and intestinal epithelium. Substitution of most of the potentially autophosphorylated residues for alanines largely abolished the cGMP stimulation of the autophosphorylation. Prolonged autophosphorylation of purified recombinant cGK II in vitro resulted in a 40-50% increase in basal kinase activity, but its maximal cGMP-stimulated activity and the EC50 for cGMP remained unaltered. Mutation of the major phosphorylatable serines 110, 114, and 445 into "phosphorylation-mimicking" glutamates had no effect on the kinetic parameters of cGK II. However, replacing the slowly autophosphorylated residue Ser126 by Glu rendered cGK II constitutively active. These results show that the fast phase of cyclic nucleotide-stimulated autophosphorylation of cGK II has a relatively small feed forward effect on its activity, whereas the secondary phase, presumably involving Ser126 phosphorylation, may generate a constitutively active form of the enzyme.
Highlights
Cyclic nucleotides are shown to stimulate the autophosphorylation of type II cGMP-dependent protein kinase on multiple sites
Replacing the slowly autophosphorylated residue Ser126 by Glu rendered cGMP-dependent protein kinase (cGK) II constitutively active. These results show that the fast phase of cyclic nucleotide-stimulated autophosphorylation of cGK II has a relatively small feed forward effect on its activity, whereas the secondary phase, presumably involving Ser126 phosphorylation, may generate a constitutively active form of the enzyme
The incorporation of phosphate into cGK II was due to intramolecular autophosphorylation, because it was minimally affected by a 10-fold dilution of the enzyme or by immobilization of the His-tagged cGK II on a nickel column
Summary
Hexahistidine tag, was cloned into the BamHI site of pFastBac (Invitrogen), yielding the cGK II bacmid transfer vector pFB1His6cGK II. Expressed rat cGK II and cGK II mutant proteins solubilized from COS cell membranes (0.4 –1 mg of protein/ml) were incubated for 2 min at 30 °C in 25 mM Tris/HCl, pH 7.4, 10 mM MgCl2, 5 mM -mercaptoethanol, 0.1 mM 3-isobutyl-1-methylxanthine, 25 mM sodium -glycerophosphate, 1% Triton X-100, 0.15 M NaCl, and 10 M [␥-32P]ATP (3 Ci/nmol). Protein kinase activity was determined by incubation of the samples (4 ng of control or autophosphorylated His-cGK II or 4 –10 g of COS membrane protein in case of transiently expressed rat cGK II or mutants thereof) at 30 °C for 4 min in 40 l of 20 mM Tris/HCl, pH 7.4, 0.15 M NaCl, 10 mM MgCl2, 5 mM -mercaptoethanol, 0.1 mM 3-isobutyl-1-methylxanthine, 25 mM. After removal of the cGMP analogue the decay of the ISC back to basal levels was monitored
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