Autophosphorylation of cGMP-dependent protein kinase type II.
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.
Vaandrager
Arie B
AB
Department of Biochemistry, Erasmus University Medical Center Rotterdam 3000 DR, The Netherlands. A.B.Vaandrager@vet.uu.nl
Hogema
Boris M
BM
Edixhoven
Marcel
M
van den Burg
Caroline M M
CM
Bot
Alice G M
AG
Klatt
Peter
P
Ruth
Peter
P
Hofmann
Franz
F
Van Damme
Jozef
J
Vandekerckhove
Joel
J
de Jonge
Hugo R
HR
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001665:Binding Sites; D019556:COS Cells; D062967:Cyclic GMP-Dependent Protein Kinase Type II; D017869:Cyclic GMP-Dependent Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D004848:Epithelium / Q000201:enzymology; D015870:Gene Expression; D006801:Humans; D007422:Intestines / Q000201:enzymology; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D051381:Rats; D011993:Recombinant Fusion Proteins; D012694:Serine / Q000378:metabolism; D018411:Spodoptera / Q000378:metabolism; D014162:Transfection
D011993:Recombinant Fusion Proteins; D012694:Serine; D062967:Cyclic GMP-Dependent Protein Kinase Type II; D017869:Cyclic GMP-Dependent Protein Kinases; C569729:PRKG2 protein, human; C569730:Prkg2 protein, mouse; C569732:Prkg2 protein, rat